Perspectives in Enzymology of Membrane Proteins by Solid-State NMR

Abstract

Membrane proteins catalyze reactions at the cell membrane and facilitate thetransport of molecules or signals across the membrane. Recently researchers have made great progress in understanding the structural biology of membrane proteins, mainly based on X-ray crystallography. In addition, the application of complementary spectroscopic techniques has allowed researchers to develop a functional understanding of these proteins. Solid-state NMR has become an indispensable tool for the structure-function analysis of insoluble proteins and protein complexes. It offers the possibility of investigating membrane proteins directly in their environment, which provides essential information about the intrinsic coupling of protein structure and functional dynamics within the lipid bilayer. However, to date, researchers have hardly explored the enzymology of mem-brane proteins. In this Account, we review the perspectives for investigating membrane-bound enzymes by solid-state NMR. Understanding enzyme mechanisms requires access to kinetic parameters, structural analysis of the catalytic center, knowledge of the 3D structure and methods to follow the structural dynamics of the enzyme during the catalytic cycle. In principle, solid-state NMR can address all of these issues. Researchers can characterize the enzyme kinetics by observing substrate turnover within the membrane or at the membrane interphase in a time-resolved fashion as shown for diacylglycerol kinase. Solid-state NMR has also provided a mechanistic understanding of soluble enzymes including triosephosphate isomerase (TIM) and different metal-binding proteins, which demonstrates a promising perspective also for membrane proteins. The increasing availability of high magnetic fields and the development of new experimental schemes and computational protocols have made it easier to determine 3D structure using solid-state NMR. Dynamic nuclear polarization, a key technique to boost sensitivity of solid-state NMR at low temperatures, can help with the analysis of thermally trapped catalytic intermediates, while methods to improve signal-to-noise per time unit enable the real-time measurement of kinetics of conformational changes during the catalytic cycle.

NMR of sedimented, fibrillized, silica-entrapped and microcrystalline (metallo)proteins

“…Membrane proteins are also extremely appealing targets for SSNMR since the growth of single crystals for X-ray diffraction experiments is challenging [4][5][6][63][64][65]. SSNMR can access these systems in microcrystalline form as well as in their native lipid environment.…”

Protein dynamics and function from solution state NMR spectroscopy

“…We therefore do not discuss the use of NMR to study nucleic acid dynamics (Kimsey et al 2015) and structure (Keane et al 2015), or quantitative analysis of protein dynamics using solid state NMR spectroscopy (Lewandowski, 2013;Ullrich & Glaubitz, 2013). We also do not cover the use of NMR to study protein folding or intrinsically disordered proteins Wright & Dyson, 2015), although it is important to note that both fields are relevant to protein function.…”

Probing Conformational Changes during the Gating Cycle of a Potassium Channel in Lipid Bilayers

“…Previous electron paramagnetic resonance experiments showed that they rotate upon activation in a counterclockwise direction, thereby opening the conduction pathway (15,16). Furthermore, solid-state NMR (ssNMR), which can be applied in lipid bilayers (17)(18)(19)(20), provided detailed insights into the structural basis of the coupling of KcsA activation and inactivation gates during channel gating (21)(22)(23).…”