Proteins can be viewed as small-world networks of amino acid residues connected through noncovalent interactions. Nuclear magnetic resonance chemical shift covariance analyses were used to identify long-range amino acid networks in the α subunit of tryptophan synthase both for the resting state (in the absence of substrate and product) and for the working state (during catalytic turnover). The amino acid networks observed stretch from the surface of the protein into the active site and are different between the resting and working states. Modification of surface residues on the network alters the structural dynamics of active-site residues over 25 Å away and leads to changes in catalytic rates. These findings demonstrate that amino acid networks, similar to those studied here, are likely important for coordinating structural changes necessary for enzyme function and regulation.

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Conformational changes in the b2a2 and b6a6 loops in the alpha subunit of tryptophan synthase (aTS) are important for enzyme catalysis and coordinating substrate channeling with the beta subunit (bTS). It was previously shown that disrupting the hydrogen bond interactions between these loops through the T183V substitution on the b6a6 loop decreases catalytic efficiency and impairs substrate channeling. Results presented here also indicate that the T183V substitution decreases catalytic efficiency in Escherchia coli aTS in the absence of the bTS subunit. Nuclear magnetic resonance (NMR) experiments indicate that the T183V substitution leads to local changes in the structural dynamics of the b2a2 and b6a6 loops. We have also used NMR chemical shift covariance analyses (CHESCA) to map amino acid networks in the presence and absence of the T183V substitution. Under conditions of active catalytic turnover, the T183V substitution disrupts long-range networks connecting the catalytic residue Glu49 to the aTS-bTS binding interface, which might be important in the coordination of catalytic activities in the tryptophan synthase complex. The approach that we have developed here will likely find general utility in understanding long-range impacts on protein structure and dynamics of amino acid substitutions generated through protein engineering and directed evolution approaches, and provide insight into disease and drug-resistance mutations.

Tryptophan synthase is a model system for understanding allosteric regulation within enzyme complexes. Amino acid interaction networks were previously delineated in the isolated alpha subunit (αTS) in the absence of the beta subunit (βTS). The amino acid interaction networks were different between the ligand-free enzyme and the enzyme actively catalyzing turnover. Previous X-ray crystallography studies indicated only minor localized changes when ligands bind αTS, and so, structural changes alone could not explain the changes to the amino acid interaction networks. We hypothesized that the network changes could instead be related to changes in conformational dynamics. As such, we conducted nuclear magnetic resonance relaxation studies on different substrate- and products-bound complexes of αTS. Specifically, we collected 15N R2 relaxation dispersion data that reports on microsecond-to-millisecond timescale motion of backbone amide groups. These experiments indicated that there are conformational exchange events throughout αTS. Substrate and product binding change specific motional pathways throughout the enzyme, and these pathways connect the previously identified network residues. These pathways reach the αTS/βTS binding interface, suggesting that the identified dynamic networks may also be important for communication with the βTS subunit.

The synaptic vesicle protein Synaptotagmin I is the calcium ion sensor for neurotransmitter release. Synaptotagmins are comprised of C2 domains, calcium ion dependent, membrane-binding domains. They contain multiple C2