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15N-detection and other NMR method developments
for investigations of IDPs
Helena Kovacs, Rainer Kümmerle,
Bruker Biospin AG Fällanden
Acknowledgement: Nathan Jespersen, Elisar Barbar Oregon State University, Corvallis,OR, USA
Acknowledgement : Tanja Mittag, Eric Martin, Jacklyn Cika, Richard Kriwacki St. Jude Children’s Research Hospital, Memphis, TN, USA Wolfgang Bermel, Bruker Germany Helena Kovacs, Bruker Switzerland
December 9, 2016 2
Introduction 1H-detected assigment of disordered protein regions
15N-detection
15N-detection and other NMR methods for
investigations of IDPs - intrinsically disordered proteins
Introduction
3 3
• 20% of human genome is IDPs • 50% of structured proteins have IDP streches or flexible linkers and termini - long (>30 residue) disordered segments occur in 2% of archaean, 4 % of bacterial and 33 % of eukaryotic proteins Ward, J. J.; Sodhi, J. S.; McGuffin, L. J.; Buxton, B. F.; Jones, D. T. (2004). "Prediction and functional analysis of native disorder in proteins from the three kingdoms of life". Journal of Molecular Biology 337: 635–45.
• IDPs are highly abundant among disease-related proteins Tompa, P(2012) "Intrinsically Disordered Proteins a 10 year recap". TIBS 931:1-8..
NMR spectroscopy is well suited for
study of IDPs on atomic level
December 9, 2016 4
In solution IDPs or IDRs (intrinsically disordered proteins or protein regions) exist as dynamic ensembles of interconverting conformers, possibly with transient time-averaged conformational preferences. From the NMR point of view, this leads to:
sharp signals, narrow line widths
310-residue full length protein, dimer 68kDa
(50% IDR)
consisting of
150-residue C-terminal domain, monomer in free form
(30% IDR)
160-residue N-terminal domain, dimer in free form
(70% IDR)
low amide 1H and 15N resolution in IDRs
Disordered regions, low dispersion
full length protein
TROSY
C-terminal
HSQC
N-terminal
TROSY
Nathan E. Jespersen, Elisar Barbar, Oregon State University, Corvallis,OR, USA
1H
1H
- two different time scales for structured vs. disordered regions
Dynamics from 15N T1 and T2 relaxation
comparison of C-terminus vs. a structured protein (SNase) both ca 150 residues, 17kDa
C-terminus 30% IDR
SNASE folded
T1 = 1.0 sec T2 = 40-60 msec in structured regions
T1 = 0.6 sec T2 = 200-400 msec in unstructured regions
- T2 relaxation is a good indicator of structure/disorder
Dynamics from 15N T1 and T2 relaxation
comparison of C-terminus vs. a structured protein (SNase) both ca 150 residues, 17kDa
T2=63ms structured parts => 15N-line width= 5Hz T2=233ms IDR => 15N-line width= 1.3Hz
December 9, 2016
1H-detected amide-to-amide connectivities
HNcocaNNH
HNcaNNH
Two correlations
for each amide
frequency.
Pairwise correlations
of amide
frequencies.
