Facile Backbone Structure Determination of Human Membrane Proteins by NMR Spectroscopy

Overview

Journal Nat Methods

Specialties Biomedical Engineering
Pathology

Date 2012 May 22

PMID 22609626

Citations 41

Authors

Christian Klammt

Innokentiy Maslennikov

Monika Bayrhuber

Cedric Eichmann

Navratna Vajpai

Ellis Jeremy Chua Chiu

Katherine Y Blain

Luis Esquivies

June Hyun Jung Kwon

Bartosz Balana

Ursula Pieper

Andrej Sali

Paul A Slesinger

Witek Kwiatkowski

Roland Riek

Senyon Choe

Affiliations

Soon will be listed here.

Abstract

Although nearly half of today's major pharmaceutical drugs target human integral membrane proteins (hIMPs), only 30 hIMP structures are currently available in the Protein Data Bank, largely owing to inefficiencies in protein production. Here we describe a strategy for the rapid structure determination of hIMPs, using solution NMR spectroscopy with systematically labeled proteins produced via cell-free expression. We report new backbone structures of six hIMPs, solved in only 18 months from 15 initial targets. Application of our protocols to an additional 135 hIMPs with molecular weight <30 kDa yielded 38 hIMPs suitable for structural characterization by solution NMR spectroscopy without additional optimization.

Citing Articles

Broken silence: 22,841 predicted deleterious synonymous variants identified in the human exome through computational analysis.

Mello A, Leao D, Dias L, Colombelli F, Recamonde-Mendoza M, Turchetto-Zolet A Genet Mol Biol. 2024; 46(3 Suppl 1):e20230125.

PMID: 38259032 PMC: 10804382. DOI: 10.1590/1678-4685-GMB-2023-0125.

Transmembrane protein 14A protects glomerular filtration barrier integrity.

Khalil R, Bonnemaijer J, Kreutz R, Spaink H, Hogendoorn P, Baelde H Physiol Rep. 2023; 11(23):e15847.

PMID: 38054547 PMC: 10698812. DOI: 10.14814/phy2.15847.

TmAlphaFold database: membrane localization and evaluation of AlphaFold2 predicted alpha-helical transmembrane protein structures.

Dobson L, Szekeres L, Gerdan C, Lango T, Zeke A, Tusnady G Nucleic Acids Res. 2022; 51(D1):D517-D522.

PMID: 36318239 PMC: 9825488. DOI: 10.1093/nar/gkac928.

Fatty acid export (FAX) proteins contribute to oil production in the green microalga .

Peter J, Huleux M, Spaniol B, Sommer F, Neunzig J, Schroda M Front Mol Biosci. 2022; 9:939834.

PMID: 36120551 PMC: 9470853. DOI: 10.3389/fmolb.2022.939834.

Systematic in silico discovery of novel solute carrier-like proteins from proteomes.

Gyimesi G, Hediger M PLoS One. 2022; 17(7):e0271062.

PMID: 35901096 PMC: 9333335. DOI: 10.1371/journal.pone.0271062.

References

Wuu J, Swartz J . High yield cell-free production of integral membrane proteins without refolding or detergents. Biochim Biophys Acta. 2008; 1778(5):1237-50. DOI: 10.1016/j.bbamem.2008.01.023. View

Savage D, Anderson C, Robles-Colmenares Y, Newby Z, Stroud R . Cell-free complements in vivo expression of the E. coli membrane proteome. Protein Sci. 2007; 16(5):966-76. PMC: 2206641. DOI: 10.1110/ps.062696307. View

Liang B, Bushweller J, Tamm L . Site-directed parallel spin-labeling and paramagnetic relaxation enhancement in structure determination of membrane proteins by solution NMR spectroscopy. J Am Chem Soc. 2006; 128(13):4389-97. PMC: 3199951. DOI: 10.1021/ja0574825. View

Klammt C, Srivastava A, Eifler N, Junge F, Beyermann M, Schwarz D . Functional analysis of cell-free-produced human endothelin B receptor reveals transmembrane segment 1 as an essential area for ET-1 binding and homodimer formation. FEBS J. 2007; 274(13):3257-69. DOI: 10.1111/j.1742-4658.2007.05854.x. View

Pervushin K, Riek R, Wider G, Wuthrich K . Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci U S A. 1997; 94(23):12366-71. PMC: 24947. DOI: 10.1073/pnas.94.23.12366. View

