The long-term goal of this research is to understand the coupling of molecular dynamics to ATPase enzyme action, focussing on the two principal ATP-driven energy transduction systems in vertebrate skeletal muscle: the myosin-actin system (contraction, force-generation), and the Ca-ATPase of sarcoplasmic reticulum (relaxation, active Ca transport). Because these processes are essentially dynamic, we focus on direct detection of protein motions, using site-specific spectroscopic probes: electron paramagnetic resonance (EPR) with nitroxide spin labels, and optical anisotropy with fluorescent and phosphorescent dyes. These methods are chosen because they have sufficient sensitivity and selectivity to detect specific molecular motions in a complex system under physiological conditions. The present project focuses on spectroscopic measurements during the transient phase of the ATPase reaction cycle. This is accomplished by initiating spectroscopic data acquisition, then using laser flash photolysis of """"""""caged"""""""" compounds (e.g., caged ATP, caged Ca), then observing the millisecond-time-resolved spectroscopic response to the resulting step change in ligand (e.g., ATP, Ca) concentration. The following specific aims will be pursued: (1) Develop improved methods for obtaining and analyzing transient spectroscopic (EPR and optical) signals from muscle fibers and membranes, using flash photolysis of caged compounds. (2) Use this technology to probe molecular dynamics and interactions in purified myosin, actin, and their complexes. (3) Probe molecular orientation, motion, and interaction in skinned muscle fibers, during the transient phase of contraction. (4) Detect transient rotational dynamics of the Ca-ATPase during calcium transport in SR membranes. Collaborative studies will be carried out with experts in the transient biochemical and mechanical kinetics of both systems, in an effort to obtain direct correlations between molecular dynamics and physiological transitions. We will use several different caged compounds, to probe different parts of the ATPase cycle, and we will perform spectroscopic experiments under conditions known to affect the physiological transients. While our central goal is to provide new and essential information about the molecular events in muscle contraction and relaxation, we also hope that the technology we are developing will prove effective in a wide range of biophysical energy transduction problems.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
2R01AR039754-04
Application #
3159981
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1988-12-01
Project End
1996-01-31
Budget Start
1992-02-12
Budget End
1993-01-31
Support Year
4
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Minnesota Twin Cities
Department
Type
Schools of Medicine
DUNS #
168559177
City
Minneapolis
State
MN
Country
United States
Zip Code
55455
Mahaney, J E; Froehlich, J P; Thomas, D D (1995) Conformational transitions of the sarcoplasmic reticulum Ca-ATPase studied by time-resolved EPR and quenched-flow kinetics. Biochemistry 34:4864-79
Berger, C L; Thomas, D D (1993) Rotational dynamics of actin-bound myosin heads in active myofibrils. Biochemistry 32:3812-21
Ostap, E M; White, H D; Thomas, D D (1993) Transient detection of spin-labeled myosin subfragment 1 conformational states during ATP hydrolysis. Biochemistry 32:6712-20
Tanner, J W; Thomas, D D; Goldman, Y E (1992) Transients in orientation of a fluorescent cross-bridge probe following photolysis of caged nucleotides in skeletal muscle fibres. J Mol Biol 223:185-203
Lewis, S M; Thomas, D D (1992) Resolved conformational states of spin-labeled Ca-ATPase during the enzymatic cycle. Biochemistry 31:7381-9
Lewis, S M; Thomas, D D (1991) Microsecond rotational dynamics of spin-labeled Ca-ATPase during enzymatic cycling initiated by photolysis of caged ATP. Biochemistry 30:8331-9
Ostap, E M; Thomas, D D (1991) Rotational dynamics of spin-labeled F-actin during activation of myosin S1 ATPase using caged ATP. Biophys J 59:1235-41
Fajer, P G; Fajer, E A; Matta, J J et al. (1990) Effect of ADP on the orientation of spin-labeled myosin heads in muscle fibers: a high-resolution study with deuterated spin labels. Biochemistry 29:5865-71
Fajer, P G; Fajer, E A; Thomas, D D (1990) Myosin heads have a broad orientational distribution during isometric muscle contraction: time-resolved EPR studies using caged ATP. Proc Natl Acad Sci U S A 87:5538-42
Stein, R A; Ludescher, R D; Dahlberg, P S et al. (1990) Time-resolved rotational dynamics of phosphorescent-labeled myosin heads in contracting muscle fibers. Biochemistry 29:10023-31

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