Overview and aims: The contraction of muscle is caused by a pair of "motor proteins", actin and myosin, that convert chemical energy into mechanical work. The cyclic binding and unbinding of these proteins to one another is central to their functions in contraction. During this cycle, the actin-binding region of myosin is believed to undergo coordinated shape changes. However, the extent and functional significance of these shape changes are unknown. The purpose of the project is to test the hypothesis that the bond between actin and myosin is mechanically tuned to function under physiological conditions in living muscle cells. Dr. Guilford will measure how long it takes single actin-myosin bonds to break when subjected to external loads applied using a laser trap (analogous to the "tractor beam" of Star Trek fame). This simple yet powerful single-molecule technique will enable studies of actin and myosin that are not accessible to any other existing method. The first aim will determine the properties of the actomyosin bond in myosin from skeletal muscle. Dr. Guilford has already demonstrated that the lifetime of the bond between actin and myosin first increases and then decreases with applied load. This is called a "catch bond". The load at which bond lifetime is highest coincides with the maximum force that can be generated by the myosin motor, which is suggestive of the mechanical tuning mentioned earlier. The data also confirm that a shape change occurs in the actin-binding region of myosin when the waste products of the chemical energy consumption are expelled. These data have broad-ranging and exciting implications for muscle function. Building upon these exciting preliminary data, Dr. Guilford will next compare the mechanics of bonds between actin and myosin derived from different muscle types. Conventional experimental approaches suggest that there are major shape differences between the myosins from different muscles, specifically in their actin-binding regions. These differences may reflect their distinct physiological applications. The functional impact of these differences is uncertain, and will be probed in this work. The effects of the protein tropomyosin which interferes with myosin binding to actin and functions to turn contraction on and off, will also be considered. The final aim will place these data within the functional contexts of intact muscles. It is known that these molecules are designed to work in a single direction. Both the direction and magnitude of applied load will be varied to more fully delineate the function of actin and myosin in active contractions of living muscle. Intellectual merit: Most work on the molecular mechanics of myosin has focused on regions of the molecule that generate the actual "power stroke" responsible for motion. However, shape changes in the actin-binding region are equally important to myosin function as they determine the strength of the bond that transmits force from one protein to the other. However, few molecular mechanics studies have focused on this region, seldom in well-defined geometries, and never between different myosins or functional states. The project will thus fill a critical void in our understanding of motor protein function. While the work is focused on understanding muscle function, these data will serendipitously provide unique insights for the general field of "bond mechanics" the study of how chemical bonds respond to mechanical loads. Catch bonds are of particular interest. The functional environment of actomyosin is much better defined than for cellular systems in which other catch bonds have been identified. Dr. Guilford's research to date has led to the hypothesis that all catch bonds are tuned to their physiologic function. This work will enable further refinement and test of this exciting hypothesis. Further, the experimental design will enable examination of how and why myosin is directionally sensitive - a hitherto unexplored problem. Dr. Guilford has extensive experience in the molecular mechanics of myosin and actin, and is a recognized expert in laser traps. Indeed, the laser trap transducer in Dr. Guilford''s lab will enable all these experiments to be conducted on a single experimental platform, which is vital to interpretation of the data. Broader impact: All of the studies to date in Dr. Guilford''s lab have involved graduate students, undergraduates and high school students. Dr. Guilford has a history of undergraduate research publication and participation in national scientific meetings. The present project will be no exception, involving students from all three educational levels. Dr. Guilford directs a High School Science Training Program (HighSTeP) for disadvantaged regional high school students. Thus one or more underrepresented or underprivileged high school students will be directly involved in the project. Dr. Guilford also has a history of publication in the educational literature, and holds leadership positions in educational programs and committees that will ensure broad and timely dissemination of any educational techniques and materials that result from this work.

Project Report

The aim of this grant was to better understand the chemical bonds that hold together proteins involved in muscle contraction and cell movement, important to the beating of the heart and the crawling of cancer cells. Because they convert chemical energy in to mechanical work these proteins are also called "motor proteins." The most common are the motor proteins actin and myosin, which undergo cyclic binding interactions during which they pull against one another. During this cycle, the actin-binding surface of myosin is believed to undergo significant, coordinated changes in shape. However, the extent and impact of these shape changes were previously unknown. The purpose of the proposed study was to probe single pairs of actin and myosin (actomyosin) molecules using mechanical force to determine whether the bond between them is tuned to support muscle contraction under physiological conditions. Our powerful single-molecule techniques enabled us to identify the energy barriers to bond breakage and bond formation that were not accessible to any other method. We used a laser trap, analagous to a "tractor beam" of Star Wars and Star Trek fame, to capture and hold microscopic spheres bearing actin molecules (see figure). The laser trap consists of a highly focused laser beam. At the focal point one may capture ("trap") and hold small translucent particles. It can also be used to measure or generate forces on the scale of those experienced by single protein molecules in living cells. Our first aim was to determine the properties of the actomyosin bond in skeletal muscle myosin - the motor that drives the contraction of voluntary muscle. Our data showed that actomyosin is a "catch-slip bond" - a feature analagous to a "finger trap" in that it appears to become stronger the harder one pulls against it. We went on to study the role of the protein tropomyosin in actomyosin bond mechanics. Tropomyosin has long been known to be part of the on-off switching system for contraction of skeletal and heart muscle, but data hinted at other possible roles for this ubiquitous protein. Our data showed tropomyosin to have a more nuanced role in regulating muscle contraction by at times hindering and other times aiding bond formation between actin and myosin. Finally we sought to understand how these measurements relate to function in a living cell, which is an enviroment much more rich in potassium salt. Our most recent work shows that high salt environments cause actomyosin bonds to last longer while simultaneously slowing their formation. All of the above studies involved graduate students, undergraduates (see photo), and even high school students. Indeed, advanced training of some of the university's most talented undergraduates has been one of the most notable impacts of this study. On the 8 journal publications resulting from this award, no less than 10 undergraduates, 2 graduate students, and 1 high school student number among the co-authors. These students not only pushed forward the stated goals of this grant, but allowed us to expand into ancillary studies and related fields in molecular motors. Of particular note, an undergraduate student and a high school student are the first two authors on a publication in Proceedings of the National Academy of Science - an incredibly rare event for such young scientists. This is a testement to the tremendous opportunities offered by NSF to the development of the next generation of scientists, but also to the potential of these very same students to themselves advance scientific knowledge even while still in school. These achievements, combined with the development of a novel approach to teaching graduate students, emphasize the incredibly broad impact of this study in science, engineering, and education.

National Science Foundation (NSF)
Division of Molecular and Cellular Biosciences (MCB)
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Richard Rodewald
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University of Virginia
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