Force response of cell-adhesion complexes and helicases

Title of dissertation: FORCE RESPONSE OF CELL-ADHESION COMPLEXES AND HELICASES Shaon Chakrabarti Doctor of Philosophy, 2015 Dissertation directed by: Professors Devarajan Thirumalai and Christopher Jarzynski Institute for Physical Science and Technology Department of Chemistry and Biochemistry Rapidly increasing technological prowess has led to the development of increasingly precise experiments to track biological systems at the single molecule level. The ability to apply measured amounts of external forces in such experiments, has added an extra probe to a scientist’s arsenal of tools, allowing detailed investigations into the response of molecules that were not possible even a few years ago. However, the emerging raw single-molecule data tends to be of limited use in the absence of careful theories that can analyze and make sense of such data. This thesis focuses on understanding single-molecule force spectroscopy data on two important biological systems–cell adhesion complexes called selectins and integrins, and nucleic-acid unwinding motors known as helicases. Selectins and integrins are receptors expressed in blood vessels, that bind to specific ligands on leukocytes, initiating a process of absorption of leukocytes from the blood flow. The microscopic details of the selectin-ligand interactions that allow this process to occur, is hotly debated and a topic of intense current research. Over the last few years, it has been established that certain selectin-ligand lifetimes show a surprising ‘catch-bond’ behavior, where the lifetime under force first increases before decreasing as expected. In this thesis, we build a structural model to explain this phenomenon and quantitatively explain a number of experimental results. Our work suggests that a loop region on the selectin receptor domain undergoes an allosteric conformational change, allowing the receptor to bind more tightly to the ligand. Force enhances this allosteric conformational change, thus resulting in an initial increase in lifetime of the complex. We provide quantitative support for this model, and also precise predictions of the outcomes of multiple mutation experiments. Helicases are molecular motors that hydrolyze nucleoside triphosphate (NTP) to carry out various kinds of cellular activities related to nucleic-acid metabolism. The particular aspect of certain helicases that we focus on in this thesis, is the NTP driven unwinding of double strand nucleic acids. Based on whether or not the helicase destabilizes the duplex base pairs while unwinding, helicases are classified as ‘active’ or ‘passive’, with different physical properties associated with each type. We develop a mathematical technique to analyze the velocities and processivities of such helicases, and predict a surprising universal behavior of the processivity under external forces. Our analysis suggests that partner proteins (invariably required for efficient unwinding of nucleic acids in vivo) have coevolved with helicases to increase the processivity, as opposed to the velocity, of all types of helicases. Finally, we establish the unwinding mechanism of the T-7 helicase, thereby providing insight into the unwinding mechanisms of a whole family (SF-4) of helicases. FORCE RESPONSE OF CELL-ADHESION COMPLEXES AND HELICASES