When The Scaffold Cannot Be Ignored The Role Of The Hydrophobic Core In Ligand Binding And Specificity

When the Scaffold Cannot Be Ignored: The Role of the Hydrophobic Core in Ligand Binding and Specificity

Identification of Ligand Binding Site and Protein-Protein Interaction Area

This volume presents a review of the latest numerical techniques used to identify ligand binding and protein complexation sites. It should be noted that there are many other theoretical studies devoted to predicting the activity of specific proteins and that useful protein data can be found in numerous databases. The aim of advanced computational techniques is to identify the active sites in specific proteins and moreover to suggest a generalized mechanism by which such protein-ligand (or protein-protein) interactions can be effected. Developing such tools is not an easy task – it requires extensive expertise in the area of molecular biology as well as a firm grasp of numerical modeling methods. Thus, it is often viewed as a prime candidate for interdisciplinary research.

The Role of Conformational Energetics in Ligand Binding and Thermal Sensation

A major outstanding question in protein science is how different regions of a protein communicate with one another. Although we can identify action-at-a-distance phenomena when they occur, a generalized mechanism and an understanding sufficiently thorough as to allow for de novo design remain works in progress. This is particularly true for natively disordered proteins and channel proteins, both of which have traditionally been more technically difficult to characterize biophysically. In this work, I explore the functional relevance of their unique energetics and dynamics. The first project investigates the functional relevance of the hydrophobic core of disordered transcription factor MarA. We randomized the MarA hydrophobic core and selected for variants able to bind the consensus sequence. We find that MarA is highly intolerant of core mutation; this is in contrast to what is seen for the well-folded transcription factor [lambda]-repressor. Furthermore, core variants that do retain the ability to bind consensus sequence have differentially altered affinities for different binding partners. We propose that this can be explained by taking into account the varying energetic impact of these mutations on different MarA conformations and posit that residues distant from the active site can alter both binding affinity and specificity in natively disordered proteins. The second project aims to shed light on the mechanisms of thermosensation. Protein channel TRPA is a heat sensor in rattlesnakes but exhibits no heat-dependent activation in mammals. This discrepancy was mapped to select ankyrin repeats within the protein's cytoplasmic N-terminal domain. We hypothesized that temperature-dependent conformational changes within these repeats propagated to the pore region may be the thermosensing mechanism employed by TRPA1. The isolated repeats were purified and analyzed biophysically. Rattlesnake ankyrin repeats 3-8 are unique in that they do not appear to unfold with temperature on the timescale tested. The mechanism of this remarkable thermotolerance and its physiological relevance are currently under investigation.