Conformational Flexibility in Nucleic Acids Investigated by NMR … · 2010. 4. 15. · nucleic...

Conformational Flexibility in Nucleic Acids

Investigated by NMR Relaxation

Thomas L. James

Department of Pharmaceutical Chemistry,University of California,

San Francisco, CA 9 ^ 3 USA

I . I ntroducti on

II. Basis For Using NMR To Study Nucleic Acid MotionsA. TheoryB. Possible Molecular Motions with Nucleic AcidsC. Approach for Nucleic Acids

III. Molecular Motion In Uncompl e.xed Nucleic AcidsA. DNAB. RNA and Polyribonuc1eotidesC. tRNA

IV. Nucleic Acid-Protein ComplexesA. Nucleosomes and ChromatinB. Ribosomes and Messenger RibonucleoproteinsC. Viruses and Other Complexes

V. Nucleic Acid-Drug ComplexesVI. Conclus ions

References

Paae119

120120127129

131139H2

U8H8150151

156

I INTRODUCTION

The importance of the conformationaiflexibility of biopolymers in terms oftheir functions is gaining recognition.The central cellular functions of rep-lication, transcription, and transla-tion quite plausibly involve distortionof the conformation of DNA and RNA.*

"Abbreviations used are: DNA, deoxyri-bonucleic acid; RNA, ribonucleic acid:poly(l), polyinosinic acid; poly(C),polycytidy1ic acid; poly(G), polygua-nylic acid; poly(U), polyuridylic acid;

Vol. k, No. 3/4

All of the different steps in theseprocesses involving nucleic acids,including control steps, require recog-nition of unique sites on the nucleicacids, which may be short-lived. Thepackaging of polynuc1eotides into com-pact forms in chromatin, ribosomes, andviruses involves folding of the nucleicacids. These and other observationsindicate that the deformation of poly-nucieotide structure is intimatelyrelated to the biological role ofnuc1e i c acids.

CSA, chemical anisotropy.

In recent years the scientificcommunity has had available a staticpicture of nucleic acids provided pri-marily by x-ray diffraction studiessupplemented by spectroscopic investi-gations, chiefly NMR. Since NMR iswell-suited for study of dynamic pro-cesses, it should be a primary tool inthe development of a motion picture ofthe flexibility of nucleic acids.

Several experimental investigationsof the conformational flexibility ofnucleic acids have been carried out.Hydrodynamic studies indicate thatdouble-stranded nucleic acids withchain lengths greater than 50° A* (150base pairs, 105 daltons) are neitherfreely flexible random coils nor com-pletely stiff rods (1-3)- Rather, thesemiflexible double-stranded nucleicacids are. often described as wormlikechains {h) . Motions which permitexchange of the hydrogen-bonded protonsof polynucleotides have been studied bytritium exchange (5.6) and, in moredetail, by NMR (7,8). These methodsare useful for assessing the availabil-ity of ami no and imino protons to aque-ous solvent on the time scale of milli-seconds to seconds for NMR and of manyseconds to minutes for tritiumexchange. Temperature jump experimentsmonitoring various physical propertieshave yielded information regarding therate of base pair formation, which isin the time scale of milliseconds (3).

Assuming that intercalated dyesreflect the motional properties ofnucleic acids, it has been possible toobtain motional information from ESRand from fluorescence decay. The decayof the fluorescence anisotropy of ethi-diurn bound to DNA has implied substan-tial local motion of the bases existson the time scale of tens of nanose-conds (9.10) • Motion in that same timerange has been found via ESR for spin-labeled acridine dyes bound to DNA(11).

Recent theoretical calculations alsopertain to motions in nucleic acids.Barkley and Zimm (10) have developeddynamical equations describing thebending and twisting of elastic chainmacromolecules. From a completely

120

different viewpoint, Levitt (12) hasused empirical energy functions to cal-culate the energy for bending DNA.Further calculations have indicatedthat the ribose in polyribonuc1eotidesis conformational1y flexible (13)-

Although these other techniques haveindicated that nucleic acids are notthe static structures depicted in bio-chemistry textbooks, NMR relaxationexperiments are especially suitable forinvestigating nucleic acid motions inthe 10" 1 1 to 10~ 5 s time range; conse-quently, NMR studies are providing con-siderable information about the localconformationa1 flexibility in nucleicacids. This review examines the con-tributions of NMR to knowledge ofnucleic acid dynamics. Of prime con-sideration are structural variations,environment, drug binding, and bindingof proteins in functional nucleic acidcomplexes. The reader is referred tothe literature for recent usefulreviews pertaining to nucleic acidstructure and interactions (1^,15).including drug binding (16).

II. BASIS FOR USING NMR TO STUDYNUCLEIC ACID MOTIONS

The following discussion is con-cerned with the means by which one canextract information about motions innucleic acids from NMR relaxationparameter measurements. The variouspotential motions in nucleic acids willbe considered. Those complications andlimitations which are apparent in thisearly stage of such studies will beci ted.

A.

1. Relaxation Parameters

Motions in nucleic acids producerandom, fluctuating magnetic and elec-tric fields, some of which may influ-ence the relaxation of nuclei on thenucleic acid which are interacting withthe fluctuating fields. Depending onthe prevalent nuclear relaxation

Bulletin of Magnetic Resonance

parameters to spectral densitiesJn(a>) which contain the motionalinformation. Although other relaxationmechanisms may be important, for illus-trative purposes we can consider inmore detail the dipolar relaxation ofnuclei (other than hydrogen) which arecoupled to hydrogen nuclei; such relax-ation is very important for 1 3 C , 3 1 P ,1 5 N , and 1 9 F . In this case, theexpressions for the spin-lattice relax-ation time (TO , spin-spin relaxationtime (T2), nuclear Overhauser effect(NOE),* rotating frame spin-latticerelaxation time in an off-resonance rffield ( T l p

o f f ) , and off-resonanceintensity ratio (R)(17-20)

3 J 1 {u> |) +6 J 2 |) ]

are given by

I/T1 = K[Jo (a>H-U| |

6J 2 (a>|_j+W|) ]

1/Tz = I/2T1 + K[2Jo (0)+3Ji

= ^

NOE = I + (TH/T|)

( [6J 2 (a) +U!| ) -J 0 (u> |-

(1)

(2)

I [J 0 (tt>^-CO| ) + 3 J 1 (u)| ) + 6 J J (6J|_|+<

1/Tlpoff = Ksin*0[2Jo(«e) +

(3/2) Ji (u.'H+a>e) +

(3/2)Ji (w H-« e)] + 1/Ti

R = [Jo (wH-wj)+3Ji (a>|) +

6j 2 (a>n+W|) ] /

(3)

[2s i n +Jo

"Unless specifically stated otherwise,any mention to NOE in this reviewrefers to observation of the heteronu-cleus 1 response while applying broad-band irradiation to all protons, e.g.,the usual nomenclature for 1 3 C is1 3 C { a H } NOE.

where K = Nfi2-yH2-/| 2 / 2 0 r '

(5)

(6)

YH and ->-| are the respective gyro-magnetic ratios of hydrogen and thenucleus being relaxed, w^ anc' wlare the respective angular Larmor fre-quencies of hydrogen and the relaxingnucleus, r is the distance between thenucleus and the hydrogen causing relax-ation, N is the number of hydrogen nuc-lei effecting relaxation, and Wu isthe linewidth. The other terms are

6 = tan"1 (7|Hi/27rvoff)

and

(7)

(8)

where u>e is the angular frequencyabout the effective field produced byapplication of the rf field Hi at a

Equa-i s appli ed

>

frequency "off off-resonance.h l if Hif Hi

i.e.o

tion h is valid onlyfar off-resonance,T|Hi.

Most of the relaxation parametersare well-known in NMR spectroscopy.The off-resonance relaxation parametersT off and R are less familiar but havebeen discussed previously (19~22). Thevalue of R is obtained as the ratio ofthe intensity of a resonance in thepresence to that in the absence of anoff-resonance rf field. The R value,as the NOE, is not explicitly dependenton the internuclear distance r [viz.equat i on 53•

The expressions given above pertainto relaxation via dipolar couplingbetween unlike nuclei. Similarly,motional information can be obtainedfrom nuclei relaxing via electric quad-rupolar interactions (23.24), chemicalshift anisotropy (21,25). scalar inter-actions (23,24,26), and homonucleardipolar coupling (22-24). From analternative viewpoint, the possibilityof different mechanisms contributing tothe relaxation of a nucleus presentsconsiderable complications to theinterpretation of relaxation data. Inthis regard, a perfidious mechanism isthat of relaxation caused by the pres-ence of paramagnetic ions; it will be

Vol. h, No. 3A 121

assumed that efforts have been made toremove any paramagnetic metal ionswhich could contribute to relaxation ofnuclei on nucleic acids.

2. Spectral Densities and MolecularMotions

The rate of the molecular motioneffecting relaxation is usuallyexpressed in terms of a correlationtime {2k); so the spectral densitiesmay also be expressed in terms of cor-relation times. In the simplest case,i.e., that of random isotropic motion,the spectral densities are

Jn(w) (9)

where rr is the rotational correla-tion time. It should be apparent fromequations 1-5 that the different spec-tral densities vary in their influenceon the different relaxation parameters.This is illustrated in Figure 1 for 1 3Crelaxed by a single proton 1.11 A awayassuming the motion is random and iso-tropic. The parameters' obvious 1y varyin their sensitivity to motions withdifferent rates which are characterizedby different correlation times. It isworth noting at this point that theparameters are dependent on u accordingto equation 9. so spectrometer fre-quency is a variable which can be used.As shown in Figure 1, the off-resonanceparameters are additionally dependenton the off-resonance frequency, whichalso can be a useful variable.

Although some relaxation data fornucleic acids have been interpretedassuming the motion contributing torelaxation is simple isotropic motion,it is usually necessary to consider themotion of nucleic acids as being morecomplicated. The usual procedure is tocalculate spectral densities, and sub-sequently relaxation parameters, usingvarious reasonable motional models.The measured relaxation parameters arecompared with the calculated values tosee if the model can fit the data.Such a procedure will not necessarily

produce a unique model; however, use ofas many relaxation parameters as possi-ble measured at different spectrometerfrequencies severely constrains thepossible motional models which will fitthe data. To further clarify the type,amplitude, and frequency of motion, theresults from several nuclei on thenucleic would need to be consistent.

We can consider the form of thespectral densities for some of the pos-sible motional models. Some of themodels yield sufficiently simple mathe-matical expressions that equations willbe given. The possible use of moresophisticated models will be consideredwith reference to the literature forthe details. The simplest model, thatof random, isotropic motion, hasalready been given [viz. equation 93 •

3. Internal Rotation with Overall Iso-tropic Reorientation

The case of random rotational diffu-sion of a spin pair about an axis ofinternal rotation which itself is reo-rienting isotropically has been devel-oped by Woessner (27) and subsequentlyapplied to motions in macromolecules(17.19.25). The spectral densities forthis model are

Jn(«) = A[2ro/(l+«2To2)]

+ B[2T B / (1+« 2 T B2 ) ]

+ C[2rc/(l+w2rc2)] (TO)

wi th

A = U A ) (3cos*o-l) 2

T B = [ 1 / T O + 1 / ( 6 T J ) ] - l

B = ( 3 A ) s i n 2 2 a

rc=[l/ro + 2/Or-,)]"1 (11)

C = (3A)sin4a

where a is the angle between the I-Hinternuclear vector and the axis of

122 Bulletin of Magnetic Resonance

RELAXATIONRATE(1/S)

100

10

1

0,1

Mil

l

11 llllll

II1

lllllll

V1

1

1

1

|

1 1 =

, 5 K H ^ 1

lllllll

1

1 1

10*9 10-7rr(s)

10-5

NOE

0

I

b (3.5 KHz)I I I

c (5.0 KHz)

10-9 10-7

Tr(S)10-5

Figure 1. Theoreticalfor 13C at 25-1^ MHz,

values of the NMRcalculated using

relaxation parameters from equations 1-6a single correlation time rr for random,

isotropic motion [c_f. equation 93- The calculations assumed a vibrationally-aver-aged C-H internuclear distance of 1.11 A. The curves for 1/T^p

of^2nd R were cal-culated using an rf field strength Hi of 0.h2 G and off-resonance frequencies

of 3-5 and 5.0 KHz.voff

Vol. k, No. Ilk 123

RELAXATION OF TETRAHEDRAL 13C (<X= 70.5°)

(1/S)

100

10

1

0.1

lllll 11 1

f

I 1 1

1 1 1T.($)E

—. io-»:3 x 1Q-«_

\ ^ 10-71V v 3 x 10-7-Vs*- -=

\1O-*:

1 1 I

NOE

10-9 10-7 10-5I J L

1 1 I I —

10-9 10-7 10-5Tj(S)

10-7H

l l l l l

10-" 10-9 10-7

r,(s)1000

w./a(Hz)

_ i i i i r _

ioo F

llo-n 10-9 10-7 10 5

Figure 2. Theoretical dependence of the NMR relaxation parameters for tetrahedral1 3C at 25.1^ MHz, calculated using the two correlation time model presented in thetext [cf. equations 10 and 11] with the angle a = 109.5°. as a function of the,internal motion correlation time TJ for a series of overall tumbling times rang-ing from 10"» to 10"* s. The calculations assumed the 1 3C relaxation is due todipolar coupling to a single hydrogen at a distance of 1.11 A. The off-resonanceparameters 1/T^p

off and R are calculated for an Hi field of 0.1*2 G applied3.5 KHz off-resonance.

