Analytica Chimica Acta 564 (2006) 1–9

Review

High-resolution protein hydration NMR experiments: Probinghow protein surfaces interact with water and

other non-covalent ligands

Hao Huang, Giuseppe Melacini ∗Departments of Chemistry, Biochemistry and Biomedical Sciences, McMaster University,

1280 Main Street, W. Hamilton, Ont., Canada L8S 4M1

Received 29 July 2005; received in revised form 13 October 2005; accepted 20 October 2005Available online 28 November 2005

Abstract

High-resolution solution NMR experiments are extremely useful to characterize the location and the dynamics of hydrating water molecules atatomic resolution. However, these methods are severely limited by undesired incoherent transfer pathways such as those arising from exchange-relayed intra-molecular cross-relaxation. Here, we review several complementary exchange network editing methods that can be used in conjunction

with other types of NMR hydration experiments such as magnetic relaxation dispersion and 1JNC′ measurements to circumvent these limitations.We also review several recent contributions illustrating how the original solution hydration NMR pulse sequence architecture has inspired newapproaches to map other types of non-covalent interactions going well beyond the initial scope of hydration. Specifically, we will show howhydration NMR methods have evolved and have been adapted to binding site mapping, ligand screening, protein–peptide and peptide–lipidinteraction profiling.© 2005 Elsevier B.V. All rights reserved.

Keywords: Protein; Water; Hydration; NMR; NOE; ROE; Surface; Screening

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22. General architecture of high-resolution NMR hydration experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

2.1. The water selection block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22.2. The water–protein mixing block . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

2.2.1. Exchange network editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.3. Alternative NMR hydration experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

3. Extension to binding site screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54. Extension to ligand screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65. Extension to protein–peptide complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66. Extension to protein–lipid interactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Abbreviations: CT, constant time; CTJ, CT quadrature-free J-resolved dimension; DHPC, dihexanoyl phosphatylcholine; DMF, N,N-dimethylformamide; DMPC,dimyristoil phosphatylcholine; DMSO, dymethyl-sulfoxide; HIV, human immunodeficiency virus; IR, infra red; MD, molecular dynamics; HMQC, hetero-nuclearmultiple quantum coherence spectrum; HSQC, hetero-nuclear single quantum coherence spectrum; MRD, magnetic relaxation dispersion; MS, mass spectrom-etry; NMR, nuclear magnetic resonance; NOE, nuclear overhauser effect; NOESY, 2D-NOE spectroscopy; PDB, protein data bank; PFG, pulsed field gradient;QF, quadrature-free; ROE, rotating frame overhauser effect; ROESY, 2D-ROE spectroscopy; TROSY, transverse relaxation optimized spectroscopy; XH, protonexchangeable with water

∗ Corresponding author. Tel.: +1 905 525 9140x26959; fax: +1 905 522 2509.E-mail address: melacin@mcmaster.ca (G. Melacini).URL: http://www.chemistry.mcmaster.ca/melacini/.

0003-2670/$ – see front matter © 2005 Elsevier B.V. All rights reserved.doi:10.1016/j.aca.2005.10.049

2 H. Huang, G. Melacini / Analytica Chimica Acta 564 (2006) 1–9

7. Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1. Introduction

Water is ubiquitously present in biological systems andit has a profound influence on the structure and dynamicsof biomolecules as well as on the affinity and specificity oftheir non-covalent interactions. The biomolecule binding watermolecules can be classified according to their location andresidence-time into surface and buried waters [1–3]. Buriedwater may stabilize protein structure by bridging protein hydro-gen bonds and/or salt bridges, whereas the most functionallyrelevant surface water molecules are usually located at bindingand/or active sites playing a crucial role in ligand recognition andenzyme catalysis [4]. Surface water molecules that are releasedupon binding provide a mainly entropic driving force as is oftenthe case in protein–DNA complexes [4]. Similarly, the displace-ment of water molecules from the heat shock 70 chaperoneprotein caused by a hydrophobic ligand was suggested to pro-vide a favorable contribution to the binding free energy [5].Surface water molecules that are preserved upon binding pro-vide a mainly enthalpic interaction driving force because of theirability to screen electrostatic repulsion, to fill cavities and to actas linkers/bridges between the interacting biomolecules. Froma survey of protein–protein interfaces in homo-dimeric proteinsand protein–protein complexes available in the PDB, interfacialwater molecules were reported to form hydrogen bonds withpctobcraooo

