Probing the Conformationally Excited States of Membrane Proteins via 1H-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 1H-detected 15N-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 Ca2+ 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 Ca2+-ATPase, but also for binding to protein kinase A. By hybridizing 1H 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.

Original languageEnglish (US)
Pages (from-to)4456-4465
Number of pages10
JournalJournal of Physical Chemistry B
Volume121
Issue number17
DOIs
StatePublished - May 4 2017

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