Long-Range Dynamic Effects of Point Mutations Propagate through Side Chains in the Serine Protease Inhibitor Eglin c

Abstract

Long-range interactions are fundamental to protein behaviors such as cooperativity and allostery. In an attempt to understand the role protein flexibility plays in such interactions, the distribution of local fluctuations in a globular protein was monitored in response to localized, nonelectrostatic perturbations. Two valine-to-alanine mutations were introduced into the small serine protease inhibitor eglin c, and the (15)N and (2)H NMR spin relaxation properties of these variants were analyzed in terms of the Lipari-Szabo dynamics formalism and compared to those of the wild type. Significant changes in picosecond to nanosecond dynamics were observed in side chains located as much as 13 A from the point of mutation. Additionally, those residues experiencing altered dynamics appear to form contiguous surfaces within the protein. In the case of V54A, the large-to-small mutation results in a rigidification of connected residues, even though this mutation decreases the global stability. These findings suggest that dynamic perturbations arising from single mutations may propagate away from the perturbed site through networks of interacting side chains. That this is observed in eglin c, a classically nonallosteric protein, suggests that such behavior will be observed in many, if not all, globular proteins. Differences in behavior between the two mutants suggest that dynamic responses will be context-dependent.

Monitoring Aromatic Picosecond to Nanosecond Dynamics in Proteins via 13C Relaxation: Expanding Perturbation Mapping of the Rigidifying Core Mutation, V54A, in Eglin c

“…The previously published response to the V54A mutation showed a contiguous 'network' of methyl-bearing residues that rigidified in response to mutation (15). With the addition of aromatic probes, the scope of the ps-ns response is expanded, which now more completely agrees with the thermodynamic observations of Gribenko et al (16).…”

“…The result was the bulk rigidification of select contiguous methyl-bearing residues radiating out from the site of mutation to the periphery of the protein's structured regions. Although such a generalized rigidification might be reflected in the globally measured heat capacity (15), this was found not to be the case (16). One possibility to describe this scenario is the limited spatial distribution of methyl-bearing residues.…”

“…It is desirable to characterize dynamic networks with high resolution, both in terms of spatial coverage through the use of a large number of probes and temporal coverage in terms of sensitivity to different timescales. Here, a previously determined dynamic network in eglin c is expanded through use of 13 C relaxation in aromatic side chains and amide hydrogen exchange.In previous work, the globular protein eglin c was used as a model for characterizing dynamic and structural responses to mutation (10,15). Pathways of dynamic changes (in response to mutation) were evident even in this simple protein, with the majority of responses observed in ps-ns side-chain fluctuations, as monitored by relaxation effects.…”

A Simple Model of Backbone Flexibility Improves Modeling of Side-chain Conformational Variability

“…More accurate modeling of side-chain conformational flexibility may also be important for structure-based drug design, when a target protein changes its binding site in response to binding different small molecules 11 . Work by Ranganathan and others [12][13][14][15][16][17][18] has provided intriguing evidence for the existence of "communication pathways" in proteins to facilitate the transmission of signals between allosteric and active sites. NMR experiments on several systems suggest that side-chains may play an important role in mediating this conformational coupling 19 .…”

Hierarchical organization of eglin c native state dynamics is shaped by competing direct and water-mediated interactions

“…Eglin c is a small 70-residue serine protease inhibitor found naturally in the leech Hirudo medicinalis whose dynamics have been studied extensively by NMR relaxation experiments (8,(21)(22)(23)(24)(25). Revealing the organization of the energy landscape of eglin c is the first goal to be accomplished in this work.…”