imaaitspbmusehm

authors. We will first quickly review the general architecture ofhigh-resolution hydration NMR experiments and then we willconsider in details some of their key building blocks, such asthe water selection and the mixing periods. We will specificallyaddress some of the artifacts and limitations of these techniques,such as the exchange-relayed transfer and we will show howNMR pulse sequences can be modified to minimize these biases.We will then illustrate how the high-resolution hydration NMRexperimental design has inspired a multitude of other NMRapproaches tailored to map other types of non-covalent inter-actions, including protein–organic solvents, protein–ligand andprotein–lipid complexes.

2. General architecture of high-resolution NMRhydration experiments

The high-resolution solution NMR experiments designedto investigate macromolecule–water interactions are generallycomposed of three major building blocks (Scheme 1). First,the water magnetization is selected while the biomoleculemagnetization is suppressed; second, magnetization is trans-ferred through dipole–dipole cross-relaxation and/or chemicalexchange from water to macromolecular protons that serveas hydration probes; third, these macromolecular probe pro-tons are properly frequency labeled in one or two homo- orhawt[fiNib

2

rmsopis

S

rotein residues mainly involving backbone carbonyls and theharged side chains of Glu, Asp and Arg [6]. It is therefore clearhat interfacial water molecules are key determinants not onlyf affinity but also of specificity in the non-covalent interactionsetween biomolecules. Interfacial water molecules play a criti-al role also in the understanding of molecular mimicry and inational drug design [7–9]. For instance, Lam et al. [8] designedseries of highly effective inhibitors against the human immun-deficiency virus (HIV) protease HIV-1 by using the carbonylxygen of a cyclic urea to mimic the hydrogen-bonding featuresf a key structural water molecule within the binding site.

The increasing appreciation of the importance of hydrat-ng water molecules in protein function and drug design hasotivated a multitude of investigations on water–protein inter-

ctions [1,4,10–19]. Multiple experimental techniques are avail-ble to characterize water molecules that interact with proteins,ncluding X-ray and neutron diffraction, Brillouin and neu-ron scattering, volumetric measurements, Raman, IR and NMRpectroscopies [10,13–15]. High-resolution solution NMR isarticularly informative because it probes at atomic resolutionoth the location and the residence-time of hydrating waterolecules. In addition, solution NMR can be applied to samples

nder physiological conditions [16–18]. Since, high-resolutionolution NMR based hydration methods have been alreadyxtensively and exhaustively reviewed elsewhere [1,16,17,19],ere the scope will be limited mainly to some recent develop-ents of methods and applications related to the work of the

etero-nuclear dimensions for the purpose of facilitating theirssignment. While the third block essentially relies on very-ell previously established 1D and 2D experiments such as

he 1D-Watergate [20] and the 2D-TOCSY or the 2D-HSQC1,16,17,21,22], the first two blocks have been recently modi-ed to address technical challenges that are specific of hydrationMR pulse sequences. Here, we will therefore focus primar-

ly on the water selection and on the water–protein transferlocks.

.1. The water selection block

The first block (“water selection”) has been thoroughlyeviewed elsewhere [1] and here we will mainly consider aethod that synergistically combines previously reported water

election principles [23] to efficiently provide a clean filteringut of protein resonances, including those of H protons, whilereserving most of the water magnetization. This editing methods based on constant-time (CT) frequency labeling (Fig. 1) whichimultaneously takes advantage of several differences in the

cheme 1. General architecture of high-resolution solution NMR experiments.