- «really great» for assigning IDRs
- but, for ordered regions «lower sensitivity than HNCACB»
Nathan E. Jespersen, Biochemistry & Biophysics, Oregon State University, Corvallis,OR, USA
1H-detected amide-to-amide connectivities
HNcaNNH HNcocaNNH
two neigbouring NHs preceeding NH only
amid
e 1
5N
amide 15N
Amide 15N-15N projection planes from 3D spectra recorded on 150mM of 34 kDa N-terminal domain dimer
Nathan E. Jespersen, Elisar Barbar, Biochemistry & Biophysics, Oregon State University, Corvallis,OR, USA
amide 1H amide 1H
amide 15N
1H-detected distant amide-to-amide connectivities
HNcaNNH
neigbouring NHs
HNcocoNH
distant HN-HN connectivities
amide H amide 15N
amid
e 1
5N
- relies on MOCCA CO-CO mixing up to 6 residues - Yoshimura, Kulminskaya, Mulder, J Biomol NMR 2015
1mM ubiquitin amide 15N-15N projection planes from 3D spectra
resolves degeneracy due to low dispersion of 15N frequencies
December 9, 2016 11
observations
high mobility makes higher molecular weight proteins accessible favourable relaxation properties of IDPs (up to 90kDa in current study)
long T2s allow distant magnetization transfer
- direct amide to amide connectivities - higher dimensional experiments coding several frequencies (4D, 5D, 6D etc)
- use of fast automated acquisition techniques such as APSY
APSY = automated projection spectroscopy for multidimensional experiments - TopSPin 3.5pl6: applicable to any Bruker pulse program (3D, 4D, 5D, 6D etc) - delivers a list of resonance frequencies
Challanges in NMR of IDPs
12
Low chemical shift dispersion - severe overlap of amide 1H resonances
Few conformational constraints - few NOEs and weak 13C-chemical shift constraints Fast exchange with water - use techniques avoiding water-excitation - X-detection avoids this complication
X-detection theoretical sensitivity
December 9, 2016 13
relative sensitivity provided sample is uniformly labeled:
gH = 26,752221 [107 rad s-1 T-1]
gC = 6,728286 [107 rad s-1 T-1]
gN = -2,712619 [107 rad s-1 T-1]
signal-to-noise is proportional to g3/2
S/Ncarbon = 0,13 less than S/Nprotons
S/Nnitrogen = 0,03 less than S/Nprotons
S/Nnitrogen = 0,18 less than S/Ncarbon
Sample loss [frequency]
Solvent dependence ~n4
Salt dependence ~n2
09.12.2016 15
13C detection: CON 950 MHz TCI
C/N-labeled ubiquitin
13C detected CON
using IPAP virtual
homodecoupling
NS = 2
TD = 1k x 800
Expt. Time 32 minutes
ppm
167168169170171172173174175176177178179180 ppm
105
110
115
120
125
130
135
140
298 K
09.12.2016 16
15N detection: NCO 950 MHz TCI (active 15N)
C/N-labeled ubiquitin
15N detected NCO
NS = 48
TD = 2k x 144
Expt. Time 150 minutes
ppm
105110115120125130135140 ppm
168
169
170
171
172
173
174
175
176
177
178
179
298 K
09.12.2016 17
13C CON vs. 15N detection NCO
13C vs. 15N detection: neglecting relaxation one expects theoretically 5:1
For the CON/NCO comparison of ubiquitin at 950 MHz and 298 K one obtains ~2.5:1
15N detected NCO, 150 min
13C detected CON, 30 min
298 K
09.12.2016 18
13C CON vs. 15N detection NCO
13C vs. 15N detection: neglecting relaxation one expects theoretically 5:1
For the CON/NCO comparison of ubiquitin at 950 MHz and 278 K one obtains ~2:1
15N detected NCO, 150 min
13C detected CON, 30 min
7:1
5:1
278 K
09.12.2016 19
15N detected HX-INEPT
950 MHz TCI
C/N-labeled ubiquitin,
298 K
15N detected HX-INEPT
NS = 4
TD = 4k x 128
Expt. Time 11 minutes
ppm
105110115120125130 ppm
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
09.12.2016 20
15N detected HX-INEPT
950 MHz TCI
C/N-labeled ubiquitin,
298 K
15N detected HX-INEPT
