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

Landscape descriptions provide a framework for identifying functionally significant dynamic linkages in proteins but cannot supply details. Rate measurements of combinatorial mutations can implicate catalysis. A major difficulty is filtering from the vastly more numerous static interactions that stabilize domain folding. The Geobacillus stearothermophilus (TrpRS) D1 switch such packing motif; it links movement to catalysis and specificity. We describe Thermofluor far UV circular dichroism melting curves all 16 variants determine their higher-order impact on unliganded TrpRS stability. prominent transition at intermediate temperatures thermal denaturation molten globule formation. Combinatorial analysis transcends protein landscape four respects: (i) bioinformatic methods identify coordinates multiple conformational states. (ii) Relative mutant temperatures, δTM, are proportional free energy changes. (iii) Structural implicates unexpected coupling between regions high local frustration. Those segments develop globular characteristics point greatest complementarity chemical state first structures melt. (iv) Residue F37 stabilizes both native states; its modify relative intrinsic impacts other residues those estimated single mutants. central component an escapement mechanism essential transduction. These conclusions begin relate differential stabilities.