β-lactamase; antibiotic resistance; circular dichroism (CD); enzyme catalysis; metalloenzyme; nuclear magnetic resonance (NMR); protein folding; zinc
Abstract :
[en] Metallo-beta-lactamases catalyse the hydrolysis of most beta-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus beta-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (DeltaG degrees ) of 32 +/- 2 kJ.mol11. For holoBcII, a first non-cooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site and the protein displays a well-organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. 2D NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with DeltaG degrees value of 65 +/- 1.4 kJ.mol11. These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well-defined conformation for both active site loops in order to maintain enzymatic activity.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Montagner, Caroline ; Université de Liège > Département des sciences de la vie > Macromolécules biologiques
Nigen, Michaël
Jacquin, Olivier
Willet, Nicolas ; Université de Liège > Département de chimie (sciences) > Nanochimie et systèmes moléculaires
Dumoulin, Mireille ; Université de Liège > Département des sciences de la vie > Enzymologie et repliement des protéines
Karsisiotis, Andreas Ioannis
Roberts, Gordon C. K.
Damblon, Christian ; Université de Liège > Département de chimie (sciences) > Chimie biologique structurale
Redfield, Christina
Matagne, André ; Université de Liège > Département des sciences de la vie > Enzymologie et repliement des protéines
Language :
English
Title :
The role of active site flexible loops in catalysis and of zinc in conformational stability of Bacillus cereus 569/H/9 beta-lactamase.
Publication date :
2016
Journal title :
Journal of Biological Chemistry
ISSN :
0021-9258
eISSN :
1083-351X
Publisher :
American Society for Biochemistry and Molecular Biology, Baltimore, United States - Maryland
Volume :
291
Issue :
31
Pages :
16124-16137
Peer reviewed :
Peer Reviewed verified by ORBi
Commentary :
Copyright (c) 2016, The American Society for Biochemistry and Molecular Biology.
Waley, S. G. (1992) in The Chemistry of β-Lactams (Page, M. I., ed) pp. 198-228, Blackie, London, UK
Frère, J. M. (1995) β-Lactamases and bacterial resistance to antibiotics. Mol. Microbiol. 16, 385-395
Fink, A. L., and Page, M. I. (2012) in β-Lactamases (Frère, J. M., ed) pp. 41-77, Nova Science Publishers, Inc., New York
Poole, K. (2004) Resistance to β-lactam antibiotics. Cell. Mol. Life Sci. 61, 2200-2223
Jacoby, G. A., and Munoz-Price, L. S. (2005) The new β-lactamases. N. Engl. J. Med. 352, 380-391
Rossolini, G. M., and Docquier, J. D. (2006) New β-lactamases: a paradigm for the rapid response of bacterial evolution in clinical setting. Future Microbiol. 1, 295-308
Frère, J. M., (ed) (2012) β-Lactamases, Nova Science Publishers, Inc., New York
Matagne, A., Dubus, A., Galleni, M., and Frère, J. M. (1999) The β-lactamase cycle: a tale of selective pressure and bacterial ingenuity. Nat. Prod. Rep. 16, 1-19
Heinz, U., and Adolph, H. W. (2004) Metallo-β-lactamases: two binding sites for one catalytic metal ion? Cell. Mol. Life Sci. 61, 2827-2839
Bebrone, C. (2007) Metallo-β-lactamases (Classification, Activity, Genetic Organization, Structure, Zinc Coordination) and their superfamily. Biochem. Pharmacol. 74, 1686-1701
Palzkill, T. (2013) Metallo-β-lactamase structure and function. Ann. N.Y. Acad. Sci. 1277, 91-104
Neuwald, A. F., Liu, J. S., Lipman, D. J., and Lawrence, C. E. (1997) Extracting protein alignment models from the sequence database. Nucleic Acids Res. 25, 1665-1677
Daiyasu, H., Osaka, K., Ishino, Y., and Toh, H. (2001) Expansion of the zinc metallo-hydrolase family of the β-lactamase fold. FEBS Lett. 503, 1-6