121* Bulletin of Magnetic Resonance

II

i nternal rotation, i s thecorrelation time for the random reo-rientation of the I-H vector around theaxis of internal rotation, and TO isthe correlation time for the isotropicreorientation of the vector. Theinverses of the rotational diffusioncoefficients for the isotropic reorien-tation and the internal rotation are6ro and 6 T • , respectively.

Figure 2 illustrates the effects ofthis two correlation time model on thecalculated relaxation parameters. Thetheoretical curves show the dependenceof the relaxation parameters on theinternal motion correlation time for aseries of TO values ranging from 10"*to 10"' s. These curves are calculatedspecifically for the case of a 1 3Cnucleus relaxed by a single proton 1.11A away with rotation about a tetrahe-dral bond a's the internal motion (so a= 109-5° or 70.5°). Consequently, thecalculated parameters for the singleisotropic motion model and the two cor-relation time model can be compared inFigures 1 and 2. It is apparent thatthe values predicted by the two modelsvary considerably. Although use ofonly two measured relaxation parametersmay or may not permit a distinctionbetween the models, employment of addi-tional parameters will permit a dis-tinction between these two models.

k. Rotational Tumbling of a Rigid Rod

Woessner (28) also considered therotational reorientation of ellipsoidalmolecules. For the case of a rigid rodwith no internal motion, equation 10and modified equation 11 appropriatelydescribe the spectral densities. How-ever, in this case, the definitions ofthe angle a and the time constants mustbe changed. Angle a is the anglebetween the I-H internuclear vector andthe long axis of the rod. If theinverses of the diffusion coefficientsfor rotation about the short axis andabout the long axis of the ellipsoidare 6TJ_ and 6T||, respectively,then the expressions to use with equa-tion 10 are

= £5/(6rJ

TO = T±

- [1/(30

(12)

Of course, curves similar to thosein Figure 2 could be generated usingthis model. For sufficiently shortsegments of DNA, it may be possible toconsider the double-helix as a rigidrod and this model therefore to be use-ful (29,30). Figure 3 compares 3 1P Ti,linewidth, and 77 (=NOE-1) for the iso-tropic rotation model-and the rigid rodmodel with ?±/T« = 100.

5. Log Distribution of CorrelationTimes

For random coil polymers, the motioncan be described by a distribution ofcorrelation times (29,31)- Althoughvarious types of distribution could beused, Scheafer (31) employed the logchi-squared distribution function

F(s,p)ds = — (ps)P'1 e'Ps dsT(p) (13)

where p relates to width of the distri-bution, F(p) is a gamma function, and s= log(l + bTc/7) with T as an averagecorrelation time and b influencing thesymmetry and width of the distribution.The spectral densities are then "

00

frF(S,p)[(expb(s)-l)/(b-l)]dsfJust as the previous models had

three variables (i.e., two correlationtimes and a geometrical factor), thedistribution model also has three vari-ables T, b, and p. Figure 3 comparesthe distribution model with the singleisotropic correlation time model for acase in which p = 20 and b = 1000 whichgives a broad slightly asymmetric dis-tribution (29). It is clear that theeffect of the distribution is to (a)broaden the NMR resonance regardless of

Vol.. M, No. 125

-11 -10 -5

Logarithmic plots of

ill

Figure 3-linewidth, and NOE vs T C based ondifferent modej s at 109.3 MHz. Thesolid line represents isotropic motionwith a single correlation time, thedotted line corresponds to anisotropicrotation of an axially symmetric ellip-soidal rigid molecule [c_f. equations(1-3, 10-12] having the ratio of diffu-sion constants D|/Dj. = 100, andthe broken line represents the iso-tropic motion having a broad distribu-tion of correlation times (i.e., p = 20and b = 1000) (c_f. equations 13 and1^). Taken from reference (29)•

correlation time, (b) raise the Ti min-imum with a diminished slope, and (c)

decrease the NOE from its maximum inthe extreme narrowing limit (r « l/«)while maintaining a measurable NOE atlonger correlation times.

6. Restricted Internal Diffusion andJump Models

Internal motions in macromoleculesmight be expected to be more compli-cated than the above models. Freerotation about bonds is certainlyunfeasible for a double-stranded helix,although it could occur for a singlestrand of random coil polymer. Morerealistic models for motions in nucleicacids will restrict the amount of rota-tional freedom about particular bonds,such a model can simply impose boundaryconditions on any rotation, permittingfree diffusion within the boundaries.Alternatively, jumps between specifiedconformations also serves the purposeof limiting the motions to physicallyrealistic situations. With such mod-els, it is also possible to considerconcerted motions in which rotation ofa specified angle around one bondoccurs simultaneously with a rotationabout a second bond. This kind ofmotion can permit local motions whilemaintaining approximately the sameoverall structure. Jump models,restricted diffusion models, and multi-ple internal rotations models haverecently been described for side-chainmotions in proteins (32,33)•

7. Molecular Motions without a Physi-cal Model

The spectral density expressed inequation 10 is a linear sum of threeterms, each of which contains a coeffi-cient based on geometry (e.g., B) and atime constant (e.g., TQ) . The coef-.ficients and time constants, dependingon the type of motion, could beexpressed for example by equations 11or 12. It is obvious that if TJ «TO (or T|| « r j , equation 10 is


reduced to two terms, each of whichreflects only one of the motions. Ingeneral, as long as the motions areindependent and have widely differentcorrelation times, it is possible toexpress spectral densities as

Jn(«) (15)

wherecharacternumber of

Wt.cs the correlation timeof a motion, M is the

independent motions, and c:is a coefficient related to the geom-etry of the interaction promotingrelaxation and controls the contribu-tion of any particular motion to therelaxation.

It has recently been advocated byJardetzky and co-workers (30,34,35)that no specific motional models beassumed and that spectral densities ofthe form of equation 15 be employedwi th and as unknownparameters to be determined from themeasured NMR relaxation data; the num-ber of unknowns to be determined may bediminished if a T-. value for overalltumbling is taken from independent dataor theoretical expressions. Althoughthe C: values determined have nophysical meaning, such a procedureshould permit a determination of theminimum number of molecular motionscontributing to relaxation. It shouldbe pointed out, however, that even withthe assumption of a particular motionalmodel, in general it becomes quicklyevident whenever the data cannot be fitunless an additional motion isincluded. It may also be possible toobtain values for the correlation timesunder very special circumstances usingthis approach: it is required that (a)T J « T : + }, (b) the motions areuncorrelated, and (c) W T : > 1 . A majorproblem is that it would be difficultto know these conditions are fulfilleda pr i or i and it is not possible toascertain that the conditions are allmet if data is analyzed assuming theconditions hold.

B. POSSIBLE MOLECULAR MOTIONS WITHNUCLEIC ACIDS

If even some of the possible motionsof a double-stranded nucleic acid areoccurring simultaneously, the actualmotion picture of the nucleic acid mustbe quite complicated. The existence ofthese motions, however, could permitthe transient existence of conforma-tions required in the nucleic acid'sbiological functions. For severalyears, only two conformationai forms(the A form and the B form) of nucleicacids were generally considered asbiologically relevant. In the pastcouple years, research into the detailsof nucleic acid structure via theoreti-cal calculations (12,13). x-ray dif-fraction (36-39), NMR (39-^3) and othertechniques (44,45) has accentuated thelikely existence of variations (usuallysubtle) in DNA structure from B-DNA(46). Differences between the proposedstructures often entail low-energy con-formational changes. As a startingpoint for us, it is therefore reason-able to consider these structures andtheir low-energy variations as definingeither potential minima about whichstructural oscillations can occur or asdefining the boundaries between whichjumps can occur.

Figure 4 depicts some of the possi-ble motions in double-stranded nucleicacids which may influence NMR relaxa-tion parameters.. The end-for-end tum-bling and "speedometer cable" rotationindicated in Figure 4 become, respec-tively, rotation around the short axisand around the long axis of the ellip-soid if the nucleic acid is a. short,rigid rod as discussed above. Not muchexperimental data is available for the"speedometer cable" motion, but tran-sient electric dichroism studies ofrod-like DNA molecules have provideddata about the end-for-end rotationaldi f f us ion (47) .

The consequences of bending DNA havebeen considered theoretically (3.10).Relatively little energy is required tobend DNA. Consequently, it was pro-posed that bending motions may contrib-ute significantly to observed nuclear

Voi. 4, No. 3/4 127

relaxation in nucleic acids. It mightbe noted that since bending could occur

in any direction, it may be consideredas an isotropic motion. The low energy

(a) "SPEEDOMETER CABLE"ROTATION

TRANSITORY STRAND

SEPARATION "BREATHING*

ENO-FOR-END ROTATION

•pROPELLERl '--.TWISTING \

(b)

S'-cntf*

Figure k. Schematic representation of the motions in a segment of double-strandednucleic acid helix. In the 3'~endo conformation of the sugar ring, the 3' carbonis above the plane formed by Cl'-Q-CV.

necessary for bending has also recentlybeen manifest in the crystal structureof a DNA dodecamer which exhibits cur-vature with a radius of curvature 112Jt.

The so-called "breathing" ofdouble-stranded nucleic acids has beenamply demonstrated (5~8). This transi-tory strand separation occurs in thetime range milliseconds to seconds.

The dynamics ofbeen considered andsional rotation ofbeen developed (10) .

twisting has alsoequations for toi—

the DNA helix haveIndications are

that this motion can account for mostof the fluorescence depolarizationdecay of ethidium intercalated into DNA(10) which has a lifetime of 2k nsec(9). The relative ease of helix twist-ing may be reflected by the fact that


different values have been reported forthe number of base pairs per turn inDNA (12,38,46,51).

Of course, the motions of bending,twisting, and breathing entail somerotations about individual bonds in thenucleic acid; the effects of thesemotions will be manifest over a signif-icant segment of the helix. In addi-tion, local motions may occur which canalter the instantaneous conformationover a portion of the nucleic acidamounting to two monomeric units orless. The Cl'-N rotation in Figure 4could lead to twisting of the base pairpropeller with motions altering thepitch of the propeller. Recent theo-retical (12) and experimental 08,47)studies have indicated the existence ofa propeller twist in contrast to theclassical B-DNA structure with eachmember of a planar base-pair in thesame piane (46).

Relatively large deviations ininstantaneous local conformation can beobtained by concerted rotations aboutsome of the bonds (illustrated in Fig-ure 4) without large effect on theoverall conformation. For example, itis possible that one member of a basepair could temporarily move away fromthe other without substantially chang-ing the structure of the nucleic acid.

Most data indicate that RNA occursin the A form (with the ribose ring inthe 3'~endo conformation) and that DNAoccurs predominantly in the B form(with the deoxyribose ring in the2'-endo conformation) and sometimes inthe A form (52); these conformationsare shown in Figure 4b. A recent theo-retical calculation, however, indicatesthat the energy barrier between the3'~endo and the 2'-endo conformationsis only 0.6 kcal/mol (13) which is sig-nificantly less than single bond rota-tion in small alkanes. This suggeststhat there may be considerable mobilityin the sugar ring of nucleic acids withthe transient existence of many puck-ered conformations in the furanose ordeoxyfuranose rings which are in thenucleic acid backbone.

