Protein dynamics with multiple-structural transitions: Investigation with single-molecule FRET
EMSL Project ID
34731
Abstract
Dynamics of protein conformational changes and protein-protein interactions play critical roles in biological systems. In response to a specific signal stimulus, the functions of multi-components biological systems are typically governed by the multiple protein structural transitions or protein-protein interactions that serve as a pathway for signal transduction. Acquiring dynamics associated with each structural transition along the signal transduction pathway is of critical importance in understanding detailed mechanism underlying the functions of the biological systems. Thus, it is highly desirable to directly observe each structural transition and protein conformational change in complex multi-protein systems in response to the functional needs. However, the spatial (structural) and temporal (kinetic) inhomogeneities and non-synchronizable nature of the complex biological systems make it difficulty to acquire such information with ensemble-averaged studies. Therefore, there is an urgent need to develop a methodology to probe structural dynamics associated with each individual protein conformational change and protein-protein interactions in response to biological functions. To address the need, we propose to implement single-molecule FRET technique into our current research to investigate structural and kinetic changes associated with each structural transition of cardiac thin filament in response to Ca2+-induced activation and deactivation. The thin filament, a switching unit of the myofilament to control muscle contraction and relaxation, is a filamentous structure composed of the heterotrimeric troponin and tropomyosin bound to the double helical actin filament. A main feature of Ca2+ activation of thin filament is dynamic interactions among the thin filament proteins. To activate muscle function, Ca2+ binds to troponin which induces a series of critical intramolecular and intermolecular structural changes in the thin filament. Therefore, Ca2+-induced activation and deactivation of the thin filament can be used as a model system to understand the allosteric signal transduction in multi-component biological system. In this study we will collaborate with scientist in EMSL using smFRET measurements to acquire structural, kinetic and energetic information associated with each structural transition of the thin filament induced by Ca2+ binding and dissociation. Elucidating the mechanism of the activation-dependent regulation of the thin filament at single molecular level is important not only because it is central to muscle regulation but also because mechanical and chemical signals such as cell stretch, neurohormonal and redox regulatory pathways manipulate activation-dependent regulation to tune contractility to meet the needs of the system. To achieve the objective of this project, we will utilize the single-molecule spectroscopy facility at EMSL through the collaboration with Dr. Dehong Hu at EMSL. EMSL pioneers in single molecule spectroscopy and the facility is an excellent match for this work.Project Details
Project type
Large-Scale EMSL Research
Start Date
2009-10-01
End Date
2011-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Related Publications
Schlecht W, KL Li, D Hu, and WJ Dong. 2015. "Fluorescence Based Characterization of Calcium Sensitizer Action on the Troponin Complex." Chemical Biology and Drug Design. doi:10.1111/cbdd.12651