TY - JOUR
T1 - Mesophilic and hyperthermophilic adenylate kinases differ in their tolerance to random fragmentation
AU - Segall-Shapiro, Thomas H.
AU - Nguyen, Peter Q.
AU - Dos Santos, Edgardo D.
AU - Subedi, Saurav
AU - Judd, Justin
AU - Suh, Junghae
AU - Silberg, Jonathan J.
N1 - Funding Information:
This work was supported by the National Aeronautics and Space Administration (grant NNX08AO20G to J.J.S.), the Robert A. Welch Foundation (grant C-1614 to J.J.S.), the National Institutes of Health Biotechnology Training Grant (grant 2T32-GM008362 to J.J. and P.Q.N.), the National Science Foundation (grant 1004853 to J.J.S.), and the Hamill Foundation (to J.J.S. and J.S.).
PY - 2011/2/11
Y1 - 2011/2/11
N2 - The extent to which thermostability influences the location of protein fragmentation sites that allow retention of function is not known. To evaluate this, we used a novel transposase-based approach to create libraries of vectors that express structurally-related fragments of Bacillus subtilis adenylate kinase (BsAK) and Thermotoga neapolitana adenylate kinase (TnAK) with identical modifications at their termini, and we selected for variants in each library that complement the growth of Escherichia coli with a temperature-sensitive adenylate kinase (AK). Mutants created using the hyperthermophilic TnAK were found to support growth with a higher frequency (44%) than those generated from the mesophilic BsAK (6%), and selected TnAK mutants complemented E. coli growth more strongly than homologous BsAK variants. Sequencing of functional clones from each library also identified a greater dispersion of fragmentation sites within TnAK. Nondisruptive fission sites were observed within the AMP binding and core domains of both AK homologs. However, only TnAK contained sites within the lid domain, which undergoes dynamic fluctuations that are critical for catalysis. These findings implicate the flexible lid domain as having an increased sensitivity to fission events at physiological temperatures. In addition, they provide evidence that comparisons of nondisruptive fission sites in homologous proteins could be useful for finding dynamic regions whose conformational fluctuations are important for function, and they show that the discovery of protein fragments that cooperatively function in mesophiles can be aided by the use of thermophilic enzymes as starting points for protein design.
AB - The extent to which thermostability influences the location of protein fragmentation sites that allow retention of function is not known. To evaluate this, we used a novel transposase-based approach to create libraries of vectors that express structurally-related fragments of Bacillus subtilis adenylate kinase (BsAK) and Thermotoga neapolitana adenylate kinase (TnAK) with identical modifications at their termini, and we selected for variants in each library that complement the growth of Escherichia coli with a temperature-sensitive adenylate kinase (AK). Mutants created using the hyperthermophilic TnAK were found to support growth with a higher frequency (44%) than those generated from the mesophilic BsAK (6%), and selected TnAK mutants complemented E. coli growth more strongly than homologous BsAK variants. Sequencing of functional clones from each library also identified a greater dispersion of fragmentation sites within TnAK. Nondisruptive fission sites were observed within the AMP binding and core domains of both AK homologs. However, only TnAK contained sites within the lid domain, which undergoes dynamic fluctuations that are critical for catalysis. These findings implicate the flexible lid domain as having an increased sensitivity to fission events at physiological temperatures. In addition, they provide evidence that comparisons of nondisruptive fission sites in homologous proteins could be useful for finding dynamic regions whose conformational fluctuations are important for function, and they show that the discovery of protein fragments that cooperatively function in mesophiles can be aided by the use of thermophilic enzymes as starting points for protein design.
KW - dynamics
KW - gene fission
KW - protein design
KW - protein fragment complementation
KW - protein thermostability
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U2 - 10.1016/j.jmb.2010.11.057
DO - 10.1016/j.jmb.2010.11.057
M3 - Article
C2 - 21145325
AN - SCOPUS:79151475523
SN - 0022-2836
VL - 406
SP - 135
EP - 148
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 1
ER -