C. APPROACH FOR NUCLEIC ACIDS

To characterize the motions as com-pletely as possible, ideally severalNMR relaxation parameters should bemeasured as a function of magneticfield strength for several nuclei onthe nucleic acids. It is assumed that

remove any paramagneticwhich have a strong influ-relaxation (24), have beenOnce the relaxation mecha-

are known (videat least, simple)

efforts tometal - ions,ence on NMRsuccessful.nism or mechanismsinfra), feasible (or,motional models are proposed and corre-sponding spectral densities used in theappropriate mathematical expressionsfor the relaxation parameters. Themotional model is tested by its abilityto predict all of the parameters inagreement with the observed data. Itshould be emphasized that the NMRrelaxation data do not yield a uniquedescription of the motions. Rather,the NMR data serve to eliminate possi-ble motional models. For that reason,obtaining as much relaxation data aspossible will hopefully narrow the listof plausible models. Data from differ-ent locations or nuclei should also beconsistent, further corrstra i ni ng themotional models. Of course, it is sat-isfying to have the NMR data in accordwith the scant data from other techni-ques.

It should also be noted that one canobtain qualitative information from NMRregarding relative mobilities or eventhe existence of internal motion with-out a detailed knowledge of the dynam-ics. Semi quantitative information mayalso be obtained without knowledge ofthe precise motional amplitudes, e.g.,several models postulating differentinternal motions can all fit the datawith approximately the same correlationtime implying that a_n internal motionin that time range exists.

Proton NMR offers the ability tomonitor several points of the nucleicacid structure with high sensitivity,but suffers in the overlap of manyresonances and in the ability to inter-pret the relaxation data quantitativelydue to possible complications from spin

Vol. 4, No. 3/4 129

diffusion contributing to the relaxa-tion (53) •

Analysis of the l 3C NMR relaxationof protonated carbons is most readilyperformed since the relaxation is domi-nated by dipolar coupling to thhedirectly bonded protons. 1 3C relaxa-tion of quaternary carbons may alsohave a contribution from dipolarcoupling to 1 4N if the carbon isdirectly bonded to nitrogen and hasfewer than two hydrogens two bonds away(51). There exists also the possibil-ity of the chemical shift anisotropycontribution to 1 3C relaxation of qua-ternary carbons at higher magneticfield strengths. Consideration of1 5N NMR of nucleic acids is similar tothat of 1 3 C . 1 3C and 1 5N NMR do offerthe advantage of monitoring severalloci in the nucleic acid with a largechemical shift scale. Both also sufferthe drawback of low sensitivity, 1 5N toan extreme. It is possible to do natu-ral abundance 1 3C NMR on nucleic acids,but enriching with that isotope aidsthe NMR substantially. With 1 5N NMR,sensitivity is not good even with iso-tope labeling, but at least it is fea-sible.

For deuter i urn NMR, sensitivitynecessitates labeling. Analysis of thedeuterium NMR has the strong advantageof a single well-defined relaxationmechanism (electric quadrupolar) with aknown interaction strength. A distinctdisadvantage of doing 2H NMR on nucleicacids is the large linewidth created bythis relaxation mechanism whichincreases demands on the spectrometer.The large 1inewidth also generally pre-cludes observation of labels in morethan one location in a single experi-ment. 2H NMR has one less measurablerelaxation parameter since the NOEexists only with dipolar relaxation.But 2H NMR presents the possibi1ity ofproviding information about the order-ing in a nucleic acid fiber if quadru-pole splitting is observed.

3 1P NtlR offers the observation of asingle readily assignable resonancewith good sensitivity. [Under certaincircumstances two resonances ; areobserved (40-42) ] . *'F NMR of nucleic

acids specifically labeled with fluo-rine presents a situation similar tothat with 3 1P NMR but with better sen-sitivity and the opportunity of study-ing different sites in the biopolymer.Analysis of 3 1P and 1 SF relaxation musttake into account the contribution fromthe chemical shift anisotropy (CSA)mechanism as well as dipolar couplingto protons (e.g. 21,25,29,149). Thisadditional complication can be dealtwith by using chemical shift anisotropydata provided by solid state NMR(29»55»56) and from the frequencydependence of relaxation parameters(29,^8,i*9). Actually, since the dipo-lar and CSA mechanisms have differentorientation dependences on the motions,satisfactory separation of CSA anddipolar contributions should providecomplementary information.

With regard to any of the nuclei,contributions of chemical shift hete-rogeity to the measured linewidth canexist since the observed resonancearises from many individual nuclei.(An obvious exception to this is thestudy of certain single nuclear reso-nances in a specific tRNA.) For exam-ple, the chemical shift inequivalenceof the different phosphorus nuclei inthe double-stranded nucleic acids wasestimated to be at least 0.1+ ppm byBo 1 ton and James ( 9) and 0.-5 ppm byShindo (29)- The use of linewidths areconsidered to be qualitatively valuablebut less useful for quantitative stud-ies unless the linewidth is clearlymuch larger than the chemical shiftinequivalence.

It was mentioned above that spindiffusion (cross relaxation) can be animportant factor for >H relaxation(e.g. 5 3 ) . 1 3 C , 1 5 N , and 3 1P experi-ments with nucleic acids generally willnot suffer the complication of cross-relaxation as long as proton decouplingis employed. Cross-relaxation can beimportant, indeed uti1ized, when protoncoupling is permitted (51)• Although•1SF NMR can potential ly exhibit theeffects of cross-relaxation, it appearsthat in a practical situation, theeffects are likely to be negligible(58).


III. MOLECULAR MOT I ON J_N UNCOMPLEXEDNUCLEIC ACIDS

Obviously it is valuable to under-stand dynamics in pure nucleic acids asa baseline for comprehending anychanges that may be wrought by complex-ing of nucleic acids with appropriateproteins, drugs, or pathogenic agents.Although other NMR studies have exam-ined interesting aspects of nucleicacid structure, the following discus-sion will focus only on mobility innucleic acids.

A. DNA

3 1P NMR

Several studies have recently takenadvantage of the good sensitivity of3 1P NMR for an investigation of DNAdynamics. Most of the pertinent relax-ation data is summarized in Table 1.It might be noted that apparently con-ditions in all of the studies were usedto maintain DNA in a double-strandedhelix with no single strands or frayingof the ends of the polymer.

The study of Hanlon et al. (59)showed that the 3 1P NMR linewidthdecreased when high molecular weightDNA was sonicated. However, the linew-idth decrease by a factor of 2-3 is nottoo large considering the size of DNAwas decreased by a factor of more than60. That study also revealed a smalldependence of the linewidth on DNA con-centration (59) .

The time must have been ripe for amore complete 3 1P relaxation analysisof DNA mobility since four researchgroups independently and almost concur-rently investigated the 3 1P relaxation

behavior of DNA. Klevan et al. (60)first reported Ti and 3 1P{ 1H} NOE meas-urements on DNA of uniform chain length(IHO base pairs) obtained by nucleasedigestion of chromatin. Since theirrelaxation results were not consistentwith a single isotropic motion, theyreasoned that internal motion must alsobe occurring. Consequently, they usedthe motional model described by equa-tions 10 and 11 assuming 3 1P relaxationwas caused by dipolar coupling to threeprotons, each at a distance of 2.8 Afrom the phosphorus, and that theappropriate angle a was 6C°. Estimat-ing that TO was in the range of 10"7

sec and noting that the NOE was rela-tively insensitive to TO values in thatrange, it was concluded that an inter-nal motion correlation time TO of O.hns predicted the measured NOE and wasalso consistent with the measured Ti.It should be noted that, to a firstapproximation, the NOE is relativelyinsensitive to the P-H internucleardistance [see equation 33 and, for thatparticular combination of TO and T-values, is not very sensitive to thegeometrical details of the internalrotation.

Bolton and James (i+8,49) determinedthe off-resonance parameters T^ ° 'and R as well as Ti and NOE values for3 1P relaxation in DNA of higher molecu-lar weight but with a molecular weightdispersion. It was independently con-cluded that internal motion in the DNAbackbone existed and the modelexpressed by equations 10 and 11 wasalso utilized - the only variationbeing that the P-H distances were all2.6 A* and the effective angle of inter-nal rotation was k0° The angle a wasvaried and, although not too sensitive,the best

Vol No. 3A 131

Sample

(base pai'r cone. , mM)

Chicken erythrocyteDNA (1.5)

Calf thymusDNA (15)

Calf thymusDNA (4-12)


Calf thymus nucleosomeDNA (8)

Calf thymusDNA (0.9)


Table 1 .

Length

(base pa i rs)

145±3

7O0±30O

600±1503OO±75260±2514O±2Ol40±20

145±3

140

12800210

145 ±3

31P NMR Re

Frequency

(MHz)

109.3

81

40.5

40.5

40.3

36.4

36.4

24.3

1axat ion

Temp.

(°c)

18.5

35

2040

23

39

18.5

8

27

18.5

Parameters

Tl

(sec)

3.4

2.2

LA O

A

CM

CM

3.93.53.63-23.0

-

2.8

-

2.15

for DNA.

NOE

1.3

1.3

1.561.6

1.371.351.44

1.28

1.6

-

1.18

0000

T2(sec)

-

-

-

.012

.014

.013

.032

-

-

-

T ° f f * • R *

(sec)

-

0.9 0.371.2 0.53

-

-

-

-

Wl/2(Hz)

103

-

9545

5635392219

28

37

4117

18

Ref.

29

48,49

48,49

30,35

29

60

59

29

lan

ceR

esor

o4-1

<uO)IDX.

oc

Bu

ile

ti

*R and T ° p were determined with an rf field of 0.47 G applied 8 kHz off-resonance

fit of all the relaxation data wasobtained with a - 1*0° which is inagreement with a molecular model con-structed from X-ray data. Klevan etai. (60) and Bolton and James ( 9) dem-onstrated that protons from bound waterwere not responsible for the relaxa-tion.

A value of 0.3 ns, in agreement withKlevan et al. (60), was determined forTJ by fitting all five 31P relaxationparameters to the motional model(h8,kS) . The TJ value was indepen-dent of temperature over the range 20°to l+0°C as one might expect for aninternal motion. Although rotationsabout the 03'-P and P-05' bonds werespecifically considered, C-0 bond rota-tion and possibly others could also becontr ibut i ng.

Simultaneously, the 31P relaxationparameters were all fitted with a TOvalue of 1.0 MS at 20°C for DNA(hB,hS) • That value is dependent ontemperature, exhibiting an activationenergy of 2.8 kcal/mol. It was sug-gested that TO may be the correlationtime for bending of DNA which would beconsistent with theoretical considera-tions of the1 energy required for bend-ing DNA (3,1*0,12). It should be under-stood that the NMR does not explicitlyindicate the type of motion, only thatit is characterized by a correlationtime of 1 /us. The motion characterizedby TO is not consistent with end-for-end tumbling of DNA which would have acorrelation time longer than 20 us(3,i*7). !f the "speedometer cable"motion depicted in Figure h exists, itcould also contribute to TO. That con-sideration will be deferred until theresults of other workers are discussed.

Measurements of the 31P iinewidth,Ti, and NOE of a short segment of DNA(1 i+5 base pairs) as a function of spec-trometer frequency by Shindo (29) haveraised a very important question, i.e.,to what extent can the 31P relaxationbe ascribed to the dipolar mechanismand to the chemical shift anisotropy(CSA) mechanism. It wi11 be assumedthat paramagnetic contributions to therelaxation from transition metal ionsare negligible; the presence of

paramagnetic metal ions could influenceany of the relaxation parameters butwould be especially notable in reducingthe NOE toward 1 since the NOE existsonly with the dipolar mechanism.Unfortunately, it is not a simple mat-ter to distinguish clearly between thedipolar and CSA contributions unlessall molecular motions are so fast thatextreme narrowing conditions (r « l/a>)apply; otherwise, partitioning therelaxation between dipolar and CSAdepends on the motional model selected.The CSA contribution, unlike the dipo-lar, is dependent on the magnetic fieldstrength (2U, 29, i*8) - Measurements ofTi and NOE at different field strengthswill enable the CSA contribution to beestimated once a particular motionalmodel is assumed. Assuming that 1 5base pair DNA can be represented by arigid rod, Shindo (29) attributed 95%of the 31P relaxation to CSA at 109-3MHz, 70% at MD.3 MHz, and M 9 % at 2k.}>MHz. On the other hand, Bolton andJames (U8) attributed 35% to CSA at 81MHz and 10% at kO.S MHz when an inter-nal motion model was used; correctingfor the 10% CSA contribution at k0.~,MHz had an insignificant influence onthe results. Since the motional modelis an important factor, it should bepointed out that the model used by Bol-ton and James (48,^9) could predict allof the relaxation parameters at eachtemperature. In contrast, the rigidrod model that Shindo (29) considereddid not give a very good fit to hisdata leading him to suggest that sig-nificant torsional and bending motionsmay also take place with DNA.

