The general goal of the proposed research is to understand the role of molecular dynamics in the function of energy-transducing membrane-bound proteins. This effort involves two major components: (1) the development of spectroscopic methods for the measurement of protein and lipid motions and (2) the application of these methods to the study of specific membrane systems. (1) Electron paramagnetic resonance (EPR) techniques will be developed for the study of orientation and rotational dynamics of spin-labeled proteins and lipids, with particular emphasis on saturation transfer EPR, used for the study of microsecond motions of membrane proteins. A second major methodological area will be time-resolved optical spectroscopy, again focussing on methods for the microsecond time range, including transient phosphorescence (or absorption) anisotropy (for measuring rotational motions) and diffusion-enhanced energy transfer (for measuring translational motions and distances). The use of several complementary techniques on the same system is essential in minimizing the ambiguity of interpretation. (2) We will use these methods primarily to probe the relationship between molecular motion and energy-transduction in the Ca-ATPase of sarcoplasmic reticulum (SR), the active Ca pump that maintains the Ca gradient necessary for the function of skeletal muscle. We will perturb the system by varying conditions likely to affect molecular motions and/or function, e.g., lipid composition, temperature, anesthetics, and concentrations of substrates and other ligands. The spectroscopically detected effects on molecular motion and structure (of both lipid and protein components) will be compared with effects on defined kinetic steps in the ATPase and Ca transport reactions. Experiments will be designed to test specific models (proposed by us and others) for the mechanism of active Ca transport. We are particularly interested in the oligomeric state of the enzyme and in lipid dynamics, which may both play key roles in the mechanism. Analogous applications to other systems (cardiac SR, photoreceptor membranes, prothrombin, and cellular membranes) will be carried out through collaborations with other research groups. Besides providing essential information for the understanding of these important membrane systems, these studies, performed in the same laboratory where spectroscopic methods are being developed, should provide models for similar applications in a wide range of other systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM027906-10
Application #
3275135
Study Section
Biophysics and Biophysical Chemistry B Study Section (BBCB)
Project Start
1980-04-01
Project End
1991-03-31
Budget Start
1989-04-01
Budget End
1990-03-31
Support Year
10
Fiscal Year
1989
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
Martin, Peter D; James, Zachary M; Thomas, David D (2018) Effect of Phosphorylation on Interactions between Transmembrane Domains of SERCA and Phospholamban. Biophys J 114:2573-2583
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Nelson, Sarah E D; Ha, Kim N; Gopinath, Tata et al. (2018) Effects of the Arg9Cys and Arg25Cys mutations on phospholamban's conformational equilibrium in membrane bilayers. Biochim Biophys Acta Biomembr 1860:1335-1341
Schaaf, Tory M; Peterson, Kurt C; Grant, Benjamin D et al. (2017) Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells. SLAS Discov 22:250-261
Schaaf, Tory M; Peterson, Kurt C; Grant, Benjamin D et al. (2017) High-Throughput Spectral and Lifetime-Based FRET Screening in Living Cells to Identify Small-Molecule Effectors of SERCA. SLAS Discov 22:262-273
Rebbeck, Robyn T; Nitu, Florentin R; Rohde, David et al. (2016) S100A1 Protein Does Not Compete with Calmodulin for Ryanodine Receptor Binding but Structurally Alters the Ryanodine ReceptorĀ·Calmodulin Complex. J Biol Chem 291:15896-907
Autry, Joseph M; Thomas, David D; Espinoza-Fonseca, L Michel (2016) Sarcolipin Promotes Uncoupling of the SERCA Ca2+ Pump by Inducing a Structural Rearrangement in the Energy-Transduction Domain. Biochemistry 55:6083-6086
McCaffrey, Jesse E; James, Zachary M; Svensson, Bengt et al. (2016) A bifunctional spin label reports the structural topology of phospholamban in magnetically-aligned bicelles. J Magn Reson 262:50-56
Svensson, Bengt; Autry, Joseph M; Thomas, David D (2016) Molecular Modeling of Fluorescent SERCA Biosensors. Methods Mol Biol 1377:503-22
Espinoza-Fonseca, L Michel; Autry, Joseph M; Thomas, David D (2015) Sarcolipin and phospholamban inhibit the calcium pump by populating a similar metal ion-free intermediate state. Biochem Biophys Res Commun 463:37-41

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