assignment strategies for large membrane proteins studied by solid-state mas nmr
DESCRIPTION
Assignment Strategies for Large Membrane Proteins studied by Solid-state MAS NMR. Victoria A. Higman Leibniz-Institut für Molekulare Pharmakologie, Berlin and School of Chemistry, University of Bristol. O uter M embrane P rotein G. 281-residue membrane protein from E.Coli - PowerPoint PPT PresentationTRANSCRIPT
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