Hogan and Jardetzky (30,35) haveobtained 31P relaxation data for frac-tionated DNA of varying chain lengths.Their analysis of the shorter fractionsassumed that the dipolar contributionto relaxation dominated and that theappropriate motional model for the DNAphosphates is a rigid rod with a two-site jump as an internal motion. Thecorrelation time used for the end-to-end tumbling was that determined bytransient electric dichroism measure-ments (hj) and the correlation timeused for rotation about the long axis

Vol. i», No. 3 A 133

of the rod was that calculated from thetheoretical expression for a cylinderof length 2L and radius b due to Lamb(61,10) :

(16)

sugar puckeringaccount for the

where k is Boltzmann's constant, T isthe absolute temperature, 77 is the sol-vent viscosity, and use is made of therelation between the rotational diffu-sion coefficient and the correlationtime, i.e., 1/DB =6x (| . On thebasis of their data and model, a corre-lation time of approximately 1 ns (30)and later revised to 2.2 ns for a jumpof ±27° (35) was calculated for theinternal motion. Although the actualvalue of the internal motion correla-tion time is higher than that of Klevanet al. (60) and Bolton and JamesC*8,1+9), it is still consistent withthe important notion that there ismotion in the phosphodiester region ofthe DNA backbone on the time scale of ananosecond. The correlation time isusually defined differently in jumpmodels and diffusion models, differingby a factor of six. If that is thecase here, the results of Hogan andJardetzky are in excellent agreementwith the others (48,1+9,60) .

Recent work has utilized a moresophisticated motional model whichtakes into account the different P-Hdistances and changes in these dis-tances with conformational fluctuationsof the DNA (64). These calculationsalso account for CSA as well as dipolarcontributions to the 31P relaxation.The results of these calculations sup-port the earlier work in terms of thefrequency of motion and provide someinsight into the likely nature of themotion. Twisting (winding and unwind-ing) motions about the helix axis, basetilting motions, and base propellertwisting motions (see Figure 4A) couldnot account for the 31P relaxationdata. The experimental 3tP resultscould be predicted by a motional modelwhich entailed rotational jumps aboutthe C3'-03' bond in addition to pucker-ing motions in the sugar ring of DNA,which is also consistent with 13Crelaxation results on the deoxyribose

carbons (vide infra);motions alone could not31P data.

Hogan and Jardetzky (30) made 31PNMR relaxation measurements on DNAfractions of different lengths (seeTable 1). Their T2 measurements inparticular may help elucidate thenature of the slower motions which con-

offtr i bute especi allyand T2 relaxation.(30,35) ascribedrelaxation for 140slow end-for-enda smaller contributionabout the long axis

to R, T]Hogan and Jardetzkythe bulk of the T2base pair DNA to the

tumbl i ng (T_J wi thfrom rotation(TJ|) . On the

other hand, Bolton and James (48,49)attributed the slower motion influenc-ing their 31P relaxation measurementsin larger DNA to bending. According totheoretical considerations (3) andtransient electric dichroism studies(47), T_ is strongly dependent onDNA chain length. Likewise, accordingto equation 16 xj, is linearly depen-dent on chain length. Although thetreatment of Barkley and Zimm (10) maynot be strictly applicable, it wouldappear from their analysis that a bend-ing motion correlation time would notdepend on chain length if a DNA longerthan 600 A was used.

For motions which are in the rangeof 10-7 to 10"5 s, the spectral densityJo(0) will dominate I/T2, and I/T2should be linearly dependent on thecorrelation time. Although there isonly a limited amount of data, it willbe noted from the data of Hogan andJardetzky (30,35) listed in Table 1that the value of 1/T2 has a dependenceon chain length that is at least linearbetween 140 and 260 base pair DNA.However, within experimental error, theI/T2 values of the 260, 300, and 600base pair DNA samples were the same.This T2 data is therefore consistentwith the notion that the 140 base pairDNA behaves as a rigid rod insofar asNMR relaxation is affected, but DNAlonger than 260 base pairs is suffi-ciently flexible that the DNA cannot betreated as a rigid rod; the slowermotion influencing NMR relaxationappears to be bending. Further NMR


experiments need to be performed tosubstantiate that conclusion, but it isin accordance with sedimentation stud-ies (2) and calculations (1) that indi-cate 700 base pair DNA, for example,extends on average to only 50% of itscontour length. It should be notedthat the linewidths of the samplesstudied by Hogan and Jardetzky (30,35)did increase with chain length. Thatis likely due to increased chemicalshift dispersion with more bending per-mitted in the DNA; as Gorenstein andKar (62) have demonstrated, 3 XP chemi-cal shifts are very sensitive to tor-sion angles and bond angles in phospho-diesters.

Mariam and Wilson (63) have examined3 1P spectra of DNA in a study of thehelix-to-coi1 transition. At tempera-tures near the melting temperature,they observed four different signals.The results indicated different T1 val-ues for the various peaks, suggestingthe possibility of different mobili-ties.

Shindo et al. (41) have recentlyobserved that aouble helicalpoly(dA-dT) exhibits a 3 1P spectrumwith two resonances having the sameintensity but different linewidths.Although not studied in detail, thissuggests the possibility of differentmobilities depending on the particularphosphodiester linkage.

2. •C NMR

Although the first 1 3C spectrum ofdenatured DNA was published severalyears ago (64a), the first naturalabundance 1 3C NMR spectra of intact DNAwere recently reported by Bolton andJames (48,50). 1 3C spectra are pre-sented in Figure 5» NOE and T1 meas-urements were carried out with thedeoxyribose carbons of the mediumlength (approximately 700 base pairs)calf thymus DNA. The results are givenin Table 2 for the ribose carbons.Under the conditions used for obtainingthe relaxation data, i.e., relativelyrapid pulse rate (0.51s delay time),

the aromatic carbon resonances werebarely visible above the noise,presumably due to long T1 values andperhaps broader lines (48,50). Datatherefore were not obtained with thearomatic resonances.

Although the model is not entirelyphysically plausible, the ribose 1 3Cdata were fit by the motional modeldescribed by equations 10 and 11(48,50). The relatively narrow linesfor the protonated carbons of DNA witha half million molecular weight cer-tainly implied that internal motionexisted in the deoxyribose moiety ofDNA. The model could serve as a firstapproximation to provide some informa-tion about the internal motions; spe-cifically, it should be possible tolearn the relative mobilities of thedifferent carbons as well as the timescale of the motions.

More recently the 1 3C data in Table2 were fit by a two state jump modeldescribing the internal motion (64).The two state jump restricts the ampli-tude of the internal motion to agreater degree than does the freeinternal rotation model used earlier.The two state mode) puts more realisticlimitations on the amplitude of theinternal motion. In this case, themodel was set up such that jumps couldoccur between the 2'-endo and the3'-endo ring conformations, i.e., withfluctuations of ±37° out of the meanplane of the deoxyribose ring.

For both motional models, the valueused for TO was that fbtained from the31P NMR experiments (48,4g) . Both mod-els basically lead to the same conclu-sions (48,50): there is motion experi-enced by the deoxyribose carbons on theorder of nanoseconds and the mobilitiesof the carbons vary as 1', 3 1. 4' < 21

< 5' with the 5' carbon experiencingsomewhat less mobility than the phos-phorus (48,49). The relative mobili-ties make sense in that the 51 carbonis not in the ring with the other car-bons and the 2' carbon does not have abulky moiety attached to it as do the1', 3 1, and 4' carbons.

Vol. 4, No. 3/4 135

5'

EDTA

-NOE

-40 -20 20 40 ppm

u

160 120 8 0 40

200 50 100 50 Oppm

Figure 5- (top) Natural abundance 50-3 MHz 13C NMR spectra of the ribose carbonsfrom k}, mg/ml calf thymus DNA in 0.1 M NaCl and 10 mM phosphate buffer, pH 7.illustrating a nuclear Overhauser effect experiment. Each spectrum is the average

136 Bu l le t i n of Magnetic Resonance

of 14400 accumulations with an acquisition time ofs. Taken from reference (48).

0.20 s and a delay time of 0.51

(middle) Natural abundance 13C NMR spectrum of native DNA (50 mg/ml)at 67-9 MHz; 43 000 scans were accumulated with a cycle repeat time of 1.5 s.Chemical shifts are reported in parts per million relative to an external Me4Sistandard. Taken from reference (65) .

(bottom) Natural abundance 25.1 MHz 13C NMR spectrum of approximately260 base pair long DNA (80 mg/ml in a 0.2 M NaCl, 1 mM EDTA, 8 mM phosphate buffer,pH 7-2). The spectrum is the average of 36OOO transients obtained with a 90° pulseand a recycle time of several seconds. Proton decoupling was performed only duringthe acquisition. Taken from reference (35).

Table 2. 13C Ti and NOE values for Calf Thymus DNA at 50.3 MHz and internalMotion Correlation Times Determined from the Relaxation Dataa

Carbon no. NOE Ti(S)

(ns)

Free internaldiffusion model

Two state jump model

r21

3'

V

5'

1.2

1.4

1.2

1.2

1.6

0.1

0.1

0.1

0.1

0.1

6

2

6

6

0.9

8

k

8

8

3(3)C

"Taken from references (48) and (50). T1 is accurate to about ±0.03s and NOE to ~10%. Since 1' and 41 carbon resonances are not resolved, the data for the two aretaken as the same. bReference (64). The two site jump is +37° and -37° from themean plane of the deoxyribose ring which describes jumps between the 2'-endo and3'-endo ring conformations. cThe two site jump model would not fit the 5'~carbondata so an additional motion was invoked (see text).

Vol. 4, No. 3/4 137

It will be noted in Table 2 that thetwo site jump models would not ade-quately account for the 5' carbonrelaxation data. The data could befit, however, by imposing an additionalinternal motion, consisting of a ±10°rotational jump with a correlation timeof three nanoseconds about the C5'-CVbond, on the 2'-endo *-» 3'~endo jump.This, of course, is simply an indica-tion that the 5' carbon experiencesgreater mobility than the other deoxy-ribose carbons.

Although quantitative informationwas not obtained, it was estimated byBolton and James (48,50) that the aro-matic carbons of DNA must be experienc-ing motions with correlation timeslonger than 20 ns. Otherwise, theirresonances would have been more readilyobserved in the 13C spectra.

Rill et ai. (65) published a 13Cspectrum of 140 base pair DNA (see Fig-ure 5) using a longer delay timebetween pulses (l-5s) than Bolton andJames (48,50) and were able to see rel-atively narrow resonances in the aro-matic region as well as the riboseregion of the spectrum. The linewidthsin their spectra lead Rill et al. (65)to observe that internal mobility orlibrational motions contribute to the13C relaxation. They also noted thatthe linewidths of the protonated sugarcarbon resonances were as narrow as thenon-protonated carbon resonances fromthe aromatic bases. That is certainlynot observed with proteins and suggeststhat the sugar moiety experiences agreater degree of mobility than thearomatic bases.

Subsequently, Hogan and Jardetzky(35) reported 13C NMR experiments onDNA. By using a pulse delay time ofapproximately 2 s, they were also ableto observe aromatic resonances as wellas sugar carbon resonances (see Figure5). With the lack of resolution due tothe lower spectrometer frequency andpresumably very large exponential fil-tering, a distinction was not madebetween the various ribose carbons northe aromatic carbons. Employing thetwo site internal motion model whichwas also used in the analysis of their

31P data (vide supra), Hogan and Jar-detzky (35) concluded that the baseplanes of DNA experience an internalmotion with a displacement of ±20° anda correlation time of 1 ns. It wasconcluded that the ribose carbons expe-rience the same +20° displacement and 1ns correlation time. The conclusionsregarding the ribose moiety are roughlyin accord with those of Bolton andJames (48,50), but conclusions regard-ing the motions experienced by thebases is about an order of magnitudefaster than suggested by Bolton andJames.

