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  Heterogeneity of protein substates visualized by spin-label EPR.

Guzzi, R., Bartucci, R., & Marsh, D. (2014). Heterogeneity of protein substates visualized by spin-label EPR. Biophysical Journal, 106(3), 716-722. doi:10.1016/j.bpj.2013.12.039.

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Item Permalink: http://hdl.handle.net/11858/00-001M-0000-0015-8795-E Version Permalink: http://hdl.handle.net/11858/00-001M-0000-0028-479E-5
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 Creators:
Guzzi, R., Author
Bartucci, R., Author
Marsh, D.1, Author              
Affiliations:
1Emeritus Group of Spectroscopy and Photochemical Kinetics, MPI for Biophysical Chemistry, Max Planck Society, ou_578625              

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 Abstract: The energy landscape of proteins is characterized by a hierarchy of substates, which give rise to conformational heterogeneity at low temperatures. In multiply spin-labeled membranous Na, K-ATPase, this heterogeneous population of conformations is manifest by strong inhomogeneous broadening of the electron paramagnetic resonance (EPR) line shapes and nonexponential spin-echo decays, which undergo a transition to homogeneous broadening and exponential relaxation at higher temperatures (previous study). In this study, we apply these EPR methods to small water-soluble proteins, of the type for which the existence of conformational substates is well established. Both alpha-helical and beta-sheet aqueous proteins that are spin-labeled on a single cysteine residue display spin-echo decays with a single phase-memory time T-2M and conventional EPR line shapes with predominantly homogeneous broadening, over a broad range of temperatures from 77 K to similar to 250 K or higher. Above similar to 200 K, the residual inhomogeneous broadening is reduced almost to zero. In contrast, both the proteins and the spin label alone, when in a glycerol-water mixture below the glass transition, display heterogeneity in spin-echo phase-memory time and a stronger inhomogeneous broadening of the conventional line shapes, similar to multiply spin-labeled membranous Na, K-ATPase below 200 K. Above 200 K (or the glass transition), a single phase-memory time and predominantly homogeneous broadening are found in both spin-label systems. The results are discussed in terms of solvent-mediated protein transitions, the ability of single spin-label sites to detect conformational heterogeneity, and the desirability of exploring multiple sites for proteins with the size and complexity of the Na,K-ATPase.

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Language(s): eng - English
 Dates: 2014-02-04
 Publication Status: Published in print
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 Rev. Method: Peer
 Identifiers: DOI: 10.1016/j.bpj.2013.12.039
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Title: Biophysical Journal
Source Genre: Journal
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Pages: - Volume / Issue: 106 (3) Sequence Number: - Start / End Page: 716 - 722 Identifier: -