Thermodynamic impacts of combinatorial mutagenesis on protein conformational stability: precise, high-throughput measurement by Thermofluor

The D1 switch is a packing motif, broadly distributed in the proteome, that couples tryptophanyl-tRNA synthetase (TrpRS) domain movement to catalysis and specificity, thereby creating an escapement mechanism essential to free-energy transduction. The escapement mechanism arose from analysis of an extensive set of combinatorial mutations to this motif, which allowed us to relate mutant-induced changes quantitatively to both kinetic and computational parameters during catalysis. To further characterize the origins of this escapement mechanism in differential TrpRS conformational stabilities, we use high-throughput Thermofluor measurements for the 16 variants to extend analysis of the mutated residues to their impact on unliganded TrpRS stability. Aggregation of denatured proteins complicates thermodynamic interpretations of denaturation experiments. The free energy landscape of a liganded TrpRS complex, carried out for different purposes, closely matches the volume, helix content, and transition temperatures of Thermoflour and CD melting profiles. Regression analysis using the combinatorial design matrix accounts for u003e90% of the variance in Tms of both Thermofluor and CD melting profiles. We argue that the agreement of experimental melting temperatures with both computational free energy landscape and with Regression modeling means that experimental melting profiles can be used to analyze the thermodynamic impact of combinatorial mutations. Tertiary packing and aromatic stacking of Phenylalanine 37 exerts a dominant stabilizing effect on both native and molten globular states. The TrpRS Urzyme structure remains essentially intact at the highest temperatures explored by the simulations.