H. Huang, G. Melacini / Analytica Chimica Acta 564 (2006) 1–9 3

NMR properties of macromolecules and of water, includingresonating frequencies, homonuclear scalar coupling constants,transverse relaxation and diffusion rates. This is because at pointa in Fig. 1 the transfer function associated with the CT-periodis:

−Iycos(ΩIt1)[∏

kcos(πJikT )

]exp

(− T

T2i

)exp

× (−γ2t2G2(T − ξ)D) (1)

The first cosine term in Eq. (1) encodes for the frequencyfilter, while the term with a product of cosines accounts forthe J coupling based filter. The two exponential terms modelthe relaxation and the diffusion filters, respectively [24,25]. Ifthe CT experiment is implemented using quadrature detection,then the signal-to-noise ratio is reduced by a factor of

√2 as

compared to measurements in which water selective pulses areemployed. However, water magnetization can be set to resonateat zero frequency offset relative to the proton channel carrier fre-quency and therefore no quadrature detection is indeed neededfor the CT evolution. The quadrature-free (QF)–CT method ledin the tested cases to a sensitivity comparable to that obtainedwith selective water excitation schemes. In addition, the QF–CTwater selection scheme facilitates the implementation of waterflip-back pulses, which improve considerably the sensitivity ofNMR hydration experiments. A further advantage of the QF–CTmsomc

2

tpri

FqAsTlp

Fig. 2. Spine of hydration confirmed by solution NMR bridging two pseudo-sheet strands of the X-ray determined structure of CI2 (PDB code 2ci2). Notall the water molecules indicated within brackets are necessary to explain thedetected cross peaks to the HN atom of R67 and R81. Adapted from Melaciniet al. [23].

sign of the protein–water NOEs and ROEs provide informa-tion on the position and on the dynamics of the hydrating watermolecules. NMR investigations on the hydration of both globu-lar [1,2,31] and fibrous [32] proteins have clearly differentiatedbetween the two main types of hydration sites in solution: (a)tightly bound water molecules usually observed in the interiorof proteins with residence-times typically in the 0.1–10 s range(in this case NOEs and ROEs have opposite signs); (b) looselybound water molecules usually detected on the protein surfacewith sub-nanosecond residence-times (in this case NOEs andROEs have the same sign) [19,33].

While NOE/ROE-based high-resolution hydration NMRexperiments have provided the first detailed view of hydrationin solution, these methods still suffer from several limitations.One of the major drawbacks is caused by undesired alternativepathways that can transfer polarization from the water to theprotein protons, effectively competing with the desired directdipole–dipole cross-relaxation (i.e. NOE or ROE). The alter-native pathways originate either from proton/proton exchangebetween water and polar groups of the protein and/or fromexchange-relayed NOE/ROEs. The latter is a two-step trans-fer in which polarization is first transferred from water to theXH groups (X = N, O, S) through chemical exchange and thenfrom the XH protons to nearby protein protons through intra-molecular dipole–dipole cross-relaxation [1,34]. Typical relay-ing labile protons (XH) include carboxyl, amino, hydroxyl,i[casnrNpv

ethod is the efficient control of radiation damping during waterelection, however other methods are also available to controlr constructively use radiation damping [26–30]. The QF–CTethod was tested on lysozyme and applied to the protein CI2

learly revealing a spine of hydration between two anti-parallel-strands (Fig. 2) [23].

