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, whic h 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 eac h 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 int ermediates.
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 v ia the other regulatory proteins, troponin I, troponin T and tropomyosin. The nature of these interactions is not well understood. In solution Ca-binding protei ns 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 t hin 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 th e proteins, and correlate them with thin filament activation. The technical challenge of this area is somewhat greater than for the acto-myosin system: the vari ous components have to be isolated (or expressed) and labeled in solution followed by the reconstitution of the regulatory system. The advantages of this approa ch are many fold: (a) structural changes can be tracked through different levels of organization: isolated proteins, binary, ternary complexes, thin filaments a nd finally muscle; (b) probes are more specifically targeted including doubly labeled complexes for fluorescence energy transfer; (c) new targeting sites are in troduced 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 trans fer
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 advanta ge over NMR, (as little as 100 picomoles of sample can be used) and a spectral resolution advantage over optical techniques, (since the molecules oriented diffe rently 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