n-detection and other nmr methods for investigations of idps · 15n-detection and other nmr methods...
TRANSCRIPT
15N-detection and other NMR methods for
investigations of IDPs
Helena Kovacs Bruker Switzerland
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 Rainer Kümmerle, Bruker Switzerland
November 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
November 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
b 9 20 6
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 15
N
amide 15N
Amide 15N-15N projection planes from 3D spectra
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 15
N
- relies on MOCCA CO-CO mixing up to 6 residues - Yoshimura, Kulminskaya, Mulder, J Biomol NMR 2015
resolves degeneracy due to low dispersion of 15N frequencies
November 9, 2016 8
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
9
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
November 9, 2016 10
Sensitivity TCI
inverse TXO (13C
optimized) TXO (15N
optimized)
1H sucrose 1.0 0.7 0.7
13C ASTM 1.0 2.0 1.6
15N formamide 1.0 2.1 3.1
relative probe sensitivities cryogenic probes
New CryoProbe options 15N for–detection
/ 2.0 cold 15N-preamp option
Several factors favour X-detection for IDPs
November 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
conclusions
November 9, 2016 12
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