The objectives of this project are to develop and evaluate novel molecular modeling and computational approaches to extract accurate structural information from site-directed spin-labeling (SDSL) studies. The combination of SDSL and electron paramagnetic resonance spectroscopy (EPR) has made remarkable advances in the past 15 years (for reviews see (Hubbell and Altenbach, 1994;Hustedt and Beth, 1999;Hubbell et al., 2000; Mchaourab and Perozo, 2000;Columbus and Hubbell, 2002;Klug and Feix, 2005) and it is now widely employed to study the structures and structural transitions of proteins and large macromolecular assemblies, many of which have been refractory to structural characterization by techniques such as X-ray crystallography and NMR. Even in those cases where atomic resolution structures can be obtained, SDSL is being increasingly employed in complementary studies to elucidate the dynamics of structural transitions that determine biological function (e.g. (Dong et al., 2005). In addition, SDSL has been employed to study the assembly of macromolecular complexes composed of elements whose individual structures have been determined by NMR or X-ray crystallographic methods (e.g. (Park et al., 2006)). We propose to combine the full power of modern molecular dynamics and computational approaches in the Lybrand laboratory with advanced EPR methods in the Hustedt laboratory to develop tools that will dramatically improve the quality of the structural models that are obtained from SDSL data. Molecular dynamics (MD) and Monte Carlo (MC) modeling strategies will be developed to treat the structure and dynamics of the methanethiosulfonate spin label (MTSSL). These strategies will be used to predict both the continuous wave EPR (CW-EPR) lineshapes of singly labeled proteins and the distance distributions measured by both CWEPR and double electron-electron resonance (DEER) experiments on doubly labeled proteins. These new computational algorithms will be tested on the well-studied protein T4 lysozyme (T4L) and then used to investigate the effect of a proline to arginine mutation on the structure and dynamics of the cytoplasmic domain of the erythrocyte anion exchange protein, band 3 (CDB3) in an ongoing collaboration with Project 2 of this grant proposal.. The overall goal of the following Specific Aims is to enable the maximum structural and dynamic information to be derived from a given set of EPR data.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Program Projects (P01)
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Special Emphasis Panel (ZRG1-BCMB-P)
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Vanderbilt University Medical Center
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Stein, Richard A; Beth, Albert H; Hustedt, Eric J (2015) A Straightforward Approach to the Analysis of Double Electron-Electron Resonance Data. Methods Enzymol 563:531-67
Edwards, Sarah J; Moth, Christopher W; Kim, Sunghoon et al. (2014) Automated structure refinement for a protein heterodimer complex using limited EPR spectroscopic data and a rigid-body docking algorithm: a three-dimensional model for an ankyrin-CDB3 complex. J Phys Chem B 118:4717-26
Smith, Jarrod A; Edwards, Sarah J; Moth, Christopher W et al. (2013) TagDock: an efficient rigid body docking algorithm for oligomeric protein complex model construction and experiment planning. Biochemistry 52:5577-84
Pester, Oxana; Barrett, Paul J; Hornburg, Daniel et al. (2013) The backbone dynamics of the amyloid precursor protein transmembrane helix provides a rationale for the sequential cleavage mechanism of ?-secretase. J Am Chem Soc 135:1317-29
Barrett, Paul J; Chen, Jiang; Cho, Min-Kyu et al. (2013) The quiet renaissance of protein nuclear magnetic resonance. Biochemistry 52:1303-20
Song, Yuanli; Hustedt, Eric J; Brandon, Suzanne et al. (2013) Competition between homodimerization and cholesterol binding to the C99 domain of the amyloid precursor protein. Biochemistry 52:5051-64
Zhuang, Tiandi; Chen, Qiuyan; Cho, Min-Kyu et al. (2013) Involvement of distinct arrestin-1 elements in binding to different functional forms of rhodopsin. Proc Natl Acad Sci U S A 110:942-7
Mittendorf, Kathleen F; Deatherage, Catherine L; Ohi, Melanie D et al. (2012) Tailoring of membrane proteins by alternative splicing of pre-mRNA. Biochemistry 51:5541-56
Deatherage, Catherine L; Hadziselimovic, Arina; Sanders, Charles R (2012) Purification and characterization of the human ?-secretase activating protein. Biochemistry 51:5153-9
Ghimire, Harishchandra; Hustedt, Eric J; Sahu, Indra D et al. (2012) Distance measurements on a dual-labeled TOAC AChR M2? peptide in mechanically aligned DMPC bilayers via dipolar broadening CW-EPR spectroscopy. J Phys Chem B 116:3866-73

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