assignment strategies for large membrane proteins studied by solid-state mas nmr

Assignment Strategies for Large Membrane Proteins

studied by Solid-state MAS NMR

Victoria A. Higman

Leibniz-Institut für Molekulare Pharmakologie, Berlinand

School of Chemistry, University of Bristol

Outer Membrane Protein G

• 281-residue membrane protein from E.Coli

• -barrel fold, monomeric porin• expressed in the absence of

genes for other porins• channel for mono-, di- and tri-

saccharides• possibly pH-gated via

extracellular loop

of biotechnological interest

Mem

brane Mem

bran

e

expression in inclusion bodies

refolding into detergent containing buffer

Ultracentrifugation

concentration and addition of E.Coli lipid extract

Sample Preparation

2D crystallisation by dialysis

13C-13C Correlation

C C

C

N C CN C C

N C

C

C

13C-13C Correlation

C C

C

N C CN C C

N C

C

C

1 – all carbons (CP)

CP – cross polarisation

13C-13C Correlation

C C

C

N C CN C C

N C

C

C

C

C

1 – all carbons (CP)2 – other carbons (PDSD)

CP – cross polarisationPDSD – proton driven spin diffusion

13C-13C Correlation

Ala C

Ala C

13C-13C Correlation

C C

C

N C CN C C

N C

C

C

C

C

1 – all carbons (CP)2 – other carbons (PDSD)

200-700 ms

20 ms

13C-13C Correlation

GAFY-OmpG

PDSD 25ms

DARR 700ms

900 MHz

13 kHz MAS

NCACX

C C

C

N C CN C C

N C

C

C

NCACX

C C

C

N C CN C C

N C

C

C

1 – Nitrogen (CP)

CP – cross polarisationPDSD – proton driven spin diffusion

NCACX

C C

C

N C CN C C

N C

C

C

1 – Nitrogen (CP)2 – C (CP)

CP – cross polarisationPDSD – proton driven spin diffusion

NCACX

C C

C

N C CN C C

N C

C

C

1 – Nitrogen (CP)2 – C (CP)3 – other carbons (PDSD)

N

other carbons

C

CP – cross polarisationPDSD – proton driven spin diffusion

NCACX N

other carbons

C

NCACX

20 ms

200-500 ms

C C

C

N C CN C C

N C

C

C

N

other carbons

C

1 – Nitrogen (CP)2 – C (CP)3 – other carbons (PDSD)

CP – cross polarisationPDSD – proton driven spin diffusion

NCOCX

C C

C

N C CN C C

N C

C

C

NCOCX

C C

C

N C CN C C

N C

C

C

1 – Nitrogen (CP)

CP – cross polarisation

NCOCX

C C

C

N C CN C C

N C

C

C

1 – Nitrogen (CP)2 – CO (CP)

CP – cross polarisation

NCOCX

C C

C

N C CN C C

N C

C

C

1 – Nitrogen (CP)2 – CO (CP)3 – other carbons (PDSD)

CP – cross polarisationPDSD – proton driven spin diffusion

N

other carbons

CO

NCOCX

C C

C

N C CN C C

N C

C

C

1 – Nitrogen (CP)2 – CO (CP)3 – other carbons (PDSD)

CP – cross polarisationPDSD – proton driven spin diffusion

N

other carbons

CO

NCACB

C C

C

N C CN C C

N C

C

C

1 – Nitrogen (CP)2 – C (CP)3 – C (DREAM)

N

C

C

CP – cross polarisationDREAM – dipolar recoupling enhancement through amplitude modulation

CANCO

C C

C

N C CN C C

N C

C

C

1 – C (CP)2 – N (CP)3 – CO (CP)

CP – cross polarisation

C

CO

N

CANCOCX

C C

C

N C CN C C

N C

C

C

1 – C (CP)2 – N (CP)3 – CO (CP)4 – other carbons (PDSD)

CP – cross polarisationPDSD – proton driven spin diffusion

C

other carbons

NCO

Assignment Strategy

• Identification of Spin Systems13C-13C Correlation

NCACX

NCACB

• Linking of Spin Systems13C-13C Correlation

NCACX

NCOCX

CANCO / CANCOCX

OmpG - Initial Analysis

U-[13C-15N] OmpGPDSD (25ms)

900 MHz13 kHz MAS

Labelling with Glycerol

C

13C

OH

13C

OH

OH

[1,3-13C]- glycerol

13C

C

OH

C

OH

OH

[2-13C]- glycerol

Labelling with Glycerol

Glycerol

Ser Gly

CysVal, Leu

Phe, Tyr, Trp

Ala

AspAsn

Lys

Ile

Thr

Met

Gln

Glu

ArgPro

His

Labelling with Glycerol

• Sharper signals

• More long-range cross peaks

• Less overlap

Labelling with Glycerol

900 MHz, 13 kHz MAS

U-[13C] OmpGPDSD (25ms)

[1,3-13C]-glycerol OmpGPDSD (50ms)

