Research

Thomas Lab Theme: Molecular dynamics of energy transduction in muscle, using site-directed spectroscopic probes. Our goal is to understand the fundamental molecular motions and interactions that are responsible for cellular movement, and to determine the molecular bases of muscle disorders. We approach this multidisciplinary problem with a wide range of techniques -- physiology, enzyme kinetics, molecular genetics, peptide synthesis, computer simulation -- but our forte is site-directed spectroscopy. After attaching site-directed probes (spin labels, fluorescent dyes, phosphorescent dyes, or isotopes) to selected muscle proteins in solution or in cells, we perform magnetic resonance or optical spectroscopy to directly detect the motions of the force-generating proteins, actin and myosin, or the membrane ion pumps and channels responsible for muscle excitation and relaxation.

Actin	Myosin

Force generation by myosin and actin.  We attach probes to selected sites on myosin and actin, then use spectroscopy to detect the structural dynamics and interactions of these proteins during their ATP-dependent interaction.  Some experiments are done on purified proteins in solution, others in functioning muscle fibers.  The detected movements of normal and mutant proteins provide direct tests of proposed models of force generation.  We used EPR to show that the myosin head undergoes a disorder-to-order transition and changes its shape as it undergoes the weak-to-strong transition in force generation (Baker et al, 1998), and we resolved the two distinct steps of this process in the millisecond time range by using flash photolysis of caged ATP (LaConte et al., 2003).  We used luminescence resonance energy transfer to detect the coordinated movements of the two heads of myosin during muscle contraction (Lidke and Thomas, 2002).  We have used phosphorescence and fluorescence resonance energy transfer (FRET) to show that actin's cooperative internal dynamics are critical for the actin-myosin interaction (Prochniewicz & Thomas, 2001), and that actin undergoes its own weak-to-strong disorder-to-order transition during active interaction with myosin (Prochniewicz et al., 2004).  We used site-directed spin-labeling (Nelson et al., 2005) and molecular dynamics simulations (Espinoza-Fonseca, et al., 2007) to show that smooth muscle myosin is activated by a disorder-to-order transition in the phosphorylation domain of the regulatory light chain.  We have also applied spectroscopy to understand the structural basis of muscle degeneration in aging (Lowe et al., 2001; Prochniewicz et al., 2005) and muscular dystrophy (Lowe et al., 2006).

Active calcium transport in the heart by the Ca-ATPase (SERCA) and its regulation by phospholamban (PLB). We are exploring the molecular dynamics of active calcium transport in muscle membranes, using spectroscopic probes of both proteins and lipids. SERCA is an integral membrane protein that uses energy from ATP hydrolysis to pump calcium into the sarcoplasmic reticulum, which relaxes the muscle and provides the calcium gradient needed for the next contraction. By attaching probes to SERCA and performing EPR and phosphorescence spectroscopy, we have shown that active transport involves large-scale movements within the cytoplasmic domain, and that function can be inhibited by reagents  (e.g., anesthetics or peptides) that restrict these movements due to aggregation (Mueller et al., 2004). In the heart SERCA is regulated by phospholamban (PLB), an integral membrane protein that inhibits (and aggregates) SERCA unless PLB is phosphorylated.  We used EPR and fluorescence resonance energy transfer (FRET) to show that PLB is in a dynamic equilibrium between monomers and pentamers, but that SERCA binds preferentially to the monomer (Reddy et al, 2003). We used FRET from SERCA to PLB to detect directly the inhibitory interaction and show that relief of PLB inhibition does not require dissociation from SERCA (Mueller et al., 2004).  In collaboration with the Veglia lab, we used NMR to determine the atomic structure of the PLB monomer (Zamoon et al., 2003) and to detect the dynamic interaction between SERCA and PLB (Zamoon et al., 2005).  We used FRET, in combination with previous EPR and NMR data, to construct a model of the PLB pentamer (Robia et al., 2005). We used peptide synthesis and the TOAC spin label to detect directly the peptide backbone dynamics of PLB in lipid bilayers (Karim et al., 2004).  We found that phosphorylation induces an order-to-disorder transition in PLB, relieving inhibition without dissociating PLB from SERCA (Karim et al., 2006; Traaseth et al., 2006). Based on this work, we were invited to publish our procedure for synthesis of TOAC-PLB in Nature Protocols (Zhang et al., 2007).

Ca²⁺ release via RYR channels. Minnesota Musclers Brad Fruen and Razvan Cornea head structural/functional studies of the intracellular channels--termed ryanodine receptors, or RYRs--that mediate the release of Ca²⁺ that triggers cardiac and skeletal muscle contraction. Goals of the RYR group include 1) to determine molecular mechanisms by which drugs modulate these channels (Zhao et al., 2001), 2) to define the effects channel mutations that underlie the clinical disorder malignant hyperthermia (Louis et al., 2001), 3) to characterize the channel interactions with accessory proteins, including calmodulin and FKBP (Fruen et al., 2000; Balog et al., 2003; Fruen et al., 2005), and 4) to determine mechanisms responsible for altered channel regulation during heart disease and skeletal muscle fatigue (Balog et al., 2000). (RYR 3D reconstruction at left adapted from Samso and Wagenknecht, J. Biol. Chem. 2001).

Research Facilities and Techniques