Helicase processivity and not the unwinding velocity exhibits universal increase with force

Helicases, involved in a number of cellular functions, are motors that translocate along singlestranded nucleic acid and couple the motion to unwinding double-strands of a duplex nucleic acid. The junction between double and single strands creates a barrier to the movement of the helicase, which can be manipulated in vitro by applying mechanical forces directly on the nucleic acid strands. Single molecule experiments have demonstrated that the unwinding velocities of some helicases increase dramatically with increase in the external force, while others show little response. In contrast, the unwinding processivity always increases when the force increases. The differing responses of the unwinding velocity and processivity to force has lacked explanation. By generalizing a previous model of processive unwinding by helicases, we provide a unified framework for understanding the dependence of velocity and processivity on force and the nucleic acid sequence. We predict that the sensitivity of unwinding processivity to external force is a universal feature that should be observed in all helicases. Our prediction is illustrated using T7 and NS3 helicases as case studies. Interestingly, the increase in unwinding processivity with force depends on whether the helicase forces base pair opening by direct interaction or if such a disruption occurs spontaneously due to thermal uctuations. Based on the theoretical results, we propose that proteins like single-strand binding proteins associated with helicases in the replisome, may have co-evolved with helicases to increase the unwinding processivity even if the velocity remains unaffected

FORCE RESPONSE OF CELL- ADHESION COMPLEXES AND HELICASES

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

Protein collapse is encoded in the folded state architecture

Folded states of single domain globular proteins are compact with high packing density. The radius of gyration, Rg, of both the folded and unfolded states increase as Nν where N is the number of amino acids in the protein. The values of the Flory exponent ν are, respectively, ≈⅓ and ≈0.6 in the folded and unfolded states, coinciding with those for homopolymers. However, the extent of compaction of the unfolded state of a protein under low denaturant concentration (collapsibility), conditions favoring the formation of the folded state, is unknown. We develop a theory that uses the contact map of proteins as input to quantitatively assess collapsibility of proteins. Although collapsibility is universal, the propensity to be compact depends on the protein architecture. Application of the theory to over two thousand proteins shows that collapsibility depends not only on N but also on the contact map reflecting the native structure. A major prediction of the theory is that β-sheet proteins are far more collapsible than structures dominated by α-helices. The theory and the accompanying simulations, validating the theoretical predictions, provide insights into the differing conclusions reached using different experimental probes assessing the extent of compaction of proteins. By calculating the criterion for collapsibility as a function of protein length we provide quantitative insights into the reasons why single domain proteins are small and the physical reasons for the origin of multi-domain proteins. Collapsibility of non-coding RNA molecules is similar β-sheet proteins structures adding support to “Compactness Selection Hypothesis”

Protein Collapse is Encoded in the Folded State Architecture

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