Herzberg, O., and Fitzgerald, M. D. (2004) in Handbook of Metalloproteins (Messerschmidt, A., Bode, W., and Gygler, M., eds) Vol. 3, pp. 217-234, John Wiley & Sons, Ltd., New York
Bebrone, C., Garau, G., Garcia-Saez, I., Chantalat, L., Carfi, A., and Dideberg, O. (2012) in β-Lactamases (Frère, J. M., ed) pp. 41-77, Nova Science Publishers, Inc., New York
Baier, F., and Tokuriki, N. (2014) Connectivity between catalytic landscapes of the metallo-β-lactamase superfamily. J. Mol. Biol. 426, 2442-2456
Karsisiotis, A. I., Damblon, C. F., and Roberts, G. C. (2014) A variety of roles for versatile zinc in metallo-β-lactamases. Metallomics 6, 1181-1197
Walsh, T. R., Toleman, M. A., Poirel, L., and Nordmann, P. (2005) Metallo-β-lactamases: the quiet before the storm? Clin. Microbiol. Rev. 18, 306-325
Cornaglia, G., Giamarellou, H., and Rossolini, G. M. (2011) Metallo-β-lactamases: a last frontier for β-lactams? Lancet Infect. Dis. 11, 381-393
Felici, A., Amicosante, G., Oratore, A., Strom, R., Ledent, P., Joris, B., Fanuel, L., and Frère, J. M. (1993) An overview of the kinetic parameters of class B β-lactamases. Biochem. J. 291, 151-155
Rasmussen, B. A., and Bush, K. (1997) Carbapenem-hydrolyzing β-lactamases. Antimicrob. Agents Chemother. 41, 223-232
Spencer, J., and Walsh, T. R. (2006) A new approach to the inhibition of metallo-β-lactamases. Angew. Chem. Int. Ed. Engl. 45, 1022-1026
Drawz, S. M., and Bonomo, R. A. (2010) Three decades of β-lactamase inhibitors. Clin. Microbiol. Rev. 23, 160-201
Payne, D. J. (1993) Metallo-β-lactamases: a new therapeutic challenge. J. Med. Microbiol. 39, 93-99
Livermore, D. M., and Woodford, N. (2000) Carbapenemases: a problem in waiting? Curr. Opin. Microbiol. 3, 489-495
Oelschlaeger, P., Ai, N., Duprez, K. T., Welsh, W. J., and Toney, J. H. (2010) Evolving carbapenemases: can medicinal chemists advance one step ahead of the coming storm? J. Med. Chem. 53, 3013-3027
Patel, G., and Bonomo, R. A. (2013) "Stormy waters ahead": global emergence of carbapenemases. Front. Microbiol. 4, 48
Bush, K. (2013) Proliferation and significance of clinically relevant β-lactamases. Ann. N.Y. Acad. Sci. 1277, 84-90
Fast, W., and Sutton, L. D. (2013) Metallo-β-lactamase: inhibitors and reporter substrates. Biochim. Biophys. Acta 1834, 1648-1659
Crowder, M. W., Spencer, J., and Vila, A. J. (2006) Metallo-β-lactamases: novel weaponry for antibiotic resistance in bacteria. Acc. Chem. Res. 39, 721-728
Karsisiotis, A. I., Damblon, C. F., and Roberts, G. C. (2013) Solution structures of the Bacillus cereus metallo-β-lactamase BcII and its complex with the broad spectrum inhibitor R-thiomandelic acid. Biochem. J. 456, 397-407
King, A. M., Reid-Yu, S. A., Wang, W., King, D. T., De Pascale, G., Strynadka, N. C., Walsh, T. R., Coombes, B. K., and Wright, G. D. (2014) Aspergillomarasmine A overcomes metallo-β-lactamase antibiotic resistance. Nature 510, 503-506
Galleni, M., Lamotte-Brasseur, J., Rossolini, G. M., Spencer, J., Dideberg, O., Frère, J. M., and Metallo-beta-lactamases Working Group. (2001) Standard numbering scheme for class B β-lactamases. Antimicrob. Agents Chemother. 45, 660-663
Frère, J. M., Galleni, M., Bush, K., and Dideberg, O. (2005) Is it necessary to change the classification of β-lactamases? J. Antimicrob. Chemother. 55, 1051-1053
Page, M. I., and Badarau, A. (2008) The mechanisms of catalysis by metallo-β-lactamases. Bioinorg. Chem. Appl. 2008, 576297
Hernandez Valladares, M., Felici, A., Weber, G., Adolph, H. W., Zeppezauer, M., Rossolini, G. M., Amicosante, G., Frère, J. M., and Galleni, M. (1997) Zn(II) dependence of the Aeromonas hydrophila AE036 metallo-β-lactamase activity and stability. Biochemistry 36, 11534-11541
Sabath, L. D., and Abraham, E. P. (1966) Zinc as a cofactor for cephalosporinase from Bacillus cereus 569. Biochem. J. 98, 11C-13C.
