Mesophilic and hyperthermophilic adenylate kinases differ in their tolerance to random fragmentation

Thomas H. Segall-Shapiro, Peter Q. Nguyen, Edgardo D. Dos Santos, Saurav Subedi, Justin Judd, Junghae Suh, Jonathan J. Silberg

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)135-148
Number of pages14
JournalJournal of Molecular Biology
Volume406
Issue number1
DOIs
StatePublished - Feb 11 2011

Keywords

  • dynamics
  • gene fission
  • protein design
  • protein fragment complementation
  • protein thermostability

ASJC Scopus subject areas

  • Biophysics
  • Structural Biology
  • Molecular Biology

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