Dipolar Recoupling in Rotating Solids.

Magic angle spinning (MAS) nuclear magnetic resonance (NMR) has evolved significantly over the past three decades and established itself as a vital tool for the structural analysis of biological macromolecules and materials. This review delves into the development and application of dipolar recoupling techniques in MAS NMR, which are crucial for obtaining detailed structural and dynamic information. We discuss a variety of homonuclear and heteronuclear recoupling methods which are essential for measuring spatial restraints and explain in detail the spin dynamics that these sequences generate.

A conserved H-bond network in human aquaporin-1 is necessary for native folding and oligomerization.

Aquaporins (AQPs) are α-helical transmembrane proteins that conduct water through membranes with high selectivity and permeability. For human AQP1, in addition to the functional Asn-Pro-Ala motifs and the aromatic/Arg selectivity filter within the pore, there are several highly conserved residues that form an expansive hydrogen-bonding network. Previous solid-state nuclear magnetic resonance studies and structural conservation analysis have detailed which residues may be involved in this network.

The retinal chromophore environment in an inward light-driven proton pump studied by solid-state NMR and hydrogen-bond network analysis.

Inward proton pumping is a relatively new function for microbial rhodopsins, retinal-binding light-driven membrane proteins. So far, it has been demonstrated for two unrelated subgroups of microbial rhodopsins, xenorhodopsins and schizorhodopsins. A number of recent studies suggest unique retinal-protein interactions as being responsible for the reversed direction of proton transport in the latter group.

Probing the energy barriers and stages of membrane protein unfolding using solid-state NMR spectroscopy.

Understanding how the amino acid sequence dictates protein structure and defines its stability is a fundamental problem in molecular biology. It is especially challenging for membrane proteins that reside in the complex environment of a lipid bilayer. Here, we obtain an atomic-level picture of the thermally induced unfolding of a membrane-embedded α-helical protein, human aquaporin 1, using solid-state nuclear magnetic resonance spectroscopy.

Partial magic angle spinning NMR H, C, N resonance assignments of the flexible regions of a monomeric alpha-synuclein: conformation of C-terminus in the lipid-bound and amyloid fibril states.

Alpha-synuclein (α-syn) is a small presynaptic protein that is believed to play an important role in the pathogenesis of Parkinson's disease (PD). It localizes to presynaptic terminals where it partitions between a cytosolic soluble and a lipid-bound state. Recent evidence suggests that α-syn can also associate with mitochondrial membranes where it interacts with a unique anionic phospholipid cardiolipin (CL).

Identifying lipids tightly bound to an integral membrane protein.

Anabaena Sensory Rhodopsin (ASR) is a microbial photosensor from the cyanobacterium Anabaena sp. PCC 7120. It was found in previous studies that ASR co-purifies with several small molecules, although their identities and structural or functional roles remained unclear.

Improved Protocol for the Production of the Low-Expression Eukaryotic Membrane Protein Human Aquaporin 2 in for Solid-State NMR.

Solid-state nuclear magnetic resonance (SSNMR) is a powerful biophysical technique for studies of membrane proteins; it requires the incorporation of isotopic labels into the sample. This is usually accomplished through over-expression of the protein of interest in a prokaryotic or eukaryotic host in minimal media, wherein all (or some) carbon and nitrogen sources are isotopically labeled. In order to obtain multi-dimensional NMR spectra with adequate signal-to-noise ratios suitable for in-depth analysis, one requires high yields of homogeneously structured protein.

Molecular motifs encoding self-assembly of peptide fibers into molecular gels.

Peptides are a promising class of gelators, due to their structural simplicity, biocompatibility and versatility. Peptides were synthesized based on four amino acids: leucine, phenylalanine, tyrosine and tryptophan. These peptide gelators, with systematic structural variances in side chain structure and chain length, were investigated using Hansen solubility parameters to clarify molecular features that promote gelation in a wide array of solvents.

Solid-state NMR spectroscopy based atomistic view of a membrane protein unfolding pathway.

Membrane protein folding, structure, and function strongly depend on a cell membrane environment, yet detailed characterization of folding within a lipid bilayer is challenging. Studies of reversible unfolding yield valuable information on the energetics of folding and on the hierarchy of interactions contributing to protein stability. Here, we devise a methodology that combines hydrogen-deuterium (H/D) exchange and solid-state NMR (SSNMR) to follow membrane protein unfolding in lipid membranes at atomic resolution through detecting changes in the protein water-accessible surface, and concurrently monitoring the reversibility of unfolding.

Structure of the Functionally Important Extracellular Loop C of Human Aquaporin 1 Obtained by Solid-State NMR under Nearly Physiological Conditions.

