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Volume 10, Issue 8 (Suppl)

J Proteomics Bioinform, an open access journal

ISSN: 0974-276X

Structural Biology 2017

September 18-20, 2017

International Conference on

Structural Biology

September 18-20, 2017 Zurich, Switzerland

How does domain motion contribute to transition-state stabilization? Combinatorial thermodynamic

cycle analysis of conformational coupling during tryptophan activation

Charles W Carter

University of North Carolina at Chapel Hill, USA

nzyme mechanisms, especially those that couple NTP hydrolysis to mechanical work and information, use sophisticated

dynamic networks to transduce active-site chemistry into domain motions that change binding affinities. We measured

and cross-validated the energetics of such networks in B.

stearothermophilus

Tryptophanyl-tRNA synthetase (TrpRS) using

both multi-mutant and modular thermodynamic cycles. Coordinated domain motions develop shear in a core packing motif

conserved in >125 different protein superfamilies. Multi-dimensional combinatorial mutagenesis showed that four side chains

from this “molecular switch” move coordinately with the active-site Mg2+ ion in the transition state for amino acid activation.

A modular thermodynamic cycle consisting of full-length TrpRS, Urzyme, and Urzyme plus each of the two domains deleted

in the Urzyme gives similar energetics. These complementary experiments establish that catalysis and specificity in full-length

TrpRS are both coupled by 5 kcal/mole to: (i) the core packing region where domain movement generates shear, and (ii) the

simultaneous motion of the two domains relative to the Urzyme. Theory shows that the minimum action path algorithm

estimates thermodynamically meaningful contributions of domain movement to kinetic rates. Correlations between those

parameters, the experimental rates, and structural variations induced in the combinatorial mutants confirm that these

estimates are realistic. These results validate our previous conclusion that catalysis by Mg2+ ion is coupled to the overall

domain motion. Computational free energy surfaces demonstrate that TrpRS catalytic domain motion itself is endergonic

but is driven thermodynamically by PPi release. Comparison of the impact of combinatorial mutagenesis on pre-steady state

and steady-state rates confirm that dynamic active-site pre-organization endows TrpRS with the elusive conditionality of NTP

utilization on domain motion.

Biography

Charles W Carter is an X-ray Crystallographer who studies the origin, evolution, and structural biology of aminoacyl-tRNA synthetases. His research group

introduced the use of urzyme-highly conserved structural cores that retain large fractions of the transition-state stabilization free energies of full length enzymes as

experimental models of ancestral enzymes.

carter@med.unc.tedu

Charles W Carter, J Proteomics Bioinform 2017, 10:8(Suppl)

DOI: 10.4172/0974-276X-C1-0100

Figure1:

High Correlations between structures (yellow), steady-

state kinetics (blue) and computed trajectories (green) for WT

and 15 combinatorial variants of typtophanyl-tRNA synthetase.