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Department of Biological Science

at Florida State University

Biological Science Faculty Member

Dr. Peter G. Fajer

  • Office: 506 Kasha Laboratory Building
  • Office: (850) 645-1337
  • Lab: Kasha Laboratory Building
  • Lab: (850) 645-1335
  • Fax: (850) 644-0481
  • Mail code: 4380
  • E-mail: pfajer@fsu.edu

Personal Home Page

Professor
FSU Developing Scholar (1998)
FSU Teaching Award (1998)
Director of NSF Training Grant in Structural Biology
Ph.D., University of Leeds, England 1983
Graduate Faculty Status

Research and Professional Interests:

My general research interest is in molecular structure-function relationships as applied to motility. The organizational level is that of large macromolecular complexes--the interface of single-particle biochemistry/biophysics and cell biology.

The particular areas of research are:

  • Energy transduction: the molecular mechanism of muscle contraction
  • Signal transduction: the initiation of muscle contraction by Ca2+
  • Physical biochemistry: the development of optical and magnetic spectroscopies to study the molecular dynamics and orientation
A.  Force generation in muscle: energy transduction

The long-term goal of this project is to understand the energy transduction of ATP into mechanical work in muscle. The atomic structure of the myosin head, which was recently determined by Ivan Rayment and coworkers, provides a structural framework within which structure-function questions can be asked. As in any motor, it is reasonable to expect that some parts of the system will be moving, and that some of these motions will be directly resulting in the generation of force. The problem might be trivially summarized as where, when and whether?

  • Where in the myosin or actin molecules do the motions occur?  There are many subdomains of the proteins involved and they can move independently of each other--we attach specific probes to a variety of labeling sites giving us multiple vantage points to observe protein behavior.
  • When are these motions induced?  The energy of ATP is released in a series of reactions comprising an acto-myosin ATPase cycle. In order to identify at which steps the structural changes are talking place we arrest the cycle using nucleotide analogs or perturb the cycle increasing populations of one or two intermediates.
  • Are these motions are coupled to force generation?  Not all structural changes have to be coupled with the force generation. To identify which ones are coupled, we correlate structural changes with the time-course of the force development.

Current projects include:

  • Determination of the internal shape changes within the myosin head by fluorescence energy transfer
  • Modulation of the head dynamics and shape by light chain phosphorylation, saturation transfer EPR and FRET
  • Kinetics of the structural changes--caged ATP, caged Ca with EPR and FRET

B.  Ca2+ activation of muscle contraction: signal transduction

Muscle contraction is initiated by the binding of Ca2+ to the thin filament. This signal somehow results in a changed interaction between the myosin head and actin. Since Ca2+ binds to troponin C, a protein which is not in direct contact with either myosin or actin, the signal must be propagated via the other regulatory proteins, troponin I, troponin T and tropomyosin. The nature of these interactions is not well understood. In solution Ca-binding proteins containing E-F hands analogous to troponin C undergo large structural changes. The conformational freedom of troponin C in complex with the proteins of the thin filament is greatly restricted and changes are more subtle. They involve changes in the mobility of constituent proteins and their relative geometries. The questions asked and the general strategy to answer them is similar to those described above for energy transduction--identify and describe the motions within the proteins, and correlate them with thin filament activation. The technical challenge of this area is somewhat greater than for the acto-myosin system: the various components have to be isolated (or expressed) and labeled in solution followed by the reconstitution of the regulatory system. The advantages of this approach are many fold: (a) structural changes can be tracked through different levels of organization: isolated proteins, binary, ternary complexes, thin filaments and finally muscle; (b) probes are more specifically targeted including doubly labeled complexes for fluorescence energy transfer; (c) new targeting sites are introduced by site specific mutagenesis.

Current projects include:

  • Description of differences in structural changes as induced by crossbridge binding in contrast to those following Ca binding--EPR, fluorescence energy transfer
  • Kinetics of distance changes between actin and troponin I--caged Ca and transient FRET
  • Site directed mutagenesis of troponin C

C.  Instrumental and Methodology Development

The EPR technique is unique insofar that it provides orientational and motional information about specific sites within a molecule. It has a sensitivity advantage over NMR, (as little as 100 picomoles of sample can be used) and a spectral resolution advantage over optical techniques, (since the molecules oriented differently in space are resonating at different magnetic fields). My lab is involved in the development of various aspects of EPR:

  • Development of theoretical prediction (simulation) of experimental spectra
  • Development of statistical methods to fit the simulations to experimental spectra
  • Development of new techniques, time-resolved EPR and the high field EPR

Current projects include:

  • Simulation of the spectra predicted by the known crystal structures
  • Development of the transient EPR at very high fields

Selected Publications:

Sár, C. P., J. Jek, P. G. Fajer, and K. Hideg. 1999. Synthesis and reactions of new alkynyl substituted nitroxide radicals. Synthesis 6: 1039-1045.

Adhikari, B., J. Somerset, J. T. Stull, and P. G. Fajer. 1999. Dynamic modulation of regulatory domain of myosin heads by pH, ionic strength and RLC phosphorylation in synthetic myosin filaments. Biochemistry 38: 3127-2132.

Palm, T., K. Sale, L. Brown, B. Hambly, H.-C. Li, and P. G. Fajer. 1999. Intradomain distances in regulatory domain of myosin head in prepower and postpower stroke states: fluorescence energy transfer. Biochemistry 38: 13026-13034.

Sienkiewicz, A., M. Jaworski, B. G. Smith, P. G. Fajer, and C. Scholes. 2000. Dielectric resonator-based side-access probe for muscle fiber EPR study. J. Magnet. Reson. 143: 144-152.

Fajer, P. G. 2000. EPR of proteins and peptides. In Encyclopedia of Analytical Chemistry. R. Meyers, ed. Wiley, and Sons, London.

Fajer, P. G. 2000. EPR spin labeling, In Encyclopedia of Life Sciences. Macmillan.

Brown, L. J., N. Klonis, W. H. Sawyer, P. G. Fajer, and B. D. Hambly. 2001. Independent movement of the regulatory and catalytic domains of myosin heads revealed by phosphorescence anisotropy. Biochemistry 40: 8283-8291.

Brown, L., L. Singh, K. Sale, B. Yu, R. Trent, P. G. Fajer, and B. D. Hambly. Functional and spectroscopic studies of an FHC mutation in motif X of cardiac myosin binding protein-C. Submitted to Eur. J. Biochem.


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