Jiang Z, Gui S, Zhang Y . Analysis of differential gene expression by fiber-optic BeadArray and pathway in prolactinomas. Endocrine. 2010; 38(3):360-8. DOI: 10.1007/s12020-010-9389-2. View

Yabuki T, Kigawa T, Dohmae N, Takio K, Terada T, Ito Y . Dual amino acid-selective and site-directed stable-isotope labeling of the human c-Ha-Ras protein by cell-free synthesis. J Biomol NMR. 1998; 11(3):295-306. DOI: 10.1023/a:1008276001545. View

Riek R, Fiaux J, Bertelsen E, Horwich A, Wuthrich K . Solution NMR techniques for large molecular and supramolecular structures. J Am Chem Soc. 2002; 124(41):12144-53. DOI: 10.1021/ja026763z. View

Rual J, Hirozane-Kishikawa T, Hao T, Bertin N, Li S, Dricot A . Human ORFeome version 1.1: a platform for reverse proteomics. Genome Res. 2004; 14(10B):2128-35. PMC: 528929. DOI: 10.1101/gr.2973604. View

10.

Cheriyath V, Leaman D, Borden E . Emerging roles of FAM14 family members (G1P3/ISG 6-16 and ISG12/IFI27) in innate immunity and cancer. J Interferon Cytokine Res. 2010; 31(1):173-81. PMC: 6468951. DOI: 10.1089/jir.2010.0105. View

11.

Klammt C, Schwarz D, Fendler K, Haase W, Dotsch V, Bernhard F . Evaluation of detergents for the soluble expression of alpha-helical and beta-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system. FEBS J. 2005; 272(23):6024-38. DOI: 10.1111/j.1742-4658.2005.05002.x. View

12.

Ishihara G, Goto M, Saeki M, Ito K, Hori T, Kigawa T . Expression of G protein coupled receptors in a cell-free translational system using detergents and thioredoxin-fusion vectors. Protein Expr Purif. 2005; 41(1):27-37. DOI: 10.1016/j.pep.2005.01.013. View

13.

Gautier A, Mott H, Bostock M, Kirkpatrick J, Nietlispach D . Structure determination of the seven-helix transmembrane receptor sensory rhodopsin II by solution NMR spectroscopy. Nat Struct Mol Biol. 2010; 17(6):768-74. PMC: 2923064. DOI: 10.1038/nsmb.1807. View

14.

Page R, Lee S, Moore J, Opella S, Cross T . Backbone structure of a small helical integral membrane protein: A unique structural characterization. Protein Sci. 2009; 18(1):134-46. PMC: 2708045. DOI: 10.1002/pro.24. View

15.

Bedo G, Vargas M, Ferreiro M, Chalar C, Agrati D . Characterization of hypoxia induced gene 1: expression during rat central nervous system maturation and evidence of antisense RNA expression. Int J Dev Biol. 2005; 49(4):431-6. DOI: 10.1387/ijdb.041901gb. View

16.

Roosild T, Greenwald J, Vega M, Castronovo S, Riek R, Choe S . NMR structure of Mistic, a membrane-integrating protein for membrane protein expression. Science. 2005; 307(5713):1317-21. DOI: 10.1126/science.1106392. View

17.

Sobhanifar S, Schneider B, Lohr F, Gottstein D, Ikeya T, Mlynarczyk K . Structural investigation of the C-terminal catalytic fragment of presenilin 1. Proc Natl Acad Sci U S A. 2010; 107(21):9644-9. PMC: 2906861. DOI: 10.1073/pnas.1000778107. View

18.

Junge F, Luh L, Proverbio D, Schafer B, Abele R, Beyermann M . Modulation of G-protein coupled receptor sample quality by modified cell-free expression protocols: a case study of the human endothelin A receptor. J Struct Biol. 2010; 172(1):94-106. DOI: 10.1016/j.jsb.2010.05.004. View

19.

Dalton J, Michalopoulos I, Westhead D . Calculation of helix packing angles in protein structures. Bioinformatics. 2003; 19(10):1298-9. DOI: 10.1093/bioinformatics/btg141. View

20.

Diercks T, Coles M, Kessler H . An efficient strategy for assignment of cross-peaks in 3D heteronuclear NOESY experiments. J Biomol NMR. 2010; 15(2):177-80. DOI: 10.1023/A:1008367912535. View