Meini, M. R., Llarrull, L. I., and Vila, A. J. (2014) Evolution of metallo-β-lactamases: trends revealed by natural diversity and in vitro evolution. Antibiotics 3, 285-316
Carfi, A., Pares, S., Duée, E., Galleni, M., Duez, C., Frère, J. M., and Dideberg, O. (1995) The 3-D structure of a zinc metallo-β-lactamase from Bacillus cereus reveals a new-type of protein fold. EMBO J. 14, 4914-4921
Carfi, A., Duée, E., Galleni, M., Frère, J. M., and Dideberg, O. (1998) 1.85 Å resolution structure of zinc β-lactamase from Bacillus cereus. Acta Crystallogr. D Biol. Crystallogr. 54, 313-323
Fabiane, S. M., Sohi, M. K., Wan, T., Payne, D. J., Bateson, J. H., Mitchell, T., and Sutton, B. J. (1998) Crystal structure of the zinc-dependent β-lactamase from Bacillus cereus at 1.9 Å resolution: binuclear active site with features of a mononuclear enzyme. Biochemistry 37, 12404-12411
Garau, G., García-Sáez, I., Bebrone, C., Anne, C., Mercuri, P., Galleni, M., Frère, J. M., and Dideberg, O. (2004) Update of the standard numbering scheme for class B β-lactamases. Antimicrob. Agents Chemother. 48, 2347-2349
Paul-Soto, R., Bauer, R., Frère, J. M., Galleni, M., Meyer-Klaucke, W., Nolting, H., Rossolini, G. M., de Seny, D., Hernandez-Valladares, M., Zeppezauer, M., and Adolph, H. W. (1999) Mono and binuclear Zn2+-β-lactamase. Role of the conserved cysteine in the catalytic mechanism. J. Biol. Chem. 274, 13242-13249
Wommer, S., Rival, S., Heinz, U., Galleni, M., Frere, J. M., Franceschini, N., Amicosante, G., Rasmussen, B., Bauer, R., and Adolph, H. W. (2002) Substrate-activated zinc binding of metallo-β-lactamases-physiological importance of the mononuclear enzymes. J. Biol. Chem. 277, 24142-24147
Rasia, R. M., and Vila, A. J. (2002) Exploring the role and the binding affinity of a second zinc equivalent in B. cereus metallo-β-lactamase. Biochemistry 41, 1853-1860
Jacquin, O., Balbeur, D., Damblon, C., Marchot, P., De Pauw, E., Roberts, G. C., Frère, J. M., and Matagne, A. (2009) Positively cooperative binding of zinc ions to Bacillus cereus 569/H/9 β-lactamase II suggests that the binuclear enzyme is the only relevant form for catalysis. J. Mol. Biol. 392, 1278-1291
Wang, Z., Fast, W., and Benkovic, S. J. (1999) On the mechanism of the metallo-β-lactamase from Bacteroides fragilis. Biochemistry 38, 10013-10023
Wang, Z., Fast, W., Valentine, A. M., and Benkovic, S. J. (1999) Metallo-β-lactamase: structure and mechanism. Curr. Opin. Chem. Biol. 3, 614-622
Frère, J. M. (ed) (2012) in β-Lactamases, pp. 41-77, Nova Science Publishers, Inc., New York
Hemmingsen, L., Damblon, C., Antony, J., Jensen, M., Adolph, H. W., Wommer, S., Roberts, G. C., and Bauer, R. (2001) Dynamics of mononuclear cadmium β-lactamase revealed by the combination of NMR and PAC spectroscopy. J. Am. Chem. Soc. 123, 10329-10335
Redfield, C. (1999) Molten globules. Curr. Biol. 9, R313
Kuwajima, K. (1989) The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure. Protein Struct. Funct. Gen. 6, 87-103
Ptitsyn, O. B., Pain, R. H., Semisotnov, G. V., Zerovnik, E., and Razgulyaev, O. I. (1990) Evidence for a molten globule state as a general intermediate in protein folding. FEBS Lett. 262, 20-24
