The development of a general kinetic model for protein nucleation with structural considerations

The cause-effect relationship between conformational changes of proteins in solution and their aggregation does not, at present, have an obvious correlation. Past investigations have examined this relationship by focusing on the effect that aggregation has on a protein's conformation. This approach is sufficient if the observed conformational changes result from aggregation. However, aggregation itself may result from prior conformational changes of proteins in solution. This dissertation investigates the thesis that conformational changes of proteins in solution induce the formation of nuclei for aggregation. To better understand this link between protein conformational changes and aggregation, the well studied aggregation of rabbit muscle myosin was monitored over a comprehensive set of environmental conditions (pH = 6-8 ionic strength = 0.1-0.4 M KCl 0-300 rpm) using a newly developed turbidimetric technique.This non-invasive technique is capable of monitoring a protein's chromophoric composition, size, and conformation using only uv-vis spectroscopy. Previous efforts fashioned to use uv-vis spectroscopy in this way have suffered from an inability to adequately account for the scattering of light by proteins. To overcome this problem, Mie theory has been incorporated into a model to account for the absorption and scattering of energy by the protein molecules and their aggregates.This model forms the basis of a quantitative tool capable of accurately reconstructing the uv-vis spectra of proteins over the wavelength range of 240-900 nm. Model molecules selected to represent the absorption spectra of tyrosine and cysteine also provide information about the conformation of proteins in the region near these residues. This information on conformational changes can be inferred from the degree of ionization of the tyrosyl residues as provided by the analysis of the uv-vis spectra using the model. The changing degree of ionization suggests that the tyrosyl residues are in a new environment and that this environment has associated with it certain species that may be able to hydrogen bond. A study comparing the differences between the denaturation of two different collagen extracts demonstrates this tool's capabilities in the analysis of proteins.The results of the aggregation study suggest that myosin's conformation near the tyrosyl residues stabilizes before myosin reaches a final state of aggregation. This suggests that the conformational changes of myosin are completed prior to completion of the aggregation process.