The major aim of this Core is to provide a facility for novel optical instrumentation to the investigators of this Program Project. The primary function of the Core will be to assist in experiments that require expertise and specialized equipment not available in the individual sub- project laboratories. The members of the Core will provide guidance and expert help to the different members of the Program Project in using Optical Trapping and Total Internal Reflection Polarized Fluorescence Microscopy (TIRPFM). This state of the art shared facility provides methods for measuring the mechanical properties of individual motor proteins and the spatial orientation of fluorescent probes linked to subdomains of individual engineered proteins. The core will further develop these techniques to better control the position of protein molecules in three dimensions and to improve the time resolution for measuring the orientation of single molecules as they proceed through their enzymatic cycles. In vitro analysis of single normal and engineered motor molecules will unambiguously measure fundamental cross-bridge properties such as stiffness, orientation changes, unitary force, step length and kinetics as a function of strain and relate these properties to structural features of this protein.
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