Luorescence (ex = 295 nm; em = 340 nm) was followed using a FP-8300 Jasco fluorescence spectrophotometer. Enzyme Assays on Purified Enzymes. Initial velocity was measured spectrophotometrically at 486 nm using the chromogenic substrate nitrocefin (32 M) within the selection of 27 to 67 using a 5 interval. Enzyme Assays on Cell Extracts. TEM-1 and its variants have been grown overnight in 96-deep-wellplates,and cellswerelysedusingCellCultureLysisReagent(Promega). Lysates were diluted in potassium phosphate buffer pH 7.25 containing nitrocefin (50 g/mL) inside microtiter plates. Initial velocity was measured spectrophotometrically at 486 nm working with a Tecan infinite 96-well plate reader. Maximum Development Rate Determination. Growth curves were performed at 37 in 96-well microtiter plates containing 200 L MH broth supplemented with 6 or one hundred mg/L of amoxicillin, using a Tecan infinite 96-well plate reader. The Maximum Growth Price was determined because the maximum worth on the derivative from the logOD600, applying R application. ACKNOWLEDGMENTS. We thank Emmanuelle Cambau for discussions; Erick Denamur, Daniel Weinreich, and Scott Wylie for essential reading of your manuscript; and Christine Lazennec-Schurdevin, Michel Panvert, and Magali Fasseu for great technical help. This work was supported by Agence Nationale de la Recherche, Programme G omique Grant ANR-08-GENM-023-001; and European Analysis Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC Grant 310944.22. Sideraki V, Huang W, Palzkill T, Gilbert HF (2001) A secondary drug resistance mutation of TEM-1 beta-lactamase that suppresses misfolding and aggregation. Proc Natl Acad Sci USA 98(1):283?88. 23. Kather I, Jakob RP, Dobbek H, Schmid FX (2008) Enhanced folding stability of TEM-1 beta-lactamase by in vitro selection. J Mol Biol 383(1):238?51. 24. Brown NG, Pennington JM, Huang W, Ayvaz T, Palzkill T (2010) Many international suppressors of protein stability defects facilitate the evolution of extended-spectrum TEM -lactamases. J Mol Biol 404(five):832?46. 25. Bradford PA (2001) Extended-spectrum beta-lactamases inside the 21st century: Characterization, epidemiology, and detection of this significant resistance threat. Clin Microbiol Rev 14(four):933?51. 26. Weinreich DM, Delaney NF, Depristo MA, Hartl DL (2006) Darwinian evolution can stick to only very handful of mutational paths to fitter proteins. Science 312(5770):111?14. 27. Kawashima S, et al. (2008) AAindex: Amino acid index database, progress report 2008.106-86-5 supplier Nucleic Acids Res 36(Database situation):D202 205. 28. Henikoff S, Henikoff JG (1992) Amino acid substitution matrices from protein blocks. Proc Natl Acad Sci USA 89(22):10915?0919. 29. Altschul SF, et al. (1997) Gapped BLAST and PSI-BLAST: A brand new generation of protein database search applications.4-Iodobenzene-1,2-diol uses Nucleic Acids Res 25(17):3389?402.PMID:33461388 30. Schymkowitz J, et al. (2005) The FoldX web server: A web-based force field. Nucleic Acids Res 33(Net server situation):W382 388. 31. Dehouck Y, Kwasigroch JM, Gilis D, Rooman M (2011) PoPMuSiC 2.1: A net server for the estimation of protein stability modifications upon mutation and sequence optimality. BMC Bioinformatics 12:151. 32. Gros PA, Tenaillon O (2009) Choice for chaperone-like mediated genetic robustness at low mutation rate: Influence of drift, epistasis and complexity. Genetics 182(two): 555?64. 33. Khan S, Vihinen M (2010) Efficiency of protein stability predictors. Hum Mutat 31(6):675?84. 34. Tokuriki N, Stricher F, Serrano L, Tawfik DS (2008) How protein.