High-throughput thermal denaturation of tryptophanyl-tRNA synthetase combinatorial mutants reveals high-order energetic coupling determinants of conformational stability.

ABSTRACT: Landscape descriptions provide a framework for identifying functionally significant dynamic linkages in proteins but cannot supply details. Rate measurements of combinatorial mutations can implicate dynamic linkages in catalysis. A major difficulty is filtering dynamic linkages from the vastly more numerous static interactions that stabilize domain folding. The Geobacillus stearothermophilus (TrpRS) D1 switch is such a dynamic packing motif; it links domain movement to catalysis and specificity. We describe Thermofluor and far UV circular dichroism melting curves for all 16 D1 switch variants to determine their higher-order impact on unliganded TrpRS stability. A prominent transition at intermediate temperatures in TrpRS thermal denaturation is molten globule formation. Combinatoria

SUBMITTER: Weinreb V 

PROVIDER: S-EPMC10449480 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature