[en] Brucella melitensis is a gram-negative bacteria known to cause brucellosis and to produce severe infections in humans. Whilst brucella's outer membrane proteins have been extensively studied due to their potential role as antigens or virulence factors, their function is still poorly understood at the structural level, as the 3D structure of Brucella beta-barrel membrane proteins are still unknown. In this context, the B. melitensis trimeric Omp2a porin has been overexpressed and refolded in n-dodecyl-beta-d-maltopyranoside. We here show that this refolding process is insensitive to urea but is temperature- and ionic strength-dependent. Reassembled species were characterized by fluorescence, size-exclusion chromatography and circular dichroism. A refolding mechanism is proposed, suggesting that Omp2a first refolds under a monomeric form and then self-associates into a trimeric state. This first complete in vitro refolding of a membrane protein from B. melitensis shall eventually lead to functional and 3D structure determination.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Roussel, G
Matagne, André ; Université de Liège - ULiège > Département des sciences de la vie > Enzymologie et repliement des protéines
De Bolle, X
Perpete, EA
Michaux, Catherine
Language :
English
Title :
Purification, refolding and characterization of the trimeric Omp2a outer membrane porin from Brucella melitensis.
Publication date :
2012
Journal title :
Protein Expression and Purification
ISSN :
1046-5928
eISSN :
1096-0279
Publisher :
Academic Press, Orlando, United States - Florida
Volume :
83
Issue :
2
Pages :
198-204
Peer reviewed :
Peer Reviewed verified by ORBi
Commentary :
Copyright (c) 2012 Elsevier Inc. All rights reserved.
M.L. Boschiroli, V. Foulongne, and D. O'Callaghan Brucellosis: a worldwide zoonosis Current Opinion in Microbiology 4 2001 58 64
N. Vizcaino, A. Cloeckaert, J. Verger, M. Grayon, and L. Fernandez-Lago DNA polymorphism in the genus Brucella Microbes and Infection/Institut Pasteur 2 2000 1089 1100
R. Benz, and K. Bauer Permeation of hydrophilic molecules through the outer membrane of gram-negative bacteria. Review on bacterial porins European Journal of Biochemistry/FEBS 176 1988 1 19
R. Benz Structure and function of porins from gram-negative bacteria Annual Review of Microbiology 42 1988 359 393
A.H. Delcour Solute uptake through general porins Frontiers in Bioscience:A Journal and Virtual Library 8 2003 d1055 d1071
W.V. Alphen, and B. Lugtenberg Influence of osmolarity of the growth medium on the outer membrane protein pattern of Escherichia coli Journal of Bacteriology 131 1977 623 630
G. Dubray, and G. Bezard Isolation of three Brucella abortus cell-wall antigens protective in murine experimental brucellosis Annals of Veterinary Research 11 1980 367 373
D.R. Verstreate, M.T. Creasy, N.T. Caveney, C.L. Baldwin, M.W. Blab, and A.J. Winter Outer membrane proteins of Brucella abortus: isolation and characterization Infection and Immunity 35 1982 979 989
J.T. Douglas, E.Y. Rosenberg, H. Nikaido, D.R. Verstreate, and A.J. Winter Porins of Brucella species Infection and Immunity 44 1984 16 21
J.Y. Paquet, C. Vinals, J. Wouters, J.J. Letesson, and E. Depiereux Topology prediction of Brucella abortus Omp2b and Omp2a porins after critical assessment of transmembrane beta strands prediction by several secondary structure prediction methods Journal of Biomolecular Structure & Dynamics 17 2000 747 757
P.J. Booth The trials and tribulations of membrane protein folding in vitro Biochimica et Biophysica Acta 1610 2003 51 56