.2. The water–protein mixing block

High-resolution hydration NMR experiments are based onhe transfer of magnetization from water to nearby proberotein protons through dipole–dipole cross-relaxation. Cross-elaxation can occur either in the laboratory frame (NOEs) orn the rotating frame (ROEs). The assignment and the relative

ig. 1. Quadrature-free constant-time hydration NMR pulse sequence. Theuadrature-free constant-time water selection block is boxed in a rectangle.dapted from Melacini et al. [23], where further details are available. For the

ake of simplicity gradient and phase cycling information has been omitted here.he magnetization transfer pathway is shown under the pulse sequence. A simi-

ar pulse sequence architecture can be applied to on-/off-resonance ROE mixingeriods.

midazole, guanidinium and amide protons depending on the pH16,35,36]. The direct cross-relaxation and the direct exchangeontributions result in ROESY cross-peaks with opposite signsnd therefore they can be efficiently separated. However, theignals originating from exchange-relayed cross-relaxation areot easily distinguished from those generated by direct cross-elaxation because these two pathways result in very similarOESY/ROESY-patterns. This is a major drawback since bothroton exchange and intra-molecular cross-relaxation are oftenery efficient processes and therefore the indirect exchange-

4 H. Huang, G. Melacini / Analytica Chimica Acta 564 (2006) 1–9

relayed peaks are frequently more intense than the direct inter-molecular NOE/ROE peaks. As a result, the exchange-relayedpeaks usually obscure the more informative signals originat-ing from the direct transfer. The only case in which directand exchange-relayed cross-relaxation contributions can be eas-ily separated is when the protein protons used to probe thehydration events are far enough from exchangeable groups thatthe exchange-relayed effects are negligible. The cut-off dis-tance from XH protons beyond which exchange-relay effectsbecome insignificant has been recently increased to ∼6 A, withthe result of excluding more than ∼80% of non-labile protonsas possible probes of hydration water molecules [19]. Further-more, this conservative approach to the exchange-relay prob-lem is reliably applicable only to systems with well-defined3D structures. The ambiguity between direct and exchange-relayed cross-relaxation has therefore severely hampered theextensive application of high-resolution NMR hydration exper-iments to molecular systems that are highly dynamic and/orare rich in polar groups. These include for instance: (a) thesurface of proteins where polar side chains are usually abun-dant and quite flexible; (b) intrinsically unstructured proteins;(c) amyloid forming polypeptides; (d) carbohydrates; (e) RNA.In some cases, the effect of exchange-relay artifacts in experi-ments designed to probe surface hydration can be minimized byextensive mutations of exchange-relaying residues without sig-nificantly perturbing the overall structure [37]. However, whenese

2

tdbefwsa(pm∼sco[gittcdrhc

Fig. 3. Two independent strategies to edit exchange-relayed cross-relaxation inhigh-resolution hydration NMR experiments.

ing biological relevance [30]. In other words, osmolytes mayrepresent an additional degree of freedom available to NMRspectroscopists for the optimization of hydration experiments.Another challenge of the selective inversion approach comesfrom the requirement of inverting the relaying labile protonsfaster than the chemical exchange with water and at the sametime using selective pulses long enough to obtain the necessaryselectivity required to avoid perturbations of the water mag-netization. This can be sometimes hard to achieve and evenwhen shaped pulses with optimal inversion profiles are usedcareful consideration should be given to the trajectory of thewater magnetization during the selective pulses (Fig. 5). Thecomplex trajectories of the water and protein protons during therepeated selective pulses will cause an averaging of NOE andROE cross-relaxation effects as well as the possible triggeringof radiation damping. The latter can however be effectively sup-pressed by interleaving pulse field gradients or using gradientsfor coherence selection rather than using difference methods[42,43].

Exchange network editing methods based on NOE/ROEcompensation, such as the off-resonance ROESY [46] or theCLEANEX experiments [45] have the advantage of not beinglimited to the slow regime for the exchange between water andthe relaying labile protons. However, these methods suppress notonly exchange-relayed artifacts, but also the peaks arising fromcross-relaxation between long-lived interior water moleculesasaoabtb

xchange-relay artifacts must be eliminated from the hydrationpectra of wild type proteins, spectroscopic exchange networkditing methods can provide a useful alternative.

