Assignment Strategies for Large Membrane Proteins studied by Solid-state MAS NMR

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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

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Outer Membrane Protein G

• 281-residue membrane protein from E.Coli
• b-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

Membrane
Membrane
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Sample Preparation
expression in inclusion bodies
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13C-13C Correlation
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13C-13C Correlation
1 all carbons (CP)
CP cross polarisation
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13C-13C Correlation
1 all carbons (CP) 2 other carbons (PDSD)
CP cross polarisation PDSD proton driven spin
diffusion
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13C-13C Correlation
Ala Ca
Ala Cb
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13C-13C Correlation
20 ms
C
C
200-700 ms
1 all carbons (CP) 2 other carbons (PDSD)
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13C-13C Correlation
GAFY-OmpG PDSD 25ms DARR 700ms 900 MHz 13 kHz
MAS
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NCACX
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NCACX
1 Nitrogen (CP)
CP cross polarisation PDSD proton driven spin
diffusion
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NCACX
1 Nitrogen (CP) 2 Ca (CP)
CP cross polarisation PDSD proton driven spin
diffusion
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NCACX
1 Nitrogen (CP) 2 Ca (CP) 3 other carbons
(PDSD)
CP cross polarisation PDSD proton driven spin
diffusion
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NCACX
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NCACX
20 ms
200-500 ms
1 Nitrogen (CP) 2 Ca (CP) 3 other carbons
(PDSD)
CP cross polarisation PDSD proton driven spin
diffusion
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NCOCX
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NCOCX
1 Nitrogen (CP)
CP cross polarisation
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NCOCX
1 Nitrogen (CP) 2 CO (CP)
CP cross polarisation
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NCOCX
1 Nitrogen (CP) 2 CO (CP) 3 other carbons
(PDSD)
CP cross polarisation PDSD proton driven spin
diffusion
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NCOCX
1 Nitrogen (CP) 2 CO (CP) 3 other carbons
(PDSD)
CP cross polarisation PDSD proton driven spin
diffusion
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NCACB
1 Nitrogen (CP) 2 Ca (CP) 3 Cb (DREAM)
CP cross polarisation DREAM dipolar
recoupling enhancement through amplitude
modulation
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CANCO
1 Ca (CP) 2 N (CP) 3 CO (CP)
CP cross polarisation
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CANCOCX
CO
Ca
N
other carbons
1 Ca (CP) 2 N (CP) 3 CO (CP) 4 other
carbons (PDSD)
CP cross polarisation PDSD proton driven spin
diffusion
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Assignment Strategy

• Identification of Spin Systems
• 13C-13C Correlation
• NCACX
• NCACB
• Linking of Spin Systems
• 13C-13C Correlation
• NCACX
• NCOCX
• CANCO / CANCOCX

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OmpG - Initial Analysis
U-13C-15N OmpG PDSD (25ms) 900 MHz 13 kHz MAS
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Labelling with Glycerol
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Labelling with Glycerol
Glycerol
His
Ser
Gly
Phe, Tyr, Trp
Cys
Val, Leu
Ala
Asp
Asn
Glu
Pro
Gln
Arg
Lys
Met
Thr
Ile
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Labelling with Glycerol

• Sharper signals
• More long-range cross peaks
• Less overlap

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Labelling with Glycerol
U-13C OmpG PDSD (25ms)
1,3-13C-glycerol OmpG PDSD (50ms)
900 MHz, 13 kHz MAS
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Labelling with Glycerol
U-13C OmpG PDSD (25ms)
1,3-13C-glycerol OmpG PDSD (50ms)
900 MHz, 13 kHz MAS
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Labelling with Glycerol
U-13C OmpG PDSD (25ms)
2-13C-glycerol OmpG PDSD (50ms)
900 MHz, 13 kHz MAS
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Identifying Spin Systems
2-13C-glycerol OmpG, PDSD (50ms) 900 MHz,
13kHz MAS
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Identifying Spin Systems
2-13C-glycerol OmpG, PDSD (50ms) 900 MHz,
13kHz MAS
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Identifying Spin Systems
2-13C-glycerol OmpG, PDSD (50ms) 900 MHz,
13kHz MAS
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Identifying Spin Systems
2-13C-glycerol OmpG, PDSD (125ms) 900 MHz,
13kHz MAS
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Identifying Spin Systems
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Identifying Spin Systems
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Identifying Spin Systems
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Identifying Spin Systems
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Linking Spin Systems
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Linking Spin Systems
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Linking Spin Systems
1,3-13C-labelled NCACX / NCOCX
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Linking Spin Systems
1,3-13C-labelled NCACX / NCOCX
2-13C-labelled NCACX / NCOCX
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Linking Spin Systems
1,3-13C-labelled NCACX / NCOCX
2-13C-labelled NCACX / NCOCX
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Linking Spin Systems
1,3-13C-labelled NCACX / NCOCX
2-13C-labelled NCACX / NCOCX
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Assignment
2-13C-glycerol OmpG, NCOCX (50ms), 400 MHz, 8
kHz MAS
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Assignment SH3
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Assignment aB-crystallin
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Labelling with Glycerol
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2,3-13C Labelling GAFY
GAFY
Phe Tyr
Gly Ala
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2,3-13C Labelling GAFY
GAFY-OmpG PDSD (25ms, 700ms) 900 MHz 13 kHz MAS
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2,3-13C Labelling GAFY
OmpG-Uni PDSD OmpG-GAFY PDSD
900 MHz, 13 kHz MAS
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2,3-13C Labelling GAFY
OmpG-Uni PDSD OmpG-GAFY PDSD
OmpG-Uni DREAM OmpG-GAFY DREAM
900 MHz, 13 kHz MAS
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Forwards Labelling GAVLS

• GAVLS
• unlabelled medium
• add labelled GAVLS
• add unlabelled CDEFHIKM
• NPQRTWY

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Forwards Labelling GAVLS
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Reverse Labelling TEMPQANDSG

• TEMPQANDSG
• labelled medium
• add unlabelled CFHKLRI
• VWYSAG

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Reverse Labelling TEMPQANDSG
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Assignment OmpG
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Data Management

• Remember
• Assignment is a giant Sudoku!
• Spreadsheet of Spin Systems
• Sequence sheets

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Data Management
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Data Management

• Remember
• Assignment is a giant Sudoku!
• Spreadsheet of Spin Systems
• Sequence sheets
• Amino acid type sheets

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Data Management
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
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
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Thank you!
Matthias Hiller Sebastian Fiedler Anne
Diehl Stefan Markovic Stefan Jehle Johanna
Becker Barth-Jan van Rossum Hartmut Oschkinat