NS = 4
TD = 4k x 128
Expt. Time 11 minutes
105110115120125130 ppm
F2 (15N) projection of 2D
TROSY: 1H & 15N Linewidth
800 kD
150 kD
50 kD
Linewidth 1H Linewidth 15N
09.12.2016 22
15N detected coupled INEPT - TROSY
TCI 950 MHz
C/N-labeled ubiquitin, 298 K
15N detected HX-INEPT,
fully coupled: illustrating the
TROSY effect
NS = 64
TD = 4k x 256
Expt. Time 7 hours
ppm
101.4101.6101.8102.0102.2102.4102.6102.8103.0103.2103.4 ppm
7.90
7.95
8.00
8.05
8.10
8.15
298 K
09.12.2016 23
15N detected coupled INEPT - TROSY
TCI 950 MHz
C/N-labeled ubiquitin, 278 K
15N detected HX-INEPT,
fully coupled: illustrating the
TROSY effect
NS = 32
TD = 4k x 256
Expt. Time 3.5 hours
ppm
101.8102.0102.2102.4102.6102.8103.0 ppm
7.80
7.82
7.84
7.86
7.88
7.90
7.92
7.94
7.96
7.98
8.00
8.02
278 K
09.12.2016 24
15N detected coupled INEPT - TROSY
950 MHz TCI
C/N-labeled ubiquitin
15N detected HX-INEPT
fully coupled: illustrating
the TROSY effect as a
function of temperature
278 K
298 K
December 9, 2016 25
Sensitivity TCI
inverse TXO (13C
optimized) TXO (15N
optimized)
1H sucrose
1.0 0.7 0.7
13C ASTM
1.0 2.1 1.6
15N formamide
0.5 2.1 3.1
relative probe sensitivities cryogenic probes
New CryoProbe options 15N for–detection
/ 1.0 cold 15N-preamp option
Several factors favour X-detection for IDPs
December 9, 2016
narrow lines of 15N and larger chemical shift dispersion 13C
high salt-tolerance of X-nuclei, minor losses even in high salt buffers
absence of 1H signals - IDPs often contain many prolines that lack amide-1H - conformational exchange may lead to complete loss of 1H signals
cryogenic probes’ power handling: longer spin locks and spin echos, but, weaker spinlock power needed for the narrower band widths of 15N and 13C
multi receiver experiments optimal as the relative sensitivity between the nuclei (15N, 13C) is balanced
compensate for inherently lower sensitivity of 15N and 13C compared to 1H
Line narrowing in 15N-detected experiments
December 9, 2016 27
refs: Hari Arthanari, personal communication Takeuchi, Arthanari, Imai, Wagner, Shimada. J Biomol NMR (2016) 64:143-151 Takeuchi, Arthanari, Shimada, Wagner. J Biomol NMR (2015) 323-331
50 100 150 kDa
in H2O
in D2O -deuteration of the protein is not needed (long-range dipolar interactions are negligible)
Jacklyn Cika, Richard Kriwacki, St. Jude Children’s Research Hospital, Memphis, TN, USA
09.12.2016
1H-detected vs. 15N-detected TROSY
1H
15N
15N
1H
Pentamer, 160kDa in total in H2O, 200mM NaCl
15N detected TROSY 3 hours
1H detected TROSY 40 min
09.12.2016
13C-detected CON vs. 15N detected NCO
15N detected NCO CO-start exp. time 17 hours
13C detected CON HA-start Rapid scanning BASH 13C-13C homodecoupling exp. time 2 hours
prolines prolines
13C vs. 15N detection: neglecting relaxation one expects theoretically 5:1
Tanja Mittag, Eric Martin, St. Jude Children’s Research Hospital, Memphis, TN, USA
8.6kDa, in D2O 700MHz TXO CryoProbe
13C
15N
15N
13C
15N-detected hCAN
proline Cd
proline Ca
Tanja Mittag, Eric Martin, St. Jude Children’s Research Hospital, Memphis, TN, USA
8.6kDa, in D2O 700MHz TXO CryoProbe HA-start
13C
15N
conclusions
December 9, 2016 31
15N-detection: sensitivity can be enhanced
15N-detected INEPT-based experiments benefit from line narrowing through deuteration of the attached amide proton by using D2O instead of H2O buffers. 15N-detected TROSY compensates for low sensitivity of 15N through line narrowing for large proteins at high magnetic field strengths and in high salt buffers.
Ideal at higher NMR fields and using optimized cryogenic probes