.2.1. Exchange network editingAs discussed above, one of the major unsolved problems in

he field of high-resolution NMR hydration is the separation ofirect and exchange-relayed cross-relaxation effects. A possi-le initial route to the solution of this problem lies in the use ofxchange network editing techniques [38–40] that select onlyor the desired direct polarization transfer pathway from theater to the protein protons [32,41–44]. The selective suppres-

ion of the exchange-relayed cross-relaxation transfer can bechieved through two main strategies: NOE/ROE compensationFig. 3a) or selective repeated inversions of the relaying labilerotons (Fig. 3b). The NOE/ROE compensation can be imple-ented using for instance off-resonance ROESY methods at a35.5 angle or tailored pulse trains such as the CLEANEX

equence [32,45,46]. The selective repeated inversion strategyan be implemented using either selective irradiation or a trainf repeated Gaussian cascade band-selective pulses (Fig. 4)42–44]. The existence of these two independent editing strate-ies is very useful for validation purposes, but the selectivenversion approach is somewhat limited by the requirement thathe exchange between water and the relaying exchangeable pro-ons be slow in the chemical-shift time-scale. This conditionan be fulfilled by carefully choosing the experimental con-itions (temperature, pH, buffer) so that the proton exchangeates are minimized [32,34]. Recently, we have also illustratedow naturally occurring osmolytes can be effectively used toonstructively modify solution conditions, while still preserv-

nd protein protons. As a result the NOE/ROE compensationtrategy is best suited for probing surface water molecules thatre highly dynamic and usually give rise to cross-relaxation wellutside the spin-diffusion limit where NOE/ROE compensationpplies. However, even when investigating surface hydrationy methods based on NOE/ROE compensation care should beaken to account for potential exchange-relay leakages causedy solvent exposed polar side chains subject to local motion. Ide-

H. Huang, G. Melacini / Analytica Chimica Acta 564 (2006) 1–9 5

Fig. 5. Typical trajectories of protein and water longitudinal (a) and transverse(b) magnetization components during the Gaussian cascade pulses used in theexperiment of Fig. 4. Adapted from Ref. [43], where further details are available.

ally, the best experimental strategy is to combine when possibleboth the selective saturation and the NOE/ROE compensationcomplementary exchange network editing methods through acontrolled selection of the experimental conditions. This com-bined exchange network editing approach in conjunction withPHOGSY-like hydration experiments [47] was essential to probethe dynamics of water molecules hydrating the collagen-triplehelix, which represents a challenging application because col-lagen is very rich in hydroxyproline hydroxyls [32]. In general,the modified mixing blocks that implement exchange networkediting as described above can be employed in conjunctionwith several water selection schemes and protein detection pulsesequence blocks (i.e. 1D, HSQC, HMQC, TROSY [1,2]).

2.3. Alternative NMR hydration experiments

The hydration NMR experiments based on laboratory androtating frame cross-relaxation described above represent just

one of the possible approaches to probe hydration by NMR.Other NMR hydration experiments include magnetic relax-ation dispersion (MRD) and 1JNC′ coupling constant mea-surements. Through MRD experiments [19,33] it is possibleto accurately and quantitatively measure the number and theresidence-times of long-lived water molecules as well as themean rotational correlation time of surface water molecules.The MRD experiments have been already extensively reviewedelsewhere [19,33,48–50] and here we will only mention thatin MRD measurements artifacts caused by proton exchangeare elegantly avoided by taking advantage of the 17O isotopeto monitor hydration dynamics. The robustness of the MRDmethod has led to its extensive application to a multitude ofbiomolecular systems some of which cannot currently be inves-tigated by high-resolution NMR techniques due mainly to theexchange-relay limitations indicated above [48]. However, theMRD technique is based on measurements of average propertiesof bulk water and therefore cannot per se provide informationat atomic resolution unless a comparative difference strategy isused in which MRD experiments are repeated under differentexperimental conditions specifically chosen to perturb selectedwater molecules [49,50].