A. Jungbauer, and W. Kaar Current status of technical protein refolding Journal of Biotechnology 128 2007 587 596
J.U. Bowie Solving the membrane protein folding problem Nature 438 2005 581 589
F. Junge, B. Schneider, S. Reckel, D. Schwarz, V. Dotsch, and F. Bernhard Large-scale production of functional membrane proteins Cellular and Molecular Life Sciences 65 2008 1729 1755
R. Laage, and D. Langosch Strategies for prokaryotic expression of eukaryotic membrane proteins Traffic 2 2001 99 104
B. Schmid, M. Kromer, and G.E. Schulz Expression of porin from Rhodopseudomonas blastica in Escherichia coli inclusion bodies and folding into exact native structure FEBS Letters 381 1996 111 114
M. Bannwarth, and G.E. Schulz The expression of outer membrane proteins for crystallization Biochimica et Biophysica Acta 1610 2003 37 45
R.M. Garavito, and S. Ferguson-Miller Detergents as tools in membrane biochemistry The Journal of Biological Chemistry 276 2001 32403 32406
P. Raman, V. Cherezov, and M. Caffrey The Membrane Protein Data Bank Cellular and Molecular Life Sciences 63 2006 36 51
H. Marquis, and T.A. Ficht The omp2 gene locus of Brucella abortus encodes two homologous outer membrane proteins with properties characteristic of bacterial porins Infection and Immunity 61 1993 3785 3790
M. Manning, and W. Colon Structural basis of protein kinetic stability: resistance to sodium dodecyl sulfate suggests a central role for rigidity and a bias toward beta-sheet structure Biochemistry 43 2004 11248 11254
Y. Watanabe Effect of various mild surfactants on the reassembly of an oligomeric integral membrane protein OmpF porin Journal of Protein Chemistry 21 2002 169 175
K. Nakamura, and S. Mizushima Effects of heating in dodecyl sulfate solution on the conformation and electrophoretic mobility of isolated major outer membrane proteins from Escherichia coli K-12 Journal of Biochemistry 80 1976 1411 1422
D.P. Goldenberg, and T.E. Creighton Gel electrophoresis in studies of protein conformation and folding Analytical Biochemistry 138 1 1984 1 18
L.K. Tamm, H. Hong, and B. Liang Folding and assembly of beta-barrel membrane proteins Biochimica et Biophysica Acta 1666 2004 250 263
V. Baldwin, and M. Bhatia Folding studies of purified LamB protein, the maltoporin from the Escherichia coli outer membrane: trimer dissociation can be separated from unfolding Biochimica et Biophysica Acta 1808 9 2011 2206 2213
K.M. Sanchez, J.E. Gable, D.E. Schlamadinger, and J.E. Kim Effects of tryptophan microenvironment, soluble domain, and vesicle size on the thermodynamics of membrane protein folding: lessons from the transmembrane protein OmpA Biochemistry 47 2008 12844 12852
D.W. Bolen Effects of naturally occurring osmolytes on protein stability and solubility: issues important in protein crystallization Methods 34 2004 312 322
B. Batas, C. Schiraldi, and J.B. Chaudhuri Inclusion body purification and protein refolding using microfiltration and size exclusion chromatography Journal of Biotechnology 68 1999 149 158
M.E. Goldberg, R. Rudolph, and R. Jaenicke A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg white lysozyme Biochemistry 30 1991 2790 2797
R.S. Negin, and J.D. Carbeck Measurement of electrostatic interactions in protein folding with the use of protein charge ladders Journal of the American Chemical Society 124 2002 2911 2916
J.Y. Paquet, M.A. Diaz, S. Genevrois, M. Grayon, J.M. Verger, X. de Bolle, J.H. Lakey, J.J. Letesson, and A. Cloeckaert Molecular, antigenic, and functional analyses of Omp2b porin size variants of Brucella spp Journal of Bacteriology 183 2001 4839 4847
