Advanced Solid-State NMR Approaches for Structure Determination of Membrane Proteins and Amyloid Fibrils
ABSTRACT Solid-state NMR (SSNMR) spectroscopy has become an important technique for studying the biophysics and structure biology of proteins. This technique is especially useful for insoluble membrane proteins and amyloid fibrils, which are essential for biological functions and are associated with human diseases. In the past few years, as major contributors to the rapidly advancing discipline of biological SSNMR, we have developed a family of methods for high-resolution structure determination of microcrystalline, fibrous, and membrane proteins. Key developments include order-of-magnitude improvements in sensitivity, resolution, instrument stability, and sample longevity under data collection conditions. These technical advances now enable us to apply new types of 3D and 4D experiments to collect atomic-resolution structural restraints in a site-resolved manner, such as vector angles, chemical shift tensors, and internuclear distances, throughout large proteins. In this Account, we present the technological advances in SSNMR approaches towards protein structure determination. We also describe the application of those methods for large membrane proteins and amyloid fibrils. Particularly, the SSNMR measurements of an integral membrane protein DsbB support the formation of a charge-transfer complex between DsbB and ubiquinone during the disulfide bond transfer pathways. The high-resolution structure of the DsbA-DsbB complex demonstrates that the joint calculation of X-ray and SSNMR restraints for membrane proteins with low-resolution crystal structure is generally applicable. The SSNMR investigations of α-synuclein fibrils from both wild type and familial mutants reveal that the structured regions of α-synuclein fibrils include the early-onset Parkinson's disease mutation sites. These results pave the way to understanding the mechanism of fibrillation in Parkinson's disease.
SourceAvailable from: George Jiaozhi Lu[Show abstract] [Hide abstract]
ABSTRACT: Membrane proteins have always presented technical challenges for structural studies because of their requirement for a lipid environment. Multiple approaches exist including X-ray crystallography and electron microscopy that can give significant insights into their structure and function. However, nuclear magnetic resonance (NMR) is unique in that it offers the possibility of determining the structures of unmodified membrane proteins in their native environment of phospholipid bilayers under physiological conditions. Furthermore, NMR enables the characterization of the structure and dynamics of backbone and side chain sites of the proteins alone and in complexes with both small molecules and other biopolymers. The learning curve has been steep for the field as most initial studies were performed under non-native environments using modified proteins until ultimately progress in both techniques and instrumentation led to the possibility of examining unmodified membrane proteins in phospholipid bilayers under physiological conditions. This review aims to provide an overview of the development and application of NMR to membrane proteins. It highlights some of the most significant structural milestones that have been reached by NMR spectroscopy of membrane proteins, especially those accomplished with the proteins in phospholipid bilayer environments where they function.Quarterly Reviews of Biophysics 07/2014; DOI:10.1017/S0033583514000080 · 11.88 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Characterization of the molecular structure and physicochemical solid-state properties of the solids forms of pharmaceutical compounds is a key requirement for successful commercialization as potential active ingredients in drug products. These properties can ultimately have a critical effect on the solubility and bioavailability of the final drug product. Here, the desmotropy of Albendazole forms I and II was investigated at the atomic level. Ultrafast magic angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopy, together with powder X-ray diffraction, thermal analysis and Fourier Transform infrared spectroscopy, were performed on polycrystalline samples of the two solids in order to fully characterize and distinguish the two forms. High-resolution one-dimensional 1H, 13C and 15N together with two-dimensional 1H/1H single quantum - single quantum, 1H/1H single quantum - double quantum, and 1H/13C chemical shift correlation solid-state NMR experiments under MAS conditions were extensively used to decipher the intramolecular and intermolecular hydrogen bonding interactions present in both solid forms. These experiments enabled the unequivocal identification of the tautomers of each desmotrope. Our results also revealed that both solid forms may be described as dimeric structures, with different intermolecular hydrogen bonds connecting the tautomers in each dimer.Molecular Pharmaceutics 01/2015; DOI:10.1021/mp500539g · 4.57 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Solid-state NMR spectra of membrane proteins often show significant line broadening at cryogenic temperatures. Here we investigate the effects of several cryoprotectants to preserve the spectral resolution of lipid membranes and membrane peptides at temperatures down to ~200 K. Trehalose, glycerol, dimethylsulfoxide (DMSO), dimethylformamide (DMF), and polyethylene glycol (PEG), were chosen. These compounds are commonly used in protein crystallography and cryobiology. (13)C and (1)H magic-angle-spinning spectra of several types of lipid membranes show that DMSO provides the best resolution enhancement over unprotected membranes and also best retards ice formation at low temperature. DMF and PEG-400 show slightly weaker cryoprotection, while glycerol and trehalose neither prevent membrane line broadening nor prevent ice formation under the conditions of our study. Neutral saturated-chain phospholipids are the most amenable to cryoprotection, whereas negatively charged and unsaturated lipids attenuate cryoprotection. (13)C-(1)H dipolar couplings and (31)P chemical shift anisotropies indicate that high spectral resolution at low temperature is correlated with stronger immobilization of the lipids at high temperature, indicating that line narrowing results from reduction of the conformational space sampled by the lipid molecules at high temperature. DMSO selectively narrowed the linewidths of the most disordered residues in the influenza M2 transmembrane peptide, while residues that exhibit narrow linewidths in the unprotected membrane are less impacted. A relatively rigid β-hairpin antimicrobial peptide, PG-1, showed a linewidth increase of ~0.5 ppm over a ~70 K temperature drop both with and without cryoprotection. Finally, a short-chain saturated lipid, DLPE, exhibits excellent linewidths, suggesting that it may be a good medium for membrane protein structure determination. The three best cryoprotectants found in this work-DMSO, PEG, and DMF-should be useful for low-temperature membrane-protein structural studies by SSNMR without compromising spectral resolution.Journal of Biomolecular NMR 07/2014; DOI:10.1007/s10858-014-9845-z · 3.31 Impact Factor