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