3. XH NMR

Although early 1H NMR spectra ofnative DNA did not prove highly useful(66), recent experiments by Early andKearns (67) examined proton resonancebands consisting of hydrogen-bondedimino protons, aromatic protons, andsugar protons from salmon sperm andcalf thymus DNA samples of variouslengths and as a function of ionicstrength. The resonances from theimino protons are shown in Figure 6.For the shorter DNA segments, it ispossible to distinguish between thehydrogen-bonded Watson-Crick protons inAT base pairs at 13-5 ppm and in GCbase pairs at 12.5 ppm.

Early and Kearns (67) calculated theproton linewidths expected for a rigidrod assuming homonuclear dipolar relax-ation from various nearby protons.With the deviations of their observedlinewidths from the calculated linew-idths, they concluded that the DNAbackbone manifested considerable inter-nal flexibility. Further, they esti-mated that the correlation time forreorientation of the base pairs is lessthan 300 ns, while the correlation timefor motion of the sugar protons isshorter, being less than 50 ns.

Hogan and Jardetzky (35) alsoobtained the proton NMR spectrum ofDNA. Using the two state model (videsupra) and assuming the H21 proton isrelaxed simply by dipolar coupling tothe adjacent methylene proton, theycalculated from their relaxation data a


340

1422

>2O00

15 14 13ppm

— ! - • •

12

Figure 6. Lowfield 300 MH2 proton NMRspectra of native calf thymus DNAobtained at different stages of diges-tion by DNase II and SI nuclease. Thetotal time (in minutes) of reaction isindicated at the right of each spectrumand the median molecular weight, inbase pairs, is indicated to the left.All spectra were obtainedbuffer solution containingand 1.0 mM ZnCl2 at pH k.(>reference (67)•

at 28°C in a100 mM NaOAc

Taken from

11 me0

correlati ontude of ±33agreement with

of 1 ns wi th an ampli-these results are in

the l3C results

presented above. Noting the complica-tions in obtaining detailed motionalinformation from protons in the basepair, they took issue with Early andKearns (67) conclusions and maintainedthat the proton NMR results could beaccounted for by base pair motion witha correlation time shorter

More recent JH NMR workal. (67a) on a 12 base-pairfragment of DNA at 300 MHzpermitted resolution of 70Comparison of non-selectivetive Ti experimentsproblem with XH NMR

than 10 ns.by Early etrestr i ctionand 600 MHzresonances.and selec-

high-lighted ainvestigations;

they found considerable spin diffusionwith even this short segment of DNA.And spin diffusion will be more exten-sive with larger DNA fragments. Assum-ing a single isotropic rotation, a cor-relation time of 7 ns for the dodecamerwas estimated (67a).

B. RjNA AND POLYRI BONUCLEOTI DES

For several years, it has been evi-dent from NMR studies and others that agreater degree of chain flexibility isproduced by melting a double-strandedpolynucleotide. The following discus-sion, however, focuses on recentefforts to describe the flexibility ofboth single-stranded and double-stranded polynucleotides more exactly.

1. Single-Stranded Polynucleotides

a. Poly (U) . Poly(U) is a randomcoil polymer. Early 13C NMR studies(68,68a) on poly(U) found the reso-nances to be sharp with varying reso-nance intensities, implying that thenuclei had different -T1 values and pos-sibly different degrees of mobility.

31P NMR T1 and NOE measurements havebeen carried out with poly(U), poly (A)and poly(C) as a function of tempera-ture (69). Assuming that a single iso-tropic correlation time described themotion, it was estimated that dipolarcoupling to protons accounted for 80%of the 31P relaxation at 40,5 NHz and72°C.

Shindo (29) measured the . 1inewidth,Ti, and NOE of poly(U) at 109.3 MHz and

Vol . h, No. 3A 139

24.3 MHz. Since poly(U) in solutionhas a random conformation, a motionalmodel with a distribution of correla-tion times was employed as describedmathematically in equations 13 and 14.A good fit of the Ti and linewidth datawas obtained with an average correla-tion time of 3«3 ns and a distributionparameter p of 14 for a b value of1000. The poorer fit of the model tothe experimental NOE value at 109.3 MHzwas ascribed to the CSA contribution torelaxation. The value of p = 14 is inthe range usually found for syntheticrubbers by 1 3C NMR experiments (31).

B. Poly (A) . Unlike poly(U), poly(A)is known to form a single-strandedstructure in which the "bases arelargely stacked, with the extent ofstacking decreasing with temperaturenoncooperatively (70). Akasaka et al.(69) measured the 3 1P Ti and NOE ofpoly(A). On the basis of negligiblechanges in coupling constant values ofApA over a wide temperature range, itwas argued that rotamer populations inApA and poly (A) did not vary with temp-erature. Assuming the motion governing3 1P relaxation was isotropic anddescribed by a single correlation time,they determined the correlation time tobe 6 ns at 20°C with an activationenergy of 5.3-b.O kcal/mol for themotion.

Although their 3 1P Ti and NOE meas-urements were in good agreement withthose of Akasaka et al. (69), Boltonand James (48,49) found that the addi-tional information supplied by theoff-resonance relaxation parameters Rand ^lp° necessitated the use ofa two correlation time model to ade-quately fit all of the relaxationparameters. Using the model repre-sented by equations 10 and 11, it wasfound that an internal motion charac-terized by a 0.5 ns correlation timewhich was independent of temperature

and a much slower motion which dependedstrongly on temperature over the 6-40°Crange provided an excellent fit for allof the relaxation parameters. At 6°C,poly (A) is virtually 100% stacked (70)and the temperature dependence of TOwas ascribed to unstacking of thepoly (A) bases leading to a polymer witha smaller radius of gyration.

The splitting, due to coupling with3 iP, in the carbon resonances ofpoly (A) at high temperature lead Smithet al. (70a) to conclude that disor-dered poly (A) exhibits a conformationaipreference although it is quite flexi-ble.

Komoroski (71) demonstrated the fea-sibility of obtaining 1 3C Ti and NOEvalues for the ribose carbons ofpoly (A) at 15 MHz. Bolton and James(50) obtained Ti and NOE values for theribose carbons of poly (A) at 25-1 and50.3 MHz. Using the value of TOobtained in 3 1P experiments withpoly(A) (48,49), the internal motioncorrelation time may be obtained usingthe mathematical model of equations 10and 11 (50) or a two site jump model(64). The calculated correlationtimes, as well as. the relaxation data,are listed in Table 3- The basic con-clusion is that the ribose ring experi-ences a motion or motions in the nano-second time range. The calculated TOvalues for this single-stranded polyri-bonucleotide are similar to those ofdouble helical DNA.

C. Poly(C) • Alasaka et al . (69)examined the temperature dependence of3 1P NOE and Ti for poly(C). Comparingwith their studies of poly(A) andpoly(U), they concluded that the flexi-bility of these single-stranded polynu-cleotides increased in the orderpoly (A) < poly(C) < poly(U) which cor-relates with a decreased tendency forbase stacking in that order.


Table 3. 13C J\ and NOE values for PoiyA and Internal Motion Correlation TimesDetermined from the Relaxation Oataa

Carbon no. 25.1

MHz

1.2

1.8

1.5

1.5

1.6

NOE

50.3

MHz

1.2

1.4

1.2

1. 1

1.3

X, (ns)

Free internal

diffusion model

7

1

4

4

3

Two state

jump model

6

4

5

6

2

I1

2'

3'

4'

5'

0. 1

0.1

0. 1

0.1

0.06

"Taken from reference (50). Ti is accurate to about ±0.0ls and the NOE to ~ 10%.Measurements were made at 30°C. b0btained at 50.3 MHz. cReference (6^). The twosite jump is between +37° and -37° from the mean plane of the ribose ring.

Poly (G) . Yamada et al. (72) studiedthe proton and 31P NMR spectra ofpoly(G) as a function of temperature.From the slow exchange of the NH protonwith deuterium in D2O (see Figure 7).it was concluded that poly(G) is in amultistranded form in neutral solution.A large difference in 31P T2 valuedetermined from the spin-echo methodand from the linewidth gave evidence ofchemical shift heterogeneity among thephosphorus nuclei due to a distributionof rotation angles about the phospho-diester bond.

Assuming a single isotropic correla-tion time to be applicable, Yamada etal. (72) used the T1/T2 ratio to calcu-late correlation times at 27°C of 50 nsand 70 ns for the H8 and phosphorus,respectively. It was observed thatthis is much slower than previouslyobserved for poly (A), poly(C), andpoly(U) (vide supra) .

d. Poly 0 ) • Neumann and Tran-Dinh(73) have utilized a comprehensivearray of nuclei jto investigate poly(l):>H (90 and 250 MHz), 2H (13-8 MHz), 13C(75.it MHz), and 31P (36.^and 111.6MHz). Using a single isotropic corre-lation time model, it was concluded

from 31P Ti and NOE measurements thatthe chemical shift anisotropy contribu-tion to relaxation amounted to 12% at36.4 MHz and 72% at 111.6 MHz. Fromthe 31P Ti and NOE data, a temperature-dependent correlation time of ~ 1 nswas calculated. The 13C NOE values forall of the observed carbon resonancesindicated a correlation time of approx-imately 0.5 ns in agreement with thevalues estimated from the proton Ti (90MH2)/Ti (250 MHz) ratio and the deuter-ium resonance linewidth for a sample ofpoly(I) labeled with deuterium at the 8position of the base.

2. Double-Stranded Polynucleotides

Tritton and Armitage Ok) obtained31P Ti of 16S rRNA and the ^ P ^ H } NOEfor a mixture of l6S and 23S rRNAobtained from E_. col i ribosomes.

Bolton andthe

James (48,^9) measuredLP Ti, NOE, R, T 1 p

o f f, andlinewidth of poly (I)-poly(C) as a func-tion of temperature; the Ti and NOEvalues were in excellent agreement withthose found by Tritton and Armitage(7I4) for rRNA. Analysis in terms ofthe two correlation time model

Vol. h, No. 3A

Figure 7- Low-field part of the pro-ton NMR spectra of poly(G) in D20solution (pH = 5.8, [Na+] = 10"3 M)after standing for varying time inter-vals, 100, 150, 200, 280 and 310 min,measured at 99.5*4 MHz at 29.5°C. Chem-ical shifts are given downfield fromOSS. Taken from reference (72).

described by equations 10 and 11yielded very good fit of all the relax-ation data (48,i*9) . It was found thatthe internal motion correlation timeTJ had a value of 0.5 ns and wasindependent of temperature, but theslower motion correlation time TO was 1MS at 20°C and had an activation energyof 2.8 kcal/mol over the range 2O-4O°C.

The values of the internal motioncorrelation time are nearly the samefor DNA, poly (I)-poly(C), and single-stranded poly (A) showing that thismotion is not substantially coupled tothe overall conformation of the polynu-cleotide [B form DNA vs. A formpoly (I)-poly (C)] nor is the motioninfluenced by double helix formation(U8,U9). The slower motion correlationtime found for poly(I)-poly (C) had thesame value and temperature dependenceof the larger DNA, providing supportfor the suggestion that the TO repre-sented a bending motion independent of

chain length.Addition of 20 mM Mg (II) to the

poly (I)-poly(C) sample altered some ofthe 3XP NMR relaxation parameters (hS).Analysis indicated that the internalmotion was not affected, but the TOvalue increased from 1 MS to 3 MS. Theexact reason for this is not known, butit might be noted that Mg (It) does sta-bilize double-stranded polynucleotidesto thermal denaturation (75)•

Q. tRNA

As noted earlier, NMR studies haveelucidated the kinetics of tRNA folding(7 ,8,76) . Thi s mot ion i s i n the mi 11i-second to seconds time range. The fol-lowing discussion pertains to fastermotions.

J_. 31P NMR Three studies have pro-vided information pertinent to thedynamic state of aminoacyl specifictRNAs via 31P NMR. Gueron and Shulman

observed several 31P resonances inof yeast tRNAPhe and E.spectra

tRNAGlu which could be examinedwhen the tRNA was melted or Mg(ll) wasadded. On the basis of the increased31P linewidths observed in the 109 MHzspectra compared with ^0 MHz spectra,it was concluded that the chemicalshift anisotropy contribution dominatedthe relaxation at 109 MHz.