Semisotnov, G. V., Rodionova, N. A., Razgulyaev, O. I., Uversky, V. N., Gripas', A. F., and Gilmanshin, R. I. (1991) Study of the "Molten Globule" intermediate state in protein folding by a hydrophobic fluorescent probe. Biopolymers 31, 119-128
Damblon, C., Prosperi, C., Lian, L. Y., Barsukov, I., Paul-Soto, R., Galleni, M., Frère, J. M., and Roberts, G. C. (1999) 1H-15N HMQC for the identification of metal-bound histidines in 113Cd substituted Bacillus cereus zinc β-lactamase. J. Am. Chem. Soc. 121, 11575-11576
Kim, Y., Cunningham, M. A., Mire, J., Tesar, C., Sacchettini, J., and Joachimiak, A. (2013) NDM-1, the ultimate promiscuous enzyme: substrate recognition and catalytic mechanism. FASEB J. 27, 1917-1927
Eftink, M., and Ghiron, C. A. (1981) Fluorescence quenching studies with proteins. Anal. Biochem. 114, 199-227
Karsisiotis, A. I., Damblon, C., and Roberts, G. C. (2014) Complete 1H, 15N, and 13C resonance assignments of Bacillus cereus metallo-β-lactamase and its complex with the inhibitor R-thiomandelic acid. Biomol. NMR Assign. 8, 313-318
Hubbard, S. J., and Thornton, J. M. (1993) NACCESS, Version 2.1.1, University College London
Rasia, R. M., and Vila, A. J. (2004) Structural determinants of substrate binding to Bacillus cereus metallo-β-lactamase. J. Biol. Chem. 279, 26046-26051
Jacquin, O. (2011) Etude des Propriétés de Repliement et de Fixation du Zinc de la Métallo-β-lactamase BcII de Bacillus cereus 569/H/9. Ph.D. thesis, University of Liège
Myers, J. K., Pace, C. N., and Scholtz, J. M. (1995) Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci. 4, 2138-2148
Pfeil, W. (1998) Protein Stability and Folding: A Collection of Thermodynamic Data, Springer-Verlag, Berlin
Whitford, D. (2005) Proteins: Structure and Function, John Wiley & Sons, Ltd., Chichester, UK
Creighton, T. E. (2010) The Biophysical Chemistry of Nucleic Acids and Proteins, Helvetian Press
Lehninger, A. L., Nelson, D. L., and Cox, M. M. (2013) Principles of Biochemistry, 6th Ed., W. H. Freeman & Co., New York
Palm-Espling, M. E., Niemiec, M. S., and Wittung-Stafshede, P. (2012) Role of metal in folding and stability of copper proteins in vitro. Biochim. Biophys. Acta 1823, 1594-1603
Wittung-Stafshede P. (2002) Role of cofactors in protein folding. Acc. Chem. Res. 35, 201-208
Selevsek, N., Rival, S., Tholey, A., Heinzle, E., Heinz, U., Hemmingsen, L., and Adolph, H. W. (2009) Zinc ion-induced domain organization in metallo-β-lactamases. J. Biol. Chem. 284, 16419-16431
Lassaux, P., Traoré, D. A., Loisel, E., Favier, A., Docquier, J. D., Sohier, J. S., Laurent, C., Bebrone, C., Frère, J. M., Ferrer, J. L., and Galleni, M. (2011) Biochemical and structural characterization of the subclass B1 metallo-β-lactamase VIM-4. Antimicrob. Agents Chemother. 55, 1248-1255
Dragani, B., Cocco, R., Ridderström, M., Stenberg, G., Mannervik, B., and Aceto, A. (1999) Unfolding and refolding of human glyoxalase II and its single-tryptophan mutants. J. Mol. Biol. 291, 481-490
Concha, N. O., Rasmussen, B. A., Bush, K., and Herzberg, O. (1996) Crystal structure of the wide-spectrum binuclear zinc β-lactamase from Bacteroides fragilis. Structure 4, 823-836
