Structure analysis of membrane-reconstituted subunit c-ring of E. coli H+-ATP synthas

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Structure analysis of membrane-reconstituted subunit c-ring of E. coli H+-ATP synthas

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NMR processing:

MDD

NMR assignment:

Backbone:

Side-chains:

NOEs:

Structure from NMR restraints:

Ab initio:

Fragment-based:

Rosetta-NMR (Robetta)

Template-based:

GeNMR

I-TASSER

Refinement:

Amber

Structure from chemical shifts:

Fragment-based:

Homology-based:

Torsion angles from chemical shifts:

Preditor

TALOS

Promega- Proline

Secondary structure from chemical shifts:

CSI (via RCI server)

TALOS

MICS caps, β-turns

d2D

PECAN

Flexibility from chemical shifts:

RCI

Interactions from chemical shifts:

HADDOCK

Chemical shifts re-referencing:

NMR model quality:

NOEs, other restraints:

Chemical shifts:

RDCs:

Pseudocontact shifts:

Protein geomtery:

SAVES2 or SAVES4

Quality Control Check

NMR spectrum prediction:

FANDAS

MestReS

V-NMR

Flexibility from structure:

Backbone S2

Methyl S2

B-factor

Molecular dynamics:

Gromacs

Amber

Antechamber

Chemical shifts prediction:

From structure:

Shiftx2

Sparta+

Camshift

CH3shift- Methyl

ArShift- Aromatic

ShiftS

Proshift

PPM

CheShift-2- Cα

From sequence:

Shifty

Camcoil

Poulsen_rc_CS

Disordered proteins:

MAXOCC

Format conversion & validation:

CCPN

From NMR-STAR 3.1

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NMR sample preparation:

Protein disorder:

DisMeta

Protein solubility:

Isotope labeling:

Solid-state NMR:

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08-08-2010, 01:57 AM

nmrlearner

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Structure analysis of membrane-reconstituted subunit c-ring of E. coli H+-ATP synthas

Abstract The subunit c-ring of H+-ATP synthase (Fo c-ring) plays an essential role in the proton translocation across a membrane driven by the electrochemical potential. To understand its structure and function, we have carried out solid-state NMR analysis under magic-angle sample spinning. The uniformly [13C, 15N]-labeled Fo c from E. coli (EFo c) was reconstituted into lipid membranes as oligomers. Its high resolution two- and three-dimensional spectra were obtained, and the 13C and 15N signals were assigned. The obtained chemical shifts suggested that EFo c takes on a hairpin-type helix-loop-helix structure in membranes as in an organic solution. The results on the magnetization transfer between the EFo c and deuterated lipids indicated that Ile55, Ala62, Gly69 and F76 were lined up on the outer surface of the oligomer. This is in good agreement with the cross-linking results previously reported by Fillingame and his colleagues. This agreement reveals that the reconstituted EFo c oligomer takes on a ring structure similar to the intact one in vivo. On the other hand, analysis of the 13C nuclei distance of [3-13C]Ala24 and [4-13C]Asp61 in the Fo c-ring did not agree with the model structures proposed for the EFo c-decamer and dodecamer. Interestingly, the carboxyl group of the essential Asp61 in the membrane-embedded EFo c-ring turned out to be protonated as COOH even at neutral pH. The hydrophobic surface of the EFo c-ring carries relatively short side chains in its central region, which may allow soft and smooth interactions with the hydrocarbon chains of lipids in the liquid-crystalline state.

Content Type Journal Article
DOI 10.1007/s10858-010-9432-x
Authors
- Yasuto Todokoro, Osaka University Institute for Protein Research 3-2 Yamadaoka Suita 565-0871 Japan
- Masatoshi Kobayashi, Osaka University Institute for Protein Research 3-2 Yamadaoka Suita 565-0871 Japan
- Takeshi Sato, Osaka University Institute for Protein Research 3-2 Yamadaoka Suita 565-0871 Japan
- Toru Kawakami, Osaka University Institute for Protein Research 3-2 Yamadaoka Suita 565-0871 Japan
- Ikuko Yumen, Osaka University Institute for Protein Research 3-2 Yamadaoka Suita 565-0871 Japan
- Saburo Aimoto, Osaka University Institute for Protein Research 3-2 Yamadaoka Suita 565-0871 Japan
- Toshimichi Fujiwara, Osaka University Institute for Protein Research 3-2 Yamadaoka Suita 565-0871 Japan
- Hideo Akutsu, Osaka University Institute for Protein Research 3-2 Yamadaoka Suita 565-0871 Japan

- Journal Journal of Biomolecular NMR
- Online ISSN 1573-5001
- Print ISSN 0925-2738

Source: Journal of Biomolecular NMR

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