An elegant alternative NMR measurement that probes hydra-tion without exchange-relay artifacts while still preservingat least residue-resolution is the determination of 1JNC′ cou-pling constants which are sensitive to protein–solvent hydrogenbCt(hoNo

3

sbaOsvof

F ditingc ake oM to thb es are

ig. 4. (a) Pulse sequence for hydration NMR experiments with band-selective eascades. Adapted from Ref. [43], where further details are available. For the sagnetization transfer pathway showing that water magnetization is transferred

y those labile protons which are repeatedly inverted by the band-selective puls

onds [51,52]. The 1JNC′ coupling constants probe mainly theO· · ·HOH hydrogen bonds, which are difficult to investigate

hrough the proton based NOE/ROE methods discussed abovei.e. 1JNC′ > 17 Hz indicates a high population of C O· · ·HOHydrogen bonds, while 1JNC′ < 14 Hz points to a low-populationf the same hydrogen bond) [51]. It appears therefore that waterOE/ROE, 1JNC′ coupling constants and MRD represent a setf complementary NMR hydration experiments.

. Extension to binding site screening

The hydration NMR experiments discussed above are welluited to study the dipole–dipole cross-relaxation not onlyetween proteins and water molecules but also between proteinsnd other small molecules, such as those of organic co-solutes.rganic solvents are potentially an excellent tool for binding site

creening of target proteins. This is because simple organic sol-ents often provide functional group specific binding site mapsf protein surfaces. However, the affinity of organic solventsor proteins is often weak with dissociation constants up to the

of exchange-relay transfer. Band-selective pulses are implemented as Gaussianf simplicity gradient and phase cycling information has been omitted here. (b)e probe protein protons through direct NOEs. NOEs that are exchange-relayedeliminated.

6 H. Huang, G. Melacini / Analytica Chimica Acta 564 (2006) 1–9

molar or sub-molar range. Despite the weakness of these inter-actions, Otting et al. were able to identify a binding site forN,N-dimethylformamide (DMF) and organic co-solvents withinthe specificity-determining substrate binding pockets of hen egg-white lysozyme [53,54]. Similarly, Dalvit et al. [55] used DMSOto locate a binding site in the protein FKBP12 through theapplication of hydration-like NMR experiments. This findingprompted a substructure search for other ligands with similarityto DMSO which led to the discovery of binding molecules withenhanced affinity for the FKBP12 target [55].

The pulse sequences used in the study of protein–organicsolvent interactions usually incorporate a T2-filter and/or a dif-fusion filter to eliminate potential artifacts caused by the overlapbetween the signals of the protein and of the organic solvent[54,55]. When the organic solvents are used in high concen-trations and limit the dynamic range during digitization of theNMR signal, multiple solvent suppression schemes based onexcitations sculpting are necessary [56,57]. The measurementof protein–organic co-solute NOEs and ROEs provides infor-mation on the orientation of the small ligand with respected tothe binding site as well as on the approximate residence-timeof the bound ligand [53,58]. Alternatively, NMRD can be usedto obtain more accurate information on the dynamics of boundorganic solvent molecules [59]. It should also be noticed that,unlike water, organic solvent can be titrated stepwise into proteinsolutions and the resulting protein chemical-shift changes canbmScnbvb

4

eioWttsilascbetso[m

can actually turn out to be a very useful effect in a differentcontext.