Concha, N. O., Janson, C. A., Rowling, P., Pearson, S., Cheever, C. A., Clarke, B. P., Lewis, C., Galleni, M., Frère, J. M., Payne, D. J., Bateson, J. H., and Abdel-Meguid, S. S. (2000) Crystal structure of the IMP-1 metallo β-lactamase from Pseudomonas aeruginosa and its complex with a mercaptocarboxylate inhibitor: binding determinants of a potent, broadspectrum inhibitor. Biochemistry 39, 4288-4298
Fitzgerald, P. M., Wu, J. K., and Toney, J. H. (1998) Unanticipated inhibition of the metallo-β-lactamase from Bacteroides fragilis by 4-morpholineethanesulfonic acid (MES): a crystallographic study at 1.85-A resolution. Biochemistry 37, 6791-6800
Toney, J. H., Fitzgerald, P. M., Grover-Sharma, N., Olson, S. H., May, W. J., Sundelof, J. G., Vanderwall, D. E., Cleary, K. A., Grant, S. K., Wu, J. K., Kozarich, J. W., Pompliano, D. L., and Hammond, G. G. (1998) Antibiotic sensitization using biphenyl tetrazoles as potent inhibitors of Bacteroides fragilis metallo-β-lactamase. Chem. Biol. 5, 185-196
Toney, J. H., Hammond, G. G., Fitzgerald, P. M., Sharma, N., Balkovec, J. M., Rouen, G. P., Olson, S. H., Hammond, M. L., Greenlee, M. L., and Gao, Y. D. (2001) Succinic acids as potent inhibitors of plasmid-borne IMP-1 metallo-β-lactamase. J. Biol. Chem. 276, 31913-31918
Scrofani, S. D., Chung, J., Huntley, J. J., Benkovic, S. J., Wright, P. E., and Dyson, H. J. (1999) NMR characterization of the metallo-β-lactamase from Bacteroides fragilis and its interaction with a tight-binding inhibitor: role of an active site loop. Biochemistry 38, 14507-14514
Yang, Y., Keeney, D., Tang, X., Canfield, N., and Rasmussen, B. A. (1999) Kinetic properties and metal content of the metallo-β-lactamase CcrA harboring selective amino acid substitutions. J. Biol. Chem. 274, 15706-15711
Huntley, J. J., Scrofani, S. D., Osborne, M. J., Wright, P. E., and Dyson, H. J. (2000) Dynamics of the metallo-β-lactamase from Bacteroides fragilis in the presence and absence of a tight-binding inhibitor. Biochemistry 39, 13356-13364
Huntley, J. J., Fast, W., Benkovic, S. J., Wright, P. E., and Dyson, H. J. (2003) Role of a solvent-exposed tryptophan in the recognition and binding of antibiotic substrates for a metallo-β-lactamase. Protein Sci. 12, 1368-1375
Mollard, C., Moali, C., Papamicael, C., Damblon, C., Vessilier, S., Amicosante, G., Schofield, C. J., Galleni, M., Frere, J. M., and Roberts, G. C. (2001) Thiomandelic acid, a broad spectrum inhibitor of zinc β-lactamases. J. Biol. Chem. 276, 45015-45023
Payne, D. J., Hueso-Rodríguez, J. A., Boyd, H., Concha, N. O., Janson, C. A., Gilpin, M., Bateson, J. H., Cheever, C., Niconovich, N. L., Pearson, S., Rittenhouse, S., Tew, D., Díez, E., Pérez, P., De La Fuente, J., et al. (2002) Identification of a series of tricyclic natural products as potent broad-spectrum inhibitors of metallo-β-lactamases. Antimicrob. Agents Chemother. 46, 1880-1886
Docquier, J. D., Lamotte-Brasseur, J., Galleni, M., Amicosante, G., Frère, J. M., and Rossolini, G. M. (2003) On functional and structural heterogeneity of VIM-type metallo-β-lactamases. J. Antimicrob. Chemother. 51, 257-266
Moali C., Anne, C., Lamotte-Brasseur, J., Groslambert, S., Devreese, B., Van Beeumen, J., Galleni, M., and Frère, J. M. (2003) Analysis of the importance of the metallo-β-lactamase active site loop in substrate binding and catalysis. (2003) Chem. Biol. 10, 319-329
