Solid state NMR: The essential technology for helical membrane protein structural characterization

Abstract

NMR spectroscopy of helical membrane proteins has been very challenging on multiple fronts. The expression and purification of these proteins while maintaining functionality has consumed countless graduate student hours. Sample preparations have depended on whether solution or solid-state NMR spectroscopy was to be performed – neither have been easy. In recent years it has become increasingly apparent that membrane mimic environments influence the structural result. Indeed, in these recent years we have rediscovered that Nobel laureate, Christian Anfinsen, did not say that protein structure was exclusively dictated by the amino acid sequence, but rather by the sequence in a given environment (Anfinsen, 1973) [106]. The environment matters, molecular interactions with the membrane environment are significant and many examples of distorted, non-native membrane protein structures have recently been documented in the literature. However, solid-state NMR structures of helical membrane proteins in proteoliposomes and bilayers are proving to be native structures that permit a high resolution characterization of their functional states. Indeed, solid-state NMR is uniquely able to characterize helical membrane protein structures in lipid environments without detergents. Recent progress in expression, purification, reconstitution, sample preparation and in the solid-state NMR spectroscopy of both oriented samples and magic angle spinning samples has demonstrated that helical membrane protein structures can be achieved in a timely fashion. Indeed, this is a spectacular opportunity for the NMR community to have a major impact on biomedical research through the solid-state NMR spectroscopy of these proteins.

High resolution solid-state NMR spectroscopy of the Yersinia pestis outer membrane protein Ail in lipid membranes

“…The importance of the lipid bilayer membrane for supporting the structure and function of membrane proteins has motivated the development of samples for structural studies by X-ray diffraction, electron microscopy (EM) and NMR spectroscopy, that resemble native membranes as closely as possible (Cross et al 2014; De Zorzi et al 2016; Maslennikov and Choe 2013; Moraes et al 2014). NMR has the unique advantage of being compatible with samples that are similar to the physiological protein environments (Zhou and Cross 2013).…”

High quality NMR structures: a new force field with implicit water and membrane solvation for Xplor-NIH

“…This is also observed for two of the soluble proteins (GB1 and EIN), and likely reflects the improvements in structural conformation afforded by the force field (see below) and its ability to guide structures towards their native conformation. This is particularly important for NMR structures determined with sparse restraints, such as those of membrane proteins, where the number of experimental measurements is often small due to a series of sample-specific challenges that pose limitations on the NMR experiments as well as sensitivity (Cross et al 2014; Ward et al 2015). …”

Facing and Overcoming Sensitivity Challenges in Biomolecular NMR Spectroscopy

“…[44,45] In many instances it seems that the optimal preconditioning of these systems needs to be elucidated on a case-by-case basis. However, the benefits that could result from cracking open the investigation of these ubiquitous systems seem so important that efforts to overcome these challenges are active world-wide.…”