Theoretical and Experimental Investigation of Switch-Like Responses Arising from Multisite Protein Phosphorylation.

Abstract

Switch-like responses are important regulatory features of biological processes involving binary decisions such as cell division. Multisite protein phosphorylation is a proposed mechanism for achieving switch-like behaviors. For example, it is conjectured that the G1/S transition of the yeast cell cycle occurs in a switch-like fashion due to multisite phosphorylation-triggered degradation of the Sic1 protein. The objective of this dissertation is first to acquire a quantitative and predictive understanding of switch-like behaviors arising from multisite phosphorylation in natural systems, and second to employ this knowledge for the design of synthetic protein devices. We first developed a mathematical model to investigate systematically the role of multisite phosphorylation in the phosphorylation-triggered degradation process of a protein like Sic1. We found that as the number of sites increases, a more switch-like temporal profile can be generated. The steepness is determined synergistically by various factors, including the total number of sites and kinetic parameters. To test our theoretical predictions, we examined the steady-state response of wild-type and mutant Sic1 with various numbers of phosphorylation sites. It was observed that the response of Sic1, measured by its binding to a downstream protein Cdc4 in the degradation pathway, to the Cln2-Cdc28 kinase in vitro is switch-like. Furthermore, the ultrasensitivity decreases as the number of sites decreases. We next showed, through computational analysis, that a multisite protein can exhibit sustainable and tunable oscillations when embedded in a negative feedback loop, formed via inhibition of the rst phosphorylation step. We also designed a protein degradation device based on multiple protein binding domains and carried out preliminary study of its implementation. Our work demonstrates the potential of utilizing multisite proteins or the broader design principle of intramolecular multiple interaction modules, which provides an effective and flexible means for generating high nonlinearity, in creating a wide range of synthetic biological devices. This dissertation suggests quantitative design principles for switch-like stimulusresponse relationships arising from multisite protein phosphorylation, which might be a widespread mechanism in cellular regulation. In addition, our results provide intriguing hypotheses to be investigated experimentally in future work, such as the critical effect of multi-step phosphorylation kinetics.