Krauss, M., Gresh, N., and Antony, J. (2003) Binding and hydrolysis of ampicillin in the active site of a zinc lactamase. J. Phys. Chem. B 107, 1215-1229
Tomatis, P. E., Fabiane, S. M., Simona, F., Carloni, P., Sutton, B. J., and Vila, A. J. (2008) Adaptive protein evolution grants organismal fitness by improving catalysis and flexibility. Proc. Natl. Acad. Sci. U.S.A. 105, 20605-20610
Salsbury, F. R., Jr., Crowder, M. W., Kingsmore, S. F., and Huntley, J. J. (2009) Molecular dynamic simulations of the metallo-β-lactamase from Bacteroides fragilis in the presence and absence of a tight-binding inhibitor. J. Mol. Model. 15, 133-145
González, J. M., Buschiazzo, A., and Vila, A. J. (2010) Evidence of adaptability in metal coordination geometry and active site loop conformation among B1 metallo-β-lactamases. Biochemistry 49, 7930-7938
Valdez, C. E., Sparta, M., and Alexandrova, A. N. (2013) The role of the flexible L43-S54 protein loop in the CcrA metallo-β-lactamase in binding structurally dissimilar β-lactam antibiotics. J. Chem. Theory Comput. 9, 730-737
Rydzik, A. M., Brem, J., van Berkel, S. S., Pfeffer, I., Makena, A., Claridge, T. D., and Schofield, C. J. (2014) Monitoring conformational changes in the NDM-1 metallo-β-lactamase by 19F NMR spectroscopy. Angew. Chem. Int. Ed. Engl. 53, 3129-3133
Zhang, H., and Hao, Q. (2011) Crystal structure of NDM-1 reveals a common β-lactam hydrolysis ;Mechanism. FASEB J. 25, 2574-2582
King, D. T., Worrall, L. J., Gruninger, R., and Strynadka, N. C. (2012) New Delhi metallo-β-lactamase: structural insights into β-lactam recognition and inhibition. J. Am. Chem. Soc. 134, 11362-11365
Matagne, A., Misselyn-Bauduin, A. M., Joris, B., Erpicum, T., Granier, B., and Frère, J. M. (1990) The diversity of the catalytic properties of class A β-lactamases. Biochem. J. 265, 131-146
Nozaki, Y. (1972) The preparation of guanidine hydrochloride. Method Enzymol. 26, 43-50
Lakowicz, J. R. (1999) Principles of Fluorescence Spectroscopy, 2nd Ed., pp. 237-265, Kluwert Academic/Plenum Publishers, New York
Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR. 6, 277-293
Vranken, W. F., Boucher, W., Stevens, T. J., Fogh, R. H., Pajon, A., Llinas, M., Ulrich, E. L., Markley, J. L., Ionides, J., and Laue, E. D. (2005) The CCPN Data Model for NMR Spectroscopy: development of a software pipeline. Proteins 59, 687-696
Mulder, F. A., Schipper, D., Bott, R., and Boelens, R. (1999) Altered flexibility in the substrate-binding site of related native and engineered high-alkaline Bacillus subtilisins. J. Mol. Biol. 292, 111-123
Vandenameele, J., Lejeune, A., Di Paolo, A., Brans, A., Frère, J. M., Schmid, F. X., and Matagne, A. (2010) Folding of class A β-lactamase is rate-limited by peptide bond isomerization and occurs via parallel pathways. Biochemistry 49, 4264-4275
Santoro, M. M., and Bolen, D. W. (1988) Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl α-chymotrypsin using different denaturants. Biochemistry 27, 8063-8068
Pace, C. N. (1990) Measuring and increasing protein stability. Trends Biotechnol. 8, 93-98
Dumoulin, M., Conrath, K., Van Meirhaeghe, A., Meersman, F., Heremans, K., Frenken, L. G., Muyldermans, S., Wyns, L., and Matagne, A. (2002) Single-domain antibody fragments with high conformational stability. Protein Sci. 11, 500-515