Role of Helix Formation in Mediating Protein-Protein Interactions

Rensselaer Polytechnic Institute, Troy NY

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Abstract

Th Protein-target interaction is one of the most important processes occurring in biological systems. One subgroup of such interactions takes place with a dramatic change in the secondary structure: from unstructured (coil) when it is unbound to a very characteristic structure (helix) when it is in the bound state. A detailed statistical thermodynamics study of the factors that affect this transition (from coil to helix), and how these factors affect the interaction between the protein and its target is very important as deregulation can result in a serious malfunction of the biological machinery and ultimately can be lethal. We will use the interactions between ubiquitin and ubiquitin interacting motif (UIM) as a model system to study in structural, dynamics and thermodynamics details the ubiquitin-UIM complex formation. Ubiquitin is a very small protein, yet it performs a wide variety of regulatory functions in the cell including protein degradation, trafficking, cell-cycle control, DNA repair, transcription regulation and gene silencing, stress response and signaling. All these functions are in the form of post-translational modifications of proteins via mono- or poly- ubiquitinylation. The extent and importance of ubiquitinylation as a regulatory cellular mechanism is widespread, and by some estimates, is surpassed only by protein phosphorylation. Among numerous target sequences that bind ubiquitin, the UIM is perhaps the simplest. It is a short 20-amino acid sequence that undergoes a coil-helix transition upon binding to ubiquitin. Rules that affect specificity and affinity in this type of interaction will be crucial in computational biology and bioinformatics to better predict target sequences that can interact with ubiquitin. To achieve these goals we will use both experimental and computational approaches to study ubiquitin-UIM complex formation. Experimental methods will include site-directed mutagenesis, calorimetry, fluorescence and circular dichroism spectroscopies, structural and relaxation NMR analysis. The computational approach will involve molecular dynamics simulations to model the energetics of interactions. This will allow us to establish general rules that can be used to modulate protein-helix interactions by affecting some of the properties that govern the helix-coil transition in target sequences. Such rules will lay the foundation for rational design of therapeutically effective target sequences for ubiquitin.