5. Extension to protein–peptide complexes

The quadrature-free constant-time (QF–CT) selectionmethod described before in the context of water selection (Sec-tion 2.1) has inspired a new approach for separating 13C and12C bound 1H magnetization for a wide range of hetero-nuclearcoupling constants [71]. This kind of isotope based separation(i.e. spectral filtering and editing) is a crucial step in the NMRexperiments designed to measure intra- and inter-molecularNOEs in complexes between biological molecules such as aprotein–peptide complex [2]. The necessary filtering and editingis concurrently achieved when the QF–CT approach is appliedto the J-dimension of a 2D J-resolved experiment: the protonsbound to 12C will evolve at zero frequency, whereas the protonsbound to 13C will be offset by a frequency equal to JCH/2. Thiscombination of QF–CT and J-resolution led to a pulse sequenceblock named CTJ (Fig. 6a), which can be easily and efficientlycombined into a 3D-NOESY-HSQC experiment using semi-constant time frequency labeling (Fig. 6b and c), leading to a4D-matrix where inter- and intra-molecular NOEs are resolvedthrough the JCH dimension. It should be noticed that the 3D-submatrices at +JCH/2 and at −JCH/2 are symmetrical both interms of signal and of noise so half of the 4D matrix can bed3

cufH[tfafitivn

6

awctadrdeDu

e used to locate binding sites and/or binding induced confor-ational changes as well as to compute binding constants [60].olvent mapping investigations similar to those described abovean be extended also to small gas molecules [18,28], to fluori-ated solvents for which inter-molecular 1H 19F NOEs cane measured [61] and to soluble paramagnetic spin labels pro-iding the additional advantage of probing surface accessibilityy selective relaxation enhancements as well [62–66].

. Extension to ligand screening

The hydration NMR experiments have inspired a verylegant method to screen libraries of ligands for bind-ng to a macromolecular receptor [67–69]. In this setf hydration-inspired ligand screening experiments, calledaterLOGSY (Water–Ligand observation with gradient spec-

roscopy) [67–70], the exchange-relay transfer pathways men-ioned above rather then being suppressed are instead used con-tructively in conjunction with transfer through interfacial andnternal water molecules to selectively saturate macromolecu-ar receptors. Ligands that bind the receptor with micromolarffinity are then easily identified with high sensitivity throughaturation transfer. The scope of the WaterLOGSY methodan be further expanded by combining them with competitioninding and titration experiments [68,70]. The WaterLOGSYxperiments have been already reviewed [36] and they are men-ioned here mainly to illustrate the flexibility and the widecope of the NMR experiments which were originally devel-ped to probe hydration water molecules at atomic resolution1,2]. In addition, the WaterLOGSY strategy shows that whatay be seen as an artifact with respect to some applications

iscarded without compromising the signal-to-noise ratio of theD-submatrices.

The CTJ-based experiment was initially validated on theomplex between (15N,13C) labelled dimeric RII(1–44) andnlabeled Ht31(493–515) [71] and was subsequently success-ully used by the Laue group to help solve the structure of theP1 chromodomain bound to histone H3 methylated at lysine 9

72] and by the Ruterjans group to determine the solution struc-ure of the 30 kDa polysulfide-sulfur transferase homodimerrom Wolinella succinogenes [73]. It should also be noticed thatlternative methods are also available to achieve J-compensatedltering and editing as obtained through the CTJ sequence. In

hese methods J-compensation is achieved by elegantly fine tun-ng 13C inversion adiabatic pulses to the dependence of JCHalues on the 13C chemical-shift typically found in proteins anducleic acids [74,75].

. Extension to protein–lipid interactions

As a final example of the generality and flexibility of therchitecture of high-resolution hydration NMR experiments, weill mention how a similar pulse sequence structure (Scheme 1)

an be extended to the investigation of peptide–lipid interac-ions. Lipids can indeed be very flexible and effectively behaves a quasi-liquid phase for the purposes of NMR pulse sequenceesign. Indeed lipid–protein dipolar–dipolar cross-relaxationates (NOEs) were originally reported for a protein embed-ed in micelles [76]. These pioneering investigations were laterxtended to the case of the integral membrane protein OmpX inHPC micelles and of a trans-membrane peptide embedded innaligned bicelles formed by a mixture of short and long chain