Gorenstein and Luxon (78) measuredthe linewidths and T1 values for 31Presonances of tRNAPne at )hG MHz and32 MHz (see Figure 8); Ti values at ]kGMHz ranged from 2.09 s to 3.5 s for 13different resonances observed. Assum-ing that the motions of the phosphorusnuclei could be represented by a singleisotropic correlation time, they esti-mated that > 90% of the relaxation at]hG MHz was due to the CSA mechanism.Subsequently using the expressionsappropriate for the CSA mechanism, Gor-enstein and Luxon (78) used their T1and T2 values to calculate that the 31Pcorrelation time is 11 + 2 ns and thechemical shift anisotropy is 180 ± 20ppm. This value for the CSA is some-what higher than that of 140 ppm esti-mated by Gueron and Shulman (77) ° r

120-130 ppm estimated by Hayashi et al.

11*2 Bulletin of Magnetic Resonance

the partially resolved

Figure 8. 31P NMR spectra of yeastphenylalanine tRNA (~ 33 mg/ml) in 100mM NaCl, 10 mM cacodylate, 10 mM MgCl2,1 mM EDTA, and 10% D20, pH 7.0, atindicated temperatures (°C). Theexpanded scale for the scattered peaksis shown over the normal spectrum. Asimulated stick figure spectrum is alsoshown at the bottom. Number of acqui-sitions, 8000 FID's, 1.86-S acquisi-tion, ll*5'76 MHz. Chemical shifts arein parts per million fromH3PO4. Taken from reference (78).

tRNA Pheit0.5were

(79).Two of

MHz 31P signals of yeastobserved by Hayashi et al. (79) to havedistinctively different Ti values.Also assuming a single isotropic corre-lation time, it was shown from T1 andNOE measurements that both the CSA anddipolar mechanisms contribute to relax-ation at ^0.5 MHz with the dipolar con-tribution being most important. Fromthe T1 and NOE values, it was estimatedthat the 3 1P correlation time was 3 nsin yeast tRNAPhe (79)•

The discrepancy between the 31P cor-relation time estimate of Gorensteinand Luxon (78) and that of Hayashi etal. (79) for tRNAPhe may be due tothe use of different relaxation parame-ters and the assumption of a singlecorrelation time in the two studies.The phosphorus may experience two ormore motions with ditimes, e.g., duebling of tRNA or,internal motion andtRNA. The NOE and

fferent correlationto anisotropic turn-as Iikely, due tooverall tumbling ofTi are more sensi-

tive to faster motions and will lead toa smaller estimation of the averagecorrelation time. But Ji is more sen-sitive to slower motions, so the aver-age correlation time estimated from Tiand T2 measurements would be longer.

2. Ml NMR Hayashi et al. (80) usedthe correlation time estimated in theirearlier 3 1P experiments (79) to'simu-late proton Ti values and compare thosewith the experimental ones obtained foryeast tRNAPhe in D2O at 100 MHz. TheTi simulations also employed proton-proton distances estimated from X-rayanalysis, and it was assumed thatmotion at any proton could be repre-sented by a single isotropic correla-tion time. The simulations could matchthe temperature dependence (21-86°C) ofthe ribose protons (H2'-H51-), but indi-cated a larger Ti variation with temp-erature than was experimentallyobserved for the HI1 and base protons;this suggested a larger distribution ofcorrelation times for the various aro-matic protons contributing to theobserved multiresonance signals. It

Vol. k, No. 3A 1*3

was estimated that the ribose protonshad a correlation time of 1 ns at5O~55°C and the base protons had thatcorrelation time at *>5~5O°C.

The proton NMR spectra of the car-bon-bound protons of tRNA have usuallyexhibited broad, overlapping bandswithout distinguishing features.Schmidt and coworkers (81,82), however,have recently resolved some of thoseresonances in tRNAt

tRNAPne from

dV a' from coli

and tRNAPne from yeast, includingseveral in the aromatic region (see

A. tRNAPhe!yeast>

spectra oftRNAPhe in D2O.20 mg/ml in tRNA,

NMRP h e

Pjt pH 7, 28°C.

Figure 9. 270 MHz lHtRNAt

Va1 andSolutions were ca,0.15 M NaCl, 5 mMChemical shifts were measured from theresidual HO2H peak and are referencedto DSS at 0 ppm after a correction forthe temperature dependence of the waterpeak shift. Taken from reference (82).

Figure 9). Using the interproton

1M.

distances estimated from the tRNAPne

crystal structure and assuming iso-tropic reorientation of the internu-clear vectors with a single correlationtime of 25 ns, as expected for tumblingof tRNA, the experimental linewidths ofindividual proton resonances could becorrectly predicted (82). This sug-gested to Schmidt and Edelheit (82)that rapid internal motion is not pres-ent in the tRNAphe bases, but a morelimited motion could not be precluded.

*H chemical shift values might elu-cidate the nature of any motions of thebases in tRNA (83). It was suggestedthat any large amplitude motions of thestacked bases in a direction parallelto the base plane would tend to averageout the ring current-induced chemicalshifts (83). Instead, the imino NH andaromatic CH proton resonances variouslyexhibit ring current shifts rangingfrom 0 to 2 ppm (8,15.82) which givesevidence against large amplitude slid-ing of bases relative to one another.However, propeller twisting of basepairs, discussed earlier, would haveless ring current shift averaging andcould still occur.

i- 11£ WJB Komoroski and Allerhand(84) measured " C Ti values of unfrac-tionated yeast tRNA at 15 MHz. Withinexperimental error, the T1 values ofthe base carbons were found to be equalto those of the sugar carbons. On thebasis of these values, it was foundthat a single correlation time of 30±l0ns (which is the value expected foroverall rotational tumbling) would fitall the Ti data over the temperaturerange 35~5i»0C. Further 13C NMR studiesby Komoroski and Allerhand (85) on theunfractionated yeast tRNA, however,lead to the conclusion that the dihy-drouraci-1 • r i rigs in folded tRNA undergoa fast internal motion with an esti-mated correlation time < 0.2 ns ati*l°C. This conclusion was reached by

the observed linewidth of theof the dihydrouraci1 ring (10that which would be expectedwas no internal motion (90

compar1ngC5 carbonHz) withif thereHz).

More recently, Bolton and James (50)measured the T1 value at 50.3 MHz and

Bulletin of Magnetic Resonance

the NOE values at 25.1 MHz and 50.3 MHzfor the individual ribose 1 3C reso-nances of unfractionated yeast tRNA.

The measured relaxation parameters arelisted in Table h along with internalmotion correlation times

Table k. 1 3C Tj and NOE Values for Unfractionated Yeast tRNA and internal MotionCorrelation Times Determined from the Relaxation Dataa

Carbon no.

r

21

V

k>

5'

h

0.1

0. 1

0.1

0. 1

0.07

25-1

MHz

1.1

1.3

1-3

1.2

1.5

NOE

50.3

MHz

1 .1

1 .2

1 .2

1 .2

1 .2

T

Free internal

diffusion model

7

6

6

7

j (ns)

Two

jump

state

model

8

7

7

8

2

"Taken from reference (50). The sample contains lOmM MgCl 2. 1, is accurate toabout ±0.02s and the NOE to ~ 15%. Measurements were made at 30°C. "Obtained at50.3 MHz. Reference (6*0 . The two state jump is between +37° and -37°from themean plane of the ribose ring.

determined from the data using two mod-els: free internal motion modeldescribed by equations 10 and 11 and atwo site jump model (6it) . Both modelsemploy a correlation time of 30 ns forthe overall rotational reorientation ofthe tRNA molecule. The basic conclu-sion is that the ribose carbons possessa motion on the nanosecond time scale.It appears that the carbons in the ringall have the same correlation time butthat C5' possesses a bit greater mobil-ity. Aromatic carbon resonances wereobserved, but their peak intensitiesimplied a longer Ti. Although notexamined quantitatively, this observa-tion suggests that the motions of thebases are restricted either in

amplitude or frequency relative to theriboses. A comparison of Tables 2-i+indicates that the sugar moieties ofDNA, poly (A) and tRNA possess about thesame dynamics with the p.ossible excep-tion that C2' in DNA and poly(A) hasmore mobility than the other ring car-bons. This suggests that the tertiarystructure in tRNA may inhibit the C2'motion. Otherwise, the analysis indi-cates that the internal motion is notstrongly coupled to the conformation ofthe polynuc1eotide.

The low sensitivity of 1 3C NMR haslimited the studies with this informa-tive nucleus. To overcome some of thesensitivity difficulties associatedwith natural abundance 1 3C NMR, a few

Vol . it, No. 3 A

recent investigations have utilizedtRNAs labeled with 1 3C at specificsites (86-88). Schmidt et al. (86)followed their earlier studies (e.g.89) demonstrating the efficacy of 1 3CNtfR studies of j_n vivo labeled tRNA byinvestigating unfractionated £. colitRNA with the adenine C2 labeled or theuracil and cytosine C2 positionslabeled. A comparison of the 1 3C spec-tra obtained at 26.2 MHz and 67.9 MHzindicated that the observed linewidthswere determined principally by chemicalshift heterogeneity of the variousbases in the folded tRNA (86). Thiswas confirmed by spin-echo T2 measure-ments which indicated inherent linew-idths of 4 Hz, 2-3 Hz, and 40 Hz forthe C2carbonsof cytosine, uracil, andadenine, respectively, in comparisonwith the observed linewidths at 25 MHzof 22,78, and 116 Hz. The very low NOEvalues for the unprotonated C2 carbonsof cytosine and uracil indicated thatdipolar relaxation was not predominant.The protonated C2 carbon of adenosineshould be governed largely by dipolarrelaxation, with the possibility of asmall contribution from the CSA mecha-nism.

T1 measurements for the C2 of ade-nine and the ribose carbons in the tRNAwere also made by Schmidt et al. (86).Using the Ti value (with the T2 valueas a guide) and assuming a single iso-tropic correlation time model, a corre-lation time of 16 + 4 ns was determinedfor the adenine C2 carbons in tRNA.Similarly, the single correlation timedescribing the ribose carbons was esti-mated to be 13 ± 3 ns. It might benoted that the measured correlationtimes are somewhat shorter than wouldbe expected if tRNA is a rigid rotortumbling in solution.

Hamill et al. (88) have also exam-ined the 13|c NMR of unf ract i onated tRNA(from S . typh i mur i um in this case) withlabeled uracil (90% 1 3C at C4) . 1 3C T<

values at 25-1 MHz were obtained overthe temperature range 23~6O°C. Anapproximate Ti value was obtained at90.5 MH2 and NOE values at 25.1 MHz.Using the ^ C ^ H and 1 3C- 1 4N distances,as well as a chemical shift anisotropyof 160 ppm (90), for uracil, the *Hdipolar, 1 AN dipolar and CSA contribu-tions to the 1 3C relaxation of theunprotonated C4 of uracil were calcu-lated assuming that tRNA is a rigidrotor describable by a single isotropiccorrelation time (88). Their calcula-tions indicate that the lH dipolar con-tribution to Ti dominates at 25-1 MHzwith a 10-20% contribution from CSA,and a 10-15% contribution from 1 4Ndipolar coupling. At 90 MHz, the CSArelaxation dominates Ti with 20-40%contributions from JH dipolar and ~ 5 %from-14N dipolar coupling.

Analysis of their 1 3C relaxationdata yielded a correlation time of 30ns at 37°C (88) which is in agreementwith the value obtained in some studies(82,84), but is somewhat larger thanthe values obtained in others(77-80,85,86) which all use the singleisotropic correlation time assumption.The value of 25~3O ns is expected ifthese is no internal motion, but lowervalues for the effective correlationtime would be obtained if internalmotion exists. Using motional modelswhich will permit internal motions(50,64), it has been shown that theexistence of internal motions in theribose rings is most reasonable. Theaccumulated data indicate that somedegree of mobility is afforded thenucleic acid backbone, but it is notyet clear to what extent the bases arepermitted motional' freedom in tRNA.

