N-detection and other NMR method developments for ... · PDF file15N-detection and other NMR...

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)


15N detected NCO

NS = 48

TD = 2k x 144


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


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


NS = 4

TD = 4k x 128


ppm

105110115120125130 ppm

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5

09.12.2016 20


950 MHz TCI

C/N-labeled ubiquitin,

298 K


NS = 4

TD = 4k x 128


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


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


950 MHz TCI



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


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

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