H. Huang, G. Melacini / Analytica Chimica Acta 564 (2006) 1–9 7

Fig. 6. Pulse sequences for the J compensated separation of 12C and 13C bound 1H magnetization based on the quadrature-free constant-time J-resolved (CT-J)method. (a) Basic CT-J building block. (b) 4D F1 semi-constant time, F2 CT-J, F3 13C-edited NOESY-HSQC for samples in D2O. (c) Scheme showing the keyfeatures of the coherence transfer pathways of the 4D experiment in (b). Adapted from Ref. [71], where additional details can be found. For the sake of simplicitygradient and phase cycling information has been omitted here.

phospholipids (i.e. DHPC and DMPC), which have the advan-tage of providing a more ‘native-like’ lipid bilayer still compat-ible with high-resolution NMR studies [77–79]. In this study,peptide–lipid NOEs were first used to determine whether thepeptide was indeed interacting primarily with the quasi-planarbilayer formed by the long chain phospholipids (DMPC) ratherthan with the curved rim formed by the short chain phospho-lipids (DHPC). For this purpose the peptide–lipid NOEs weremeasured using 2D-band-selective NOESY experiments appliedto several unaligned bicellar systems with different lipid deuter-ation schemes so that the contributions from peptide–peptideNOEs, DHPC and DPMC could be clearly separated (Fig. 7).

FbA

As shown in Fig. 7 only when the DMPC is protonated thelipid–peptide NOEs are significantly different from the noiseindicating that the model trans-membrane peptide used wasindeed incorporated in the less-curved region of the unalignedbicelle, as desired for the purpose of more closely mimick-ing the cellular membrane environment. In addition, when thelipid–peptide NOEs are combined with amide–water chemicalexchange measurements the position of different residues withrespect to the membrane–water interface could be determined:residues that are completely out of the bilayer display strongwater exchange peaks, but no lipid NOEs; residues at the bilayer-water interface are characterized by both water exchange andlipid NOE peaks, whereas residues that are embedded within thelipid hydrophobic core display only lipid NOEs and no exchangewith water. It is clear that this type of ‘depth mapping’ is onlyqualitative, but it provides an initial assessment of the generaltopology of membrane interacting peptides.

The bicelle lipid NOE/water chemical exchange approachhas been applied to other membrane binding peptides [80,81],including the prion protein residues 110–136, which is extremelyrich in glycines, but interestingly can still form a membranespanning helix at near neutral pH [81]. It should be noticed thatin this study the multiple glycine residues were assigned byusing a strategy in which different controlled amounts of 14Nand 15N-labeled glycines precursors were incorporated in thepeptide so that the relative intensities in the HSQC spectra couldbhs3o

ig. 7. Lipid deuteration scheme and lipid–peptide NOEs used to differentiateetween DHPC and DPMC interactions of a model trans-membrane peptide.dapted from Ref. [78].

e used for assignment purposes [82]. This assignment strategyad to be used because the large molecular weight of the bicellarystems prevented the use of other more sophisticated 2D andD NMR experiments traditionally employed for the assignmentf globular proteins [83].

8 H. Huang, G. Melacini / Analytica Chimica Acta 564 (2006) 1–9

7. Conclusions and perspectives

The body of work reviewed here shows clearly that despite thecurrent limitations of high-resolution solution NMR hydrationexperiments these methods have inspired several NMR-basedapproaches suitable to investigate diverse types of non-covalentinteractions going well beyond the initial scope of hydratingwater molecules. Indeed, hydration-like NMR pulse sequenceshave been successfully applied to binding site mapping, ligandscreening, protein–peptide and peptide–lipid complexes. It isalso anticipated that hydration NOE/ROE experiments will befurther applied to biological systems, especially if combinedwith exchange network editing methods, MRD and 1JNC′ mea-surements for the purpose of circumventing the exchange-relayproblem, which is currently one of the major limitations of high-resolution solution hydration NMR experiments.

Acknowledgments

We thank NSERC and McMaster University for financialsupport.

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