Fajer Lab Research Interests

We have laboratories in the Institute of Molecular Biophysics and at the National High Magnetic Field Laboratory (NHMFL), which is the center of research in the U.S. for the development and application of instrumentation using high magnetic fields. The NHMFL provides for large scale integration of the technical and conceptual aspects of EPR, NMR, MRI and ICR spectroscopies.

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
  • In common with other spectroscopic techniques is the ambiguity in the interpretation of signals in terms of protein behavior. Thus the development of EPR theory is paramount in research if we are going to extract fully the information contained in the spectra. Up until now the deconvolution of EPR signals was done in terms of the orientation of the spin probe in the magnetic field, rather than the orientation of the myosin head with respect to the fiber which is of more universal interest. We have completed the development of a computational algorithm based on Eulerian transformations between probe, molecular and fiber frames of reference. The application of this algorithm to samples with known head orientation revealed the position of the spin probe within the protein [20]. This information will be used in the determination of the myosin head orientation in the intermediate states of the contractile cycle.
• Development of statistical methods to fit the simulations to experimental spectra
  • The fitting of simulations to experimental spectra is a tedious task that is also very subjective. How do we know that the solution we have stumbled upon is unique ? Manual searches of N-dimensional space are beyond the scope of any individual and we have to resort to statistical methods. We are currently developing an optimization routine based on a combination of Simplex, Monte-Carlo, cross-validation and Global Analysis methods [13,21]. The application of this method, combined with the Eulerian algorithm described above, revealed an accurate description of the orientational distribution of the myosin head in the presence of ADP [21].
• Development of new techniques, time-resolved EPR and the high field EPR
  • New methodologies: We have a long standing interest in the development of EPR for the measurement of molecular dynamics. I have been involved in the early development of Saturation Transfer EPR [4-8], a steady-state technique which is now a routine measurement in many laboratories. We were involved in the development of a time-resolved technique, Saturation Recovery EPR for the measurement of molecular dynamics [10] but the prohibitive cost of such an instrument precluded its wide-spread application. The commercial availability of FT-EPR spectrometers has made it possible in the last two years to consider Fourier Transform methods as a viable route for studying protein dynamics. Preliminary experiments performed in Prof. Freed’s laboratory at Cornell are very promising and we will be continuing this collaborative effort in the future.

Current projects include:

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