It should be noted that the recent1 3C spectra obtained by Schweizer 'etal. (87) for E.. coj_i tRNA! V a 1 with[4-13C]uraci1 incorporated (see Figure10) holds the promise of actuallyobtaining specific


25MHz l3C-spectrum of 4-*3C-Uracil labeled tRNAValT from £Co/ /SO- l87

D2

^W^^m^H^

5 8

10

191 187 183 179 175 171 167

Chemical Shift, ppm, from Tetramethylsilane163 159

(b) 3-0 framework with l3C-enriched Uracilsosin tRNAValj

1 > C Stem Acceptor Stem

/iU64 / 1

Loop

DLoop

Variable -

Loop

Ant icodon

Stem

Anticodon

Figure 10. 25 MHz spectrum of |>- 1 3C] uracil labelled t R N A ^ 1 (21 mg/ml in D 20solution of 30 mM K 2HP04 (pD 7- 3 ) , 150 mt\ NaCl, 15 mM Mg C l 2 , 1-5 mM EDTA, 3mtf N a 2 S 2 0 3 , 0.03% NaN 3) , ambient temperature 32-3J4°C. 1 KHz sweep width,16,30^ transients, 90° pulse ( 5 M s e c ) , and k second acquisition time. Chemicalshifts measured from internal dioxane and converted to TMS by adding 66.3 ppm.(b) . Three dimensional framework of yeast t R N A P h e with the superposition of the1 3 C enriched uracils as found in E_. col i tRNAy a l. Taken from reference (87).

motional information-at different loci within a single well-defined tRNA.

Vol. h, No. 3A ]1*7

Some of the individual peaks areassigned and others are presently beingeluc i dated.

IV. NUCLEIC ACID z PROTEIN COMPLEXES

For the most part, the biologicalaction of nucleic acids involves inter-action with proteins in one manner oranother. The nature of these interac-tions is a matter of intense investiga-tion with studies on natural and modelsystems exploring such possible fea-tures as ionic attractions, stacking ofaromatic amino acid residues withnucleic acid bases, and topology (thebeta structure of proteins fits themajor groove of DNA quite well). NMRhas been utilized in examining some ofthe questions regarding nucleic acid-protein complexes. The following dis-cussion, however, will be limited to aconsideration of the motional proper-ties of nucleic acids in functional (ornearly so) nucleoprotein complexes.

A. NUCLEOSOMES AND CHROMATIN

Nucleosome core particles compriseone of the. two fundamental repeatingsubunits in chromatin. They areroughly disk-shaped with dimensions 57AX 110A across and are composed of a DNAhelix with several proteins (see refer-ence 91 for a review). A few studieshave recently been made using 3 1P NMRon this important and complicatednucleoprotein (59.&O.92-94). It mightbe noted that only a single 3 1P signalis observed in studies of nucleopro-teins, so any conclusions will be tem-pered by this realization.

In the earliest study by Hanlon etal. (50), it was found that the inten-sity of the 3 1P signal from two out ofthe three chromatin samples examinedhad intensities decreased by more thanhalf compared with pure DNA. It wasnoted that the decrease in intensitycorrelated with the amount of B-DNApresent, as judged from their circulardichroism spectra, and they conse-quently proposed that the B-DNA hadstructural rigidity which precluded 3 1Psignal observation.

Klevan et al. (60) measured the J 1PTi and NOE for nucleosomes (see Table5) and compared results with the DNAextracted from the nucleosomes (videsupra). Using the two correlation timemodel represented by equations 10 and11 with the tumbling time estimatedfrom electric dichroism measurementsfor the overall reorientation, theydetermined the internal motion influ-encing 3 1P relaxation to have a corre-lation time of 0.4 ns. That is thesame value as calculated for DNAprompting the conclusion that bindingof DNA to proteins in nucleosomes haslittle influence on that internalmotion (60). It may be noted fromTable 5 that substituting D2O for H2Ohas an influence on Ti, but not much onNOE, unlike 3 1P relaxation in pure DNAwhich is not altered by D2O substitu-tion.

The large NOE value obtained by Kle-van et al. (60) permitted them to maketheir analysis assuming only dipolarrelaxation. In contrast, Shindo et al.(9*0 maintained that CSA contributionswere also important inof nucleosomes. Chemicalvalence was also shown to

3 1P relaxationshift i nequi-

a largebefactor in the observed 3 1P linewidths -the chemical shift heterogeneity appar-ently is 0.77 ppm (9M in contrast to0.50 ppm in pure DNA (29). Fair agree-ment of the observed relaxation parame-ters (see Table 5) and calculatedrelaxation parameters was obtainedusing a motional model with a distribu-tion of correlation times as descibedby equations 13 and 14. The best fitwas obtained with an average correla-tion time of 30 ns and a distributionparameter p = 20, which permits thecorrelation times to range over abouttwo orders of magnitude. It will benoted that the average correlation timeis about an order of magnitude shorterthan the rotational reorientation timeof nucleosomes (18). Clearly, motionsare experienced by the phosphorus nuc-lei which are faster than the tumblingof the nucleosomes. Since a differentmotional model was employed in theaccompanying paper on DNA (29) (videsupra), a direct comparison is


Table 5. 31P NMR Relaxation Parameters for Nucleoprotelns

-4"

Solvent Frequency Temp. Ti

(MHz) ( C) (sec)

NOE _offd

Tlp(sec)

W 1/2(Hz)

Ref.

A.

B.

C.

Calf thymus nucieosome(lOmM Tris, 5mM EDTA, pH 7.6)

Chicken erythrocyte nucleosomecore (5mM Tris, O.lmM EDTA, pH 8)

+ 6 M urea

E. coli 70s ribosome(50mM Tris, lOmM MgOAc, 0.1M KC1,pH 7.5)

H20D20

H20D20

36.4

109.340.324.3

109.324.3

36.4

8

19

19

23

2.83.6

3.343.002.70

2.32.2

2.0(2.3)b

1.71.6

CM

CMCM

1

IS\1.20

1.321.08

50S ribosomal subunit(20mM Tris, 0.1M, NH4CI, MgOAc,pH 7-5)

HoO

30S ribosomal subunit H2O(lOmM Tris, 30mM NH^Cl, lOmM MgCl2,6mM mercaptoethanol, pH 7-5)

D. rat liver ribosomes (intact) D2O(50mM Tris, lOmM MgOAc, 0.1M KC1,pH 7-5)

rat liver ribosomes (limit digest D2O"core" following RNase treatment)

E. rat 1iver messenger ribonucleopro- D2Otein (0.3* SDS, 0.1M NaCl, 50mMNaoAc, lOmM EDTA, pH 5-2)

36.4

36.4

40.581

40.5

40.5

23

23

20

20

1.3

1-7

2.352.45

2.5

0.46

1.18

1.21

1.11.1

1.4

0.77

1.0

0.33

0.4

3337

1304525

20535

208

60

94

94

74

8090

60

1.5 0.05(4 KHz) 0.11(4KHz)1400.13 0.29

0.28(l6KHz) O.6O(l6KHz)

74

74

97

97 ^oz

97 J

O>

'Experiment performed with an rf field of strength 0.48 gauss applied 8.0 KHz off-resonance unless noted otherwise,value in parentheses was obtained when 0.1M EDTA was added.

The

not possible.Shindo et ai. (9M also examined the

influence of urea on the nucleosome 3 1Prelaxation (see Table 5 ) - It was con-cluded that the urea partially dis-rupted the hi stones in the nucleosomecore providing a somewhat more mobileDNA (T = 15 ns) and a broader distribu-tion of correlation times withoutincreasing the chemical shift hetero-genei ty.

Feigon and Kearns (95) have shownthat the proton NMR spectra of the lowfield imino protons are virtually iden-tical in nucleosome core particles andthe DNA extracted from the nuc1eosomes.On the basis of XH NMR linewidths, itappears that the DNA base pairs haveabout the same degree of motional free-dom in the nucleosomes as in the freeDNA.

B. RIBOSOMES AND MESSENGERRIBONUCLEOPROTEINS.

Since the ribosome is the site ofprotein synthesis in cells, a consider-able effort has been put into an under-standing of its structure and function.Undoubtedly due to its very complexnature and size, few NMR studies havebeen carried out with ribosomes. Trit-ton and Armitage (7*0 have measured the3 1P T1 and NOE for the 50S and 30Ssubunits and the 70S ribosome from £.coli (see Table 5 ) • Using the motionalmodel with two correlation times andindependent values for the rotationalreorientation of ribosomes and subunits(96), it was concluded from the NOEvalues that the correlation time forinternal motion is 3~5 ns. It isinteresting to note that the substitu-tion of D2O for H2O influences therelaxation as it did with nucleosomesbut which it did not with pure DNA.The implication of this observation isnot entirely clear at this time. The3 1P spectra published by Tritton andArmitage (7^) exhibited peaks charac-teristic of degradation products fromthe action of ribonuc1 ease.

Additional 3 1P NMR experiments with

undegraded ribosomes (rat liver, inthis case) have also been carried outwith the off-resonance relaxationparameters "H ° ^ and R being meas-ured in addition to T2, NOE, and Wi^(97); the results are listed in Table5- Using the motional model of equa-tions 10 and 11, all the relaxationparameters were consistent with aninternal motion of 3 ns, in agreementwith the conclusions of Tritton andArmitage (7^). This is a factor of sixslower than the internal motion inuncomplexed double-stranded RNA (^8,^9)(vide supra). Since the 3 1P resultsobtained for intact ribosomes representan average for RNA in contact with pro-tein and RNA spaced between proteins,it was surmised that the markeddecrease in phosphodiester mobilitywould be accentuated by removing the30% of RNA susceptible to ribonucleasecleavage since the remaining 70% of theRNA would be in intimate contact withprotein; it is known thatgest ribosome has almosttein and essentially thetation properties asribosome. Instead of finding the phos-phorus mobi1ity decreased in the limit-digest ribosome, the mobility was foundto have increased so that the correla-tion time had decreased to 0.5 ns, thesame value as the free RNA.

Recent 3 1P NMR experiments (97) havealso been carried out with messengerribonuc1eoprotein (mRNP) which is aboutthe same size as ribosomes. The T1,NOE, Tip° f f> R» and Wi, values aregiven in Table 5- It is apparent thatthe T1 value of mRNP is much lower thanthat of any other nucleic acid-proteincomplex listed; in fact, it is lowerthan one could expect for a 3 1P nucleusrelaxed solely by protons on the RNA.Consequently, selective 3 1P{ iH} NOEexperiments were carried out in whichthe 3 1P resonance intensity was moni-tored as 5 U Hz "windows" in the protonNMR spectrum were strongly irradiatedrather than irradiating all proton fre-quencies as is usually done with heter-onuclear NOE experiments. The selec-tive NOE experiments showed that thephosphorus in the mRNP was dipolar

the Iallsamethe

I imithesed

i

t-di-pro-imen-ntact


coupled to protein protons as well asribose protons. This observation cer-tainly reflects the intimate associa-tion of protein and nucleic acid inthis complex. Perhaps the most inter-esting feature revealed by the mRNPrelaxation study is that the nature ofthe complexes formed between proteinsand nucleic acids is not the same inal1 cases.

Recently, the XH NMR spectrum and1 3C NMR spectrum of E_. coli ribosomeswith 12% 1 3C labeling have beenreported (98). Although de Wit et al.(98) emphasized protein resonances intheir discussion, resonances which canprobably be ascribed to the RNA moietyappear in the spectra. It shouldtherefore be possible to use theseresonances for future studies of ribo-somes .

C. VIRUSES AND OTHER COMPLEXES

Viruses have certainly not beenintensively investigated by NMR. Thereasons are simple. The viruses arehuge (by molecular standards), compli-cated (with nucleic acid and protein,at least, and in some cases lipid andcarbohydrate as well), and quite possi-bly rigid. Although it appears thatliquid NMR techniques will be applica-ble to some viruses, solid state NMRtechniques such as dipolar decoupling,cross-polarization, and magic anglesample spinning should find utility instudies of viruses. Very recentreports have demonstrated the feasibil-ity of solid state NMR investigationsof viruses. Opel la (99) has found evi-dence that the DNA in filamentous bac-teriophage fd is substantially immobi-lized when packaged in the virus.

I 3C NMR spectra of tobacco mosaicvirus (100) have been obtained forviruses which have enhanced levels of1 3 C . Resonances from the proteinmoiety were the emphasis of the stud-ies, but smaller resonances from theRNA moiety have a possibility forfuture work.

Although NMR studies of some spe-cific proteins which interact withnucleic acids have been carried out,

there apparently has not been an NMRinvestigation of the nucleic acidmoiety in complexes with these prot-eins. Model studies have been carriedout in that NMR has been used to exam-ine binding of gene 5 protein to tetra-and octanucleotides (102,103). On thebasis of their investigation, Garssenet al. (103) postulated a protein-in-duced mechanism of unwinding determinedby fluctuations in the double helicalONA structure.

