On O, Chao L (2007) Understanding the evolutionary fate of finite populations: The dynamics of mutational effects. PLoS Biol 5(4):e94. 3. Sanju R, Moya A, Elena SF (2004) The distribution of fitness effects caused by singlenucleotide substitutions in an RNA virus. Proc Natl Acad Sci USA 101(22):8396?401. four. Domingo-Calap P, Cuevas JM, Sanju R (2009) The fitness effects of random mutations in single-stranded DNA and RNA bacteriophages. PLoS Genet 5(11):e1000742. five. Lind PA, Berg OG, Andersson DI (2010) Mutational robustness of ribosomal protein genes. Science 330(6005):825?27. six. P C, Papp B, Lercher MJ (2006) An integrated view of protein evolution. Nat Rev Genet 7(five):337?48. 7. Dean AM, Thornton JW (2007) Mechanistic approaches towards the study of evolution: The functional synthesis. Nat Rev Genet eight(9):675?88. eight. Li B, Leal SM (2008) Strategies for detecting associations with uncommon variants for popular illnesses: Application to evaluation of sequence data. Am J Hum Genet 83(three):311?21. 9. Cirulli ET, Goldstein DB (2010) Uncovering the roles of uncommon variants in popular disease via whole-genome sequencing. Nat Rev Genet 11(6):415?25. 10. Grantham R (1974) Amino acid difference formula to help clarify protein evolution. Science 185(4154):862?64. 11. Deng Z, et al. (2012) Deep sequencing of systematic combinatorial libraries reveals -lactamase sequence constraints at higher resolution. J Mol Biol 424(3-4):150?67. 12. Adzhubei IA, et al. (2010) A approach and server for predicting damaging missense mutations. Nat Strategies 7(four):248?49. 13. Kumar P, Henikoff S, Ng Computer (2009) Predicting the effects of Sorcin/SRI, Human (sf9, His-GST) coding non-synonymous variants on protein function using the SIFT algorithm. Nat Protoc four(7):1073?081. 14. Bloom JD, et al. (2005) Thermodynamic prediction of protein neutrality. Proc Natl Acad Sci USA 102(three):606?11. 15. DePristo MA, Weinreich DM, Hartl DL (2005) Missense meanderings in sequence space: A biophysical view of protein evolution. Nat Rev Genet 6(9):678?87. 16. Wylie CS, Shakhnovich EI (2011) A biophysical protein folding model accounts for most mutational fitness effects in viruses. Proc Natl Acad Sci USA 108(24):9916?921. 17. Soskine M, Tawfik DS (2010) Mutational effects and also the evolution of new protein functions. Nat Rev Genet 11(8):572?82. 18. Ramsey DC, Scherrer MP, Zhou T, Wilke CO (2011) The partnership involving relative solvent accessibility and evolutionary rate in protein evolution. Genetics 188(2): 479?88. 19. Keseler IM, et al. (2009) EcoCyc: A complete view of Escherichia coli biology. MEM Non-essential Amino Acid Solution (100��) Storage Nucleic Acids Res 37(Database problem):D464 470. 20. Salverda ML, De Visser JA, Barlow M (2010) Organic evolution of TEM-1 -lactamase: Experimental reconstruction and clinical relevance. FEMS Microbiol Rev 34(6): 1015?036. 21. Wang X, Minasov G, Shoichet BK (2002) Evolution of an antibiotic resistance enzyme constrained by stability and activity trade-offs. J Mol Biol 320(1):85?five.by IPTG (1 mM), and purified on anion-exchange column (Q Sepharose FF, GE Healthcare) followed by gel filtration (Superdex 75 column, GE Healthcare). Thermal Denaturation of Proteins. TEM-1 and its variants have been subjected to thermal denaturation (25?0 with 1.5 /min ramping rates). Intrinsic fluorescence (ex = 295 nm; em = 340 nm) was followed applying a FP-8300 Jasco fluorescence spectrophotometer. Enzyme Assays on Purified Enzymes. Initial velocity was measured spectrophotometrically at 486 nm employing the chromogenic substrate nitrocefin (32 M) inside the array of 27 to 67 w.
