Quantitative Computational and Biophysical Investigation of Multivalent Proteins
The Graduate School, Stony Brook University: Stony Brook, NY.
The valency of a biological molecule is the number of interactions that it is able to make with other molecules. Multivalency arises in proteins which oligomerize or contain tandem repeats, and commonly involves the binding of carbohydrates. Biological processes which rely on multivalency include surface interactions (e.g. virus-cell adhesion and cell-cell binding) or de-mixing phenomena. The energetics of multivalent interactions can be significantly enhanced relative to their monovalent counterparts, and numerous multivalent inhibitors of viral infection have been identified. In this dissertation, we present computational and biophysical investigations of several multivalent proteins related to human viral pathogens. The bivalent lectin Cyanovirin-N inhibits HIV infection by binding the high-mannose glycans on the surface of the viral glycoprotein gp120. We performed Poisson-Boltzmann calculations and identified adjacent serines sequestered in the protein core which form a bridging interaction. We showed that this interaction does not overcome the desolvation penalty for burying the two groups, and went on to design and characterize a series of stabilized protein variants. The tetravalent lectin MVL also neutralizes HIV, but recognizes a glycan substructure different from Cyanovirin-N. An unresolved question regarding MVL and other HIV-neutralizing agents is whether multivalency is necessary for efficient viral neutralization. We biophysically characterized individual monovalent domains of MVL. The C-terminal domain was folded and populated a monomer/dimer equilibrium at micromolar concentrations. HIV neutralization experiments revealed that the C- terminal domain alone was able to neutralize three of four viral strains with efficacy near that of the wild type protein, suggesting that multivalency is not necessary for nanomolar inhibition by this protein. The adenoviral protein E4-ORF3 forms a heterogeneous polyvalent nuclear fiber and inactivates several host responses to the infection. Using biophysical techniques we characterized a nonfunc- tional variant of E4-ORF3, revealing that a homodimer is the building block of the nuclear web. Based on a subsequently-solved X-ray structure, we propose mechanisms of how the mutation abrogates function, and how E4-ORF3 is able to capture a diverse panel of host cellular proteins.