Knowledge of the oligomeric state of a protein is essential for understanding its function. It has been estimated that more than 80% of proteins in Escherichia coli are composed of two or more subunits, and among these, ∼80% are homooligomers. The oligomerization state of some proteins can be modulated by environmental factors such as concentration, temperature, pH, ligand binding, or ionic strength. Methods such as size exclusion chromatography, analytical ultracentrifugation, and chemical cross-linking are often used to investigate the oligomeric states of proteins, but these approaches are not always adequate to identify transient intermediates. Here, I will discuss our progress in developing methodologies to characterize complex protein assembly pathways by fluorescence correlation spectroscopy (FCS). FCS relies on the measurement and analysis of the fluctuations in fluorescence intensity that arise when fluorescent molecules diffuse into and out of a femtoliter-sized optically defined volume. The temporal behavior of these fluctuations is analyzed in terms of an autocorrelation function, which decays with a characteristic time that can be analyzed in terms of the diffusion coefficients of the particles present in the sample. We have used FCS to characterize the intrinsic solution stability and oligomerization dynamics of the homodimeric Escherichia coli β and the homotrimeric Saccharomyces cerevisiae proliferating cell nuclear antigen (PCNA) sliding clamps. For E. coli β, the equilibrium dissociation constant of the dimeric protein varies with NaCl concentration as predicted by simple charge-screening models, indicating that charged amino acids contribute to the remarkable stability of the interface at physiological salt concentrations. Interestingly, experiments In KGlu suggest a previously unreported self-association mechanism in which several protein rings stack on each other with an affinity highly dependent on glutamate concentration. I will present these results with emphasis on the approaches that allow us to study how the oligomeric state of a protein is modulated by protein concentration and the Ionic composition of the buffer.

Protein oligomerization and self-assembly monitored by fluorescence correlation spectroscopy

2019 Spring National Meeting

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