H. Hong, and L.K. Tamm Elastic coupling of integral membrane protein stability to lipid bilayer forces Proceedings of the National Academy of Sciences of the United States of America 101 2004 4065 4070
G.H. Huysmans, S.E. Radford, D.J. Brockwell, and S.A. Baldwin The N-terminal helix is a post-assembly clamp in the bacterial outer membrane protein PagP Journal of Molecular Biology 373 2007 529 540
P.H. Yancey, M.E. Clark, S.C. Hand, R.D. Bowlus, and G.N. Somero Living with water stress: evolution of osmolyte systems Science 217 1982 1214 1222
T. Arakawa, and S.N. Timasheff The stabilization of proteins by osmolytes Biophysical Journal 47 1985 411 414
T.O. Street, D.W. Bolen, and G.D. Rose A molecular mechanism for osmolyte-induced protein stability Proceedings of the National Academy of Sciences of the United States of America 103 2006 13997 14002
N.K. Burgess, T.P. Dao, A.M. Stanley, and K.G. Fleming Beta-barrel proteins that reside in the Escherichia coli outer membrane in vivo demonstrate varied folding behavior in vitro The Journal of Biological Chemistry 283 2008 26748 26758
Y.K. Reshetnyak, Y. Koshevnik, and E.A. Burstein Decomposition of protein tryptophan spectra into log-normal components. III. Correlation between fluorescence and microenvironment parameters of individual tryptophan residues Biophysical Journal 81 2001 1735 1758
K. Dornmair, H. Kiefer, and F. Jahnig Refolding of an integral membrane protein The Journal of Biological Chemistry 265 1990 18907 18911
A. Veerappan, F. Cymer, N. Klein, and D. Schneider The tetrameric α-helical membrane protein GlpF unfolds via a dimeric folding intermediate Biochemistry 50 2011 10223 10230
F.N. Barrera, L.M. Renart, M.L. Molina, J.A. Poveda, J.A. Eninar, A.M. Fernandez, J.L. Neira, and J.M. Gonzalez-Ros Unfolding and refolding in vitro of a tetrameric-helical membrane protein: the prokaryotic potassium channel KcsA Biochemistry 44 2005 14344 14352
Y. Watanabe, and Y. Inoko Reassembly of an integral oligomeric membrane protein OmpF porin in n-octyl beta-d-glucopyranoside-lipids mixtures The Protein Journal 28 2009 66 73
J. Siritapetawee, H. Prinz, C. Krittanai, and W. Suginta Expression and refolding of Omp38 from Burkholderia pseudomallei and Burkholderia thailandensis, and its function as a diffusion porin The Biochemical Journal 384 2004 609 617
V. Visudtiphole, M.B. Thomas, D.A. Chalton, and J.H. Lakey Refolding of Escherichia coli outer membrane protein F in detergent creates LPS-free trimers and asymmetric dimers The Biochemical Journal 392 2005 375 381
A. Kreusch, and G.E. Schulz Refined structure of the porin from Rhodopseudomonas blastica. Comparison with the porin from Rhodobacter capsulatus Journal of Molecular Biology 243 1994 891 905
S.W. Cowan, T. Schirmer, G. Rummel, M. Steiert, R. Ghosh, R.A. Pauptit, J.N. Jansonius, and J.P. Rosenbusch Crystal structures explain functional properties of two E. coli porins Nature 358 1992 727 733
K. Seshadri, and R. Garemyr Architecture of beta-barrel membrane proteins: analysis of trimeric porins Protein Science 7 9 1998 2026 2032
C. Jansen, M. Heutink, J. Tommassen, and H. de Cock The assembly pathway of outer membrane protein PhoE of Escherichia coli European Journal of Biochemistry/FEBS 267 2000 3792 3800
W.J. Rocque, and E.J. McGroarty Isolation and preliminary characterization of wild-type OmpC porin dimers from Escherichia coli K-12 Biochemistry 28 1989 3738 3743
S.C. Gill, and P.H. von Hippel Calculation of protein extinction coefficients from amino acid sequence data Analytical Biochemistry 182 1989 319 326
N. Sreerama, and R.W. Woody Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set Analytical Biochemistry 287 2000 252 260