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      Probing the Conformationally Excited States of Membrane Proteins via (1)H-Detected MAS Solid-State NMR Spectroscopy.

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          Abstract

          Proteins exist in ensembles of conformational states that interconvert on various motional time scales. High-energy states of proteins, often referred to as conformationally excited states, are sparsely populated and have been found to play an essential role in many biological functions. However, detecting these states is quite difficult for conventional structural techniques. Recent progress in solution NMR spectroscopy made it possible to detect conformationally excited states in soluble proteins and characterize them at high resolution. As for soluble proteins, integral or membrane-associated proteins populate different structural states often modulated by their lipid environment. Solid-state NMR spectroscopy is the method of choice to study membrane proteins, as it can detect both ground and excited states in their natural lipid environments. In this work, we apply newly developed (1)H-detected (15)N-HSQC type experiments under moderate magic angle spinning speeds to detect the conformationally excited states of phospholamban (PLN), a single-pass cardiac membrane protein that regulates Ca(2+) transport across sarcoplasmic reticulum membrane. In its unbound state, the cytoplasmic domain of PLN exists in equilibrium between a T state, which is membrane bound and helical, and an R state, which is membrane detached and unfolded. The R state is important for regulation of the sarcoplasmic reticulum Ca(2+)-ATPase, but also for binding to protein kinase A. By hybridizing (1)H detected solution and solid-state NMR techniques, it is possible to detect and resolve the amide resonances of the R state of PLN in liquid crystalline lipid bilayers. These new methods can be used to study the conformationally excited states of membrane proteins in native-like lipid bilayers.

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          Author and article information

          Affiliations
          [1 ] Department of Chemistry and ‡Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota , Minneapolis, Minnesota 55455, United States.
          Journal
          J Phys Chem B
          The journal of physical chemistry. B
          American Chemical Society (ACS)
          1520-5207
          1520-5207
          May 04 2017
          : 121
          : 17
          28406633 10.1021/acs.jpcb.7b03268

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