Ith a 5 interval. Enzyme Assays on Cell Extracts. TEM-1 and its variants were grown overnight in 96-deep-wellplates,and cellswerelysedusingCellCultureLysisReagent(Promega). Lysates have been diluted in potassium phosphate buffer pH 7.25 containing nitrocefin (50 g/mL) inside microtiter plates. Initial velocity was measured spectrophotometrically at 486 nm making use of a Tecan infinite 96-well plate reader. Maximum Development Rate Determination. Development curves have been performed at 37 in 96-well microtiter plates containing 200 L MH broth supplemented with 6 or one hundred mg/L of amoxicillin, utilizing a Tecan infinite 96-well plate reader. The Maximum Development Price was determined because the maximum worth of your derivative on the logOD600, making use of R computer software. ACKNOWLEDGMENTS. We thank Emmanuelle Cambau for discussions; Erick Denamur, Daniel Weinreich, and Scott Wylie for crucial reading with the manuscript; and Christine Lazennec-Schurdevin, Michel Panvert, and Magali Fasseu for outstanding technical help. This operate was supported by Agence Nationale de la Recherche, Programme G omique Grant ANR-08-GENM-023-001; and European Investigation Council beneath 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) Increased folding stability of TEM-1 beta-lactamase by in vitro choice. J Mol Biol 383(1):238?51. 24. Brown NG, Pennington JM, Huang W, Ayvaz T, Palzkill T (2010) Several global suppressors of Semaphorin-4D/SEMA4D, Human (713a.a, HEK293, His) 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 important resistance threat. Clin Microbiol Rev 14(four):933?51. 26. Weinreich DM, Delaney NF, Depristo MA, Hartl DL (2006) Darwinian evolution can comply with only very few mutational paths to fitter proteins. Science 312(5770):111?14. 27. Kawashima S, et al. (2008) AAindex: Amino acid index database, progress report 2008. Nucleic Acids Res 36(Database challenge):D202 205. 28. Prostatic acid phosphatase/ACPP Protein site 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. Nucleic Acids Res 25(17):3389?402. 30. Schymkowitz J, et al. (2005) The FoldX internet server: A web based force field. Nucleic Acids Res 33(Internet server concern):W382 388. 31. Dehouck Y, Kwasigroch JM, Gilis D, Rooman M (2011) PoPMuSiC two.1: A internet server for the estimation of protein stability alterations 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 price: Impact of drift, epistasis and complexity. Genetics 182(two): 555?64. 33. Khan S, Vihinen M (2010) Functionality of protein stability predictors. Hum Mutat 31(six):675?84. 34. Tokuriki N, Stricher F, Serrano L, Tawfik DS (2008) How protein stability and new functions trade off. PLOS Comput Biol 4(2):e1000002. 35. Tokuriki N, Tawfik DS (2009) Chaperonin overexpression promotes genetic variation and enzyme evolution. Nature 459(7247):668?73. 36. Liberles DA, et al. (2012) The interface of protein structure, protein biop.