V. NUCLEIC ACID z DRUG COMPLEXES

An important aspect of the pharma-cology of many drugs on the molecularlevel is binding of the drugs tonucleic acids. NMR is being success-fully used to elucidate some of thedetails of drug binding, primarily viastudies with oligomers serving as DNAor RNA models. See references (16) and(10**) for recent reviews. Drug effi-cacy is not always determined by theequilibrium binding constant to nucleicacids nor by transport to the nucleicacid. Dynamics, both fast and slow,can play a major role in drug action.Nucleic acid conformationa1 changes areusually required for drug binding anddrug release. Very little is knownabout the influence of drugs on confor-mational changes and virtually nothingabout their influence on conformationa1fluctuations.

Of the few studies that have beenmade, binding of ethidium bromide to

Br"C2H5

(Ethidium Bromide)

nucleic acids has been a major focus.Examination of the low-field exchange-able proton resonances of amino acidspecific tRNAs in the presence of ethi-dium bromide lead Jones et al. (105) to

Vol. h, No. 151

conclude that the drug has a specificinterca1 ative binding site which willbind chloroquine as well. The lH NMRindicated that drug binding to this onesite did not alter the conformation normobility in the remainder of the tRNA.

Rill et al. (65) obtained a naturalabundance 1 3C NMR spectrum of DNA inthe presence of a large amount of ethi-diurn bromide (1.8 ethidium bromide perphosphate). A significant increase inlinewidth was obtained for all the DNAcarbons. Apparently, the linewidthsincreased so much for the carbons inthe aromatic rings that they were notreported; the linewidths for the ribosecarbons increased by a factor of 2-6.

Hogan and Jardetzky (106) titratedDNA with ethidium bromide and measuredthe intensity decrease for the 3 1Presonance and the low-field exchange-able proton resonances. The intensitydecrease indicated that the drug mark-edly diminishes the mobility of the DNAat the binding site resulting in reso-nances so broad that they are notdetected. It was concluded that fourphosphates were immobilized for everyethidium bound; two from the intercala-tion site and two from the adjacent5'-phosphates. The Ti of the observ-able 3 1P resonance increased propor-tionately with the amount of bound eth-idium, increasing by a factor of twowhen the ethidiurn/base pair ratio wasraised from 0 to 0.3- Likewise, the T2and Hnewidth changed with T2 decreas-ing a factor of four when the ratio was0.3. Regarding these changes to benegligible, it was concluded that theeffect of ethidium intercalation isrestricted to a two base pair longregion of DNA. If that conclusion iscorrect, the implication is that thebase pairs in DNA experience internalmotion independent of their neighbors.That may be correct even if the relaxa-tion parameters are altered to a cer-tain extent by ethidium binding.Slower long-range motions, e.g., bend-ing, could be altered by intercalative

binding of ethidium without alteringthe local motions on the DNA other thanin the vicinity of the binding site.An alteration over the long-range isconsistent with the hydrodynamic prop-erties of DNA with drug intercalation(3).

Recently, 1SF NMR has been utilizedin conjunction with fluorescence andoptical absorption spectroscopy toexamine the binding of fluorinateddrugs to nucleic acids (21,107). Someof these studies pertain to themotional properties of drug-nucleicacid complexes. Fluoroquine, an anti-malarial drug, was shown

(F 1 uoroqui ne)

to intercalate just as its better knownrelative chloroquine (21). The 1 9Frelaxation parameters, including thosefrom the off-resonance relaxationexperiments, were measured for fluoro-quine bound to DNA, poly (A) and tRNAunder conditions ' in which all the drugwas bound; the results are listed inTable 6.

Theoretical curves for the X'Frelaxation parameters were generatedusing the two correlation time modelmathematically represented by equations10 and 11 assuming that the 19F relaxa-tion has contributions both from dipo-lar coupling to the two adjacent aro-matic protons and from chemical shiftanisotropy (see Figure 11) (21). itwas found that the dipolar contributiondominates at Sk.] MHz, but the CSA con-tribution may account for nearly


Table 6. 19F Relaxation Parameters (9^-1 MHz) for Fluoroquine and Its NucleicAcid Complexes (21)

Samplec Temp. (°C) Ti(s) NOE W ] / 2 (Hz)

Fluoroquine

Fluoroquine + DNA

Fluoroquine + tRNA

Fluoroquine + polyA

25

37

25

37

10

25

2.7

O.h

0.2

0.2

0.2

0.3

1.5

0.8

0.8

0.8

1 .0

1-5

—

0.29

0.56

0.75

0.65

0.73

-

0.12

0.11

0.15

0.13

0.22

<

'V

'V

Oi

'V

o<

1

100

30

30

15

10

aSolutions contained 10 mM NaCl and 10 mM cacodylate, pH 7, in D 20. bAn rf

field of strength 0.23 gauss was applied 5-6 KHz off-resonance.- cSample was20-30 mM in DNA base pairs with one fluoroquine per 10 base pairs. "Sample waslmM in tRNA with three fluoroquines per tRNA. "Sample was 30 mM in poly (A)monomers with one fluoroquine per ten monomers.

20% of the relaxation under certaincircumstances. All of the relaxationparameters for the fluoroquine-DNA com-plex could be wel1-described by the

curves with a slower corre-TO of 0.5 MS and an inter-correlation time r- of 1

theoret ical1 at ion t imenal motionns. It is presumed that the fast cor-relation time characterizes a slidingmotion of the intercalated drug paral-lel to the plane of the base pairs withwhich it is stacked. Robinson et al(11) calculated nanosecond correlationtimes from their ESR work for smallspin-labeled dyes intercalated with DNAand suggested that the smaller dyes mayhave motion relative to the DNA.

The 19F relaxation data for tRNA andpoly (A) would not fit the proposedmodel (21). Subsequently, selective1 9F{ 1H} nuclear Overhauser effect

experiments were carried out in whichthe influence of strong irradiation atselective proton resonance frequencieson the 15F resonance intensity wasexamined. The selective NOE experi-ments showed that dipolar coupling toribose protons, as well as the aromaticdrug protons, contributed to fluorinerelaxation in fluoroquine complexeswith tRNA and poly(A) (21). In con-trast, the structure of the fluoroquinecomplex with DNA permitted dipolarcoupling of fluorine only with the aro-matic drug protons as indicated bythese selective NOE experiments.

Fluorescence, uv-visible absorption,and 19F NMR spectroscopic studies ofthe DNA, tRNA, and poly (A) complexes off1uoroquinacrine have been carried out(107).

Vol. h, No. 3/4 153

(F1uoroqu i nacr i ne)it was demonstrated that fluoroqui-

nacrine intercalates with nucleic acidsjust as its better known relative qui-nacrine. The three ring system off1uoroquinacrine should permit interac-tion with both bases in the base pairsof intercalated complexes. 19F NMR(see Figure 12) measurements of Ti,T 1 p

o f f , R, NOE, and linewidth weremade for samples in which all the drugwas complexed. The data for the DNAcomplex could all be predicted with aTO of 3 MS and an internal motion cor-relation time of 2 ns. Likewise, thepoly (A) complex could be described by aro of 0-5 MS and a T-x of 1 ns. Thedata for the tRNA complex, however,could not be fitted. Selective 1'F{1H}NOE experiments with the tRNA and DNAcomplexes of fluoroquinacrine showedthat the fluorine was dipolar coupledto the adjacent aromatic protons in theDNA complex, but the tRNA complexexhibited additional fluorine dipolarcoupling to ribose protons.

VI . CONCLUSIONS

Although the ultimate goal of deli-neating all the types, rates, andamplitudes of the motions in nucleic

acids and their complexes is far fromattainment, work from several researchgroups has provided a glimpse of thedynamic picture. In summary, NMRrelaxation results have shown that thenucleic acid backbone is relativelymobile with motions on the order ofnanoseconds in both the phosphodiesterand ribose moieties for either single-stranded or double-stranded nucleicacids. The motions depend very littleon whether DNA or RNA is examined;therefore, the overall conformation ofthe nucleic acid does not govern thelocal motions. The local motions inthe backbone can be ascribed to pucker-ing in the ribose ring and probably toa rotational wobbling in the phospho-diester. A limited amount of informa-tion does suggest that small variationsin mobility do exist from atom to atomwithin the backbone.

It is clear that the base moietiesof nucleic acids also experience inter-nal motions. There is some suggestionthat this base motion for helicalnucleic acids may be as fast as that inthe ribose and phosphodiester moieties.However, other results suggest a bitmore restricted mobility in the hydro-gen-bonded, stacked bases.

NMR relaxation data also indicatethat double-stranded nucleic acidspossess a motion in the microsecondtime range. There is a strong possi-bility that this is a bending motion,but possible torsional motions in thehelix may also be important.

Variations in the molecular motionsdue to complex formation of the nucleicacids is important from a biochemicaland pharmacological viewpoint. Thereis evidence that the fast (nanosecond)motions of

15*. Bulletin of Magnetic Resonance

"F DIPOLAR*CSA RELAXATION

10

1/T '

(sec')

0.1

0.01

A

f

V-1.5

10-" 10 ••10 •» 10-7t(sec)

(sec')

1OU

10

1

0.1

C

-b

—-<^d

c

*" •* - a

b0.5

10-1' 10-7 10 5(sec)

(Hz)

10-n 10-» lO-'t (sec)

10-5

100

10

1

It

E

^ , f

b

10' ifr» in

Figure 11. Theoretical dependence of the spin-lattice relaxation rate, nuclearOverhauser effect, off-resonance rotating frame spin-lattice relaxation rate,off-resonance intensity ratio, and linewidth for 1 9 F at 9^.1 M H 2 . The relaxationparameters were calculated assuming the two-correlation time model presented inequations 10 and 11 with an internal motion correlation time T- and an isotropicslower motion correlation TQ. The calculations account for a contribution torelaxation from chemical shift anisotropy as well as dipolar contributions from twoprotons each at a distance of 2.6 A away from the fluorine. The dipolar interac-tion is modulated by internal rotation with the angle between an F-H vector and theaxis of rotation, a = IO9 . 5 0 . The chemical shift anisotropy 6 = 51-2 ppm, asymme-try parameter 77 = -1.27 ppm, and Euler angles 6 = 19°, 7 = 3 0 ° . The off-resonanceNMR relaxation parameters were calculated assuming an rf field of strength 0.23gauss applied 5-6 KHz off-resonance. The NOE curves exhibit dipolar contributionsonly. The various plots w e r e calculated with T O values of (a) 3

x 1 0 " 8 s, (b) 1 0 " 7

s, (c) 3 X 10-7 s, (d) 10-' s, (e) 3 X 1 0 " s, and (f)ence (21) .

10-5 s. Taken from refer-

Vol. M, No. 3/4 155

Fluoroquinacrine

+DNA

+tRNA

however, that the nature of the inter-actions in the different protein-nu-cleic acid complexes varies.

Experiments with intercalating drugsindicated that the motion in thenucleic acid is strongly restricted inthe vicinity of the bound drug, butrelatively little restricted elsewhereon the helix. Other NMR relaxationexperiments indicate that small- andintermediate-sized intercalating drugsthemselves experience a motion on thenanosecond time scale as well as theslower microsecond motion of nucleicacids when drug-nucleic acid complexesare formed.

+Poly(A)

-3 -2 -I

19,F Chemical Shift (ppm)

Acknowiedqments

I especially wish to express mygratitude to Professor Aviva Lapidotand the staff of the Weizmann Insti-tute, Rehovot, Israel for their hospi-tality during the tenure of my sabbati-cal leave in which this review waswritten. Support for the author'sresearch discussed in this review wasprovided by grantsfrom the Nationaland by ResearchAward AM 00291 fromtutes of Health.

GM25O18 and RROO892nstitutes of HealthCareer Developmentthe National Insti-

Figure 12. X'F NMR resonance at 9^.1MHz of fluoroquinacrine free andentirely complexed with poly (A), tRNA,and DNA. The samples contained 10 mMNaCl and 10 mM cacodylate, pH 7, in 10%D2O. The chemical shift scale isrelative to the free drug. The phos-phate-to-drug ratios are 10 forpoly (A), kO for tRNA, and 20 for DNA.Taken from reference (107)•

nucleic acids are slowed about an orderof magnitude for nucleic acids in somenatural complexes with proteins, butnot in others. It is definitely clearfrom the NMR relaxation experiments,

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