Labelling with Glycerol

900 MHz, 13 kHz MAS

U-[13C] OmpGPDSD (25ms)

[1,3-13C]-glycerol OmpGPDSD (50ms)

Labelling with Glycerol

900 MHz, 13 kHz MAS

U-[13C] OmpGPDSD (25ms)

[2-13C]-glycerol OmpGPDSD (50ms)

Identifying Spin Systems

[2-13C]-glycerol OmpG, PDSD (50ms)

900 MHz, 13kHz MAS

Identifying Spin Systems

[2-13C]-glycerol OmpG, PDSD (50ms)

900 MHz, 13kHz MAS

Identifying Spin Systems

[2-13C]-glycerol OmpG, PDSD (50ms)

900 MHz, 13kHz MAS

Identifying Spin Systems

[2-13C]-glycerol OmpG, PDSD (125ms)

900 MHz, 13kHz MAS

Identifying Spin Systems

Identifying Spin Systems

Identifying Spin Systems

Identifying Spin Systems

Linking Spin Systems

Linking Spin Systems

Linking Spin Systems

[1,3-13C]-labelledNCACX / NCOCX

Linking Spin Systems

[1,3-13C]-labelledNCACX / NCOCX

[2-13C]-labelledNCACX / NCOCX

Linking Spin Systems

[1,3-13C]-labelledNCACX / NCOCX

[2-13C]-labelledNCACX / NCOCX

Linking Spin Systems

[1,3-13C]-labelledNCACX / NCOCX

[2-13C]-labelledNCACX / NCOCX

[2-13C]-glycerol OmpG, NCOCX (50ms), 400 MHz, 8 kHz MAS

ThrN

C

N

C

CN C C

CC

C

C

ThrLeu C

ThrC LeuCLeuC

LeuCO

Assignment

Assignment – SH3

Assignment – B-crystallin

Labelling with Glycerol

[2,3-13C] Labelling – GAFY

CN C

CN C

C

Gly Ala

CN C

C

CN C

C

Phe Tyr

Glucose

Ser Gly

Cys

Val, Leu

Phe, Tyr, Trp

Ala

AspAsn

Lys

Ile

Thr

Met

Gln

Glu

Arg

Pro

His

GAFY

GAFY-OmpGPDSD (25ms, 700ms)900 MHz13 kHz MAS

[2,3-13C] Labelling – GAFY

[2,3-13C] Labelling – GAFY

OmpG-Uni PDSDOmpG-GAFY PDSD

900 MHz, 13 kHz MAS

[2,3-13C] Labelling – GAFY

OmpG-Uni PDSDOmpG-GAFY PDSD

900 MHz, 13 kHz MAS

OmpG-Uni DREAMOmpG-GAFY DREAM

Forwards Labelling – GAVLS

GAVLS• unlabelled medium• add labelled GAVLS• add unlabelled

CDEFHIKM

NPQRTWY

Glucose

Ser Gly

Cys

Val, Leu

Phe, Tyr, Trp

Ala

AspAsn

Lys

Ile

Thr

Met

Gln

Glu

Arg

Pro

His

Forwards Labelling – GAVLS

Reverse Labelling – TEMPQANDSG

TEMPQANDSG• labelled medium• add unlabelled

CFHKLRI

VWYSAG

Glucose

Ser Gly

Cys

Val, Leu

Phe, Tyr, Trp

Ala

AspAsn

Lys

Ile

Thr

Met

Gln

Glu

Arg

Pro

His

Reverse Labelling – TEMPQANDSG

Assignment – OmpG

Data Management

Remember:

Assignment is a giant Sudoku!

• Spreadsheet of Spin Systems

• Sequence sheets

Data Management

Data Management

Remember:

Assignment is a giant Sudoku!

• Spreadsheet of Spin Systems

• Sequence sheets

• Amino acid type sheets

Data Management

G L A A L A E L E F L E G L T

K L T D L R W L S D L N G L Q

A L R Y L E Y L P P L T T L G

G L D G L F G L S S L E

1 MEERNDWHFN IGAMYEIENV EGYGEDMDGL AEPSVYFNAA NGPWRIALAY YQEGPVDYSA 60

61 GKRGTWFDRP ELEVHYQFLE NDDFSFGLTG GFRNYGYHYV DEPGKDTANM QRWKIAPDWD 120

121 VKLTDDLRFN GWLSMYKFAN DLNTTGYADT RVETETGLQY TFNETVALRV NYYLERGFNM 180

181 DDSRNNGEFS TQEIRAYLPL TLGNHSVTPY TRIGLDRWSN WDWQDDIERE GHDFNRVGLF 240

241 YGYDFQNGLS VSLEYAFEWQ DHDEGDSDKF HYAGVGVNYS F 281

Thank you!

Matthias Hiller

Sebastian Fiedler

Anne Diehl

Stefan Markovic

Stefan Jehle

Johanna Becker

Barth-Jan van Rossum

Hartmut Oschkinat