Human aquaporin 1 (hAQP1) is the first discovered selective water channel present in lipid membranes of multiple types of cells. Several structures of hAQP1 and its bovine homolog have been obtained by electron microscopy and X-ray crystallography, giving a consistent picture of the transmembrane domain with the water-conducting pore. The transmembrane domain is formed by six full helices and two half-helices, which form a central constriction with conserved asparagine-proline-alanine motifs.

Biosynthetic production of fully carbon-13 labeled retinal in E. coli for structural and functional studies of rhodopsins.

The isomerization of a covalently bound retinal is an integral part of both microbial and animal rhodopsin function. As such, detailed structure and conformational changes in the retinal binding pocket are of significant interest and are studied in various NMR, FTIR, and Raman spectroscopy experiments, which commonly require isotopic labeling of retinal. Unfortunately, the de novo organic synthesis of an isotopically-labeled retinal is complex and often cost-prohibitive, especially for large scale expression required for solid-state NMR.

Partial solid-state NMR H, C, N resonance assignments of a perdeuterated back-exchanged seven-transmembrane helical protein Anabaena Sensory Rhodopsin.

Anabaena Sensory Rhodopsin (ASR) is a unique photochromic membrane-embedded photosensor which interacts with soluble transducer and is likely involved in a light-dependent gene regulation in the cyanobacterium Anabaena sp. PCC 7120. We report partial spectroscopic H, C and N assignments of perdeuterated and back-exchanged ASR reconstituted in lipids.

Applications of solid-state NMR to membrane proteins.

Membrane proteins mediate flow of molecules, signals, and energy between cells and intracellular compartments. Understanding membrane protein function requires a detailed understanding of the structural and dynamic properties involved. Lipid bilayers provide a native-like environment for structure-function investigations of membrane proteins.

Solid-State NMR Provides Evidence for Small-Amplitude Slow Domain Motions in a Multispanning Transmembrane α-Helical Protein.

Proteins are dynamic entities and populate ensembles of conformations. Transitions between states within a conformational ensemble occur over a broad spectrum of amplitude and time scales, and are often related to biological function. Whereas solid-state NMR (SSNMR) spectroscopy has recently been used to characterize conformational ensembles of proteins in the microcrystalline states, its applications to membrane proteins remain limited.

Oligomeric Structure of Anabaena Sensory Rhodopsin in a Lipid Bilayer Environment by Combining Solid-State NMR and Long-range DEER Constraints.

Oligomerization of membrane proteins is common in nature. Here, we combine spin-labeling double electron-electron resonance (DEER) and solid-state NMR (ssNMR) spectroscopy to refine the structure of an oligomeric integral membrane protein, Anabaena sensory rhodopsin (ASR), reconstituted in a lipid environment. An essential feature of such a combined approach is that it provides structural distance restraints spanning a range of ca 3-60Å while using the same sample preparation (i.

Structure and Dynamics of Extracellular Loops in Human Aquaporin-1 from Solid-State NMR and Molecular Dynamics.

Multiple moderate-resolution crystal structures of human aquaporin-1 have provided a foundation for understanding the molecular mechanism of selective water translocation in human cells. To gain insight into the interfacial structure and dynamics of human aquaporin-1 in a lipid environment, we performed nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics simulations. Using magic angle spinning solid-state NMR, we report a near complete resonance assignment of the human aquaporin-1.

Sparse (13)C labelling for solid-state NMR studies of P. pastoris expressed eukaryotic seven-transmembrane proteins.

We demonstrate a novel sparse (13)C labelling approach for methylotrophic yeast P. pastoris expression system, towards solid-state NMR studies of eukaryotic membrane proteins. The labelling scheme was achieved by co-utilizing natural abundance methanol and specifically (13)C labelled glycerol as carbon sources in the expression medium.

Isotope labeling of eukaryotic membrane proteins in yeast for solid-state NMR.

Solid-state NMR (ssNMR) is a rapidly developing technique for exploring structure and dynamics of membrane proteins, but its progress is hampered by its low sensitivity. Despite the latest technological advances, routine ssNMR experiments still require several milligrams of isotopically labeled protein. While production of bacterial membrane proteins on this scale is usually feasible, obtaining such quantities of eukaryotic membrane proteins is often impossible or extremely costly.

Proton detection for signal enhancement in solid-state NMR experiments on mobile species in membrane proteins.

Direct proton detection is becoming an increasingly popular method for enhancing sensitivity in solid-state nuclear magnetic resonance spectroscopy. Generally, these experiments require extensive deuteration of the protein, fast magic angle spinning (MAS), or a combination of both. Here, we implement direct proton detection to selectively observe the mobile entities in fully-protonated membrane proteins at moderate MAS frequencies.

Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR.

Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from electronic spins to the nuclear spins of interest. Typically, both the protein and an exogenous source of electronic spins, such as a biradical, are either codissolved or suspended and then frozen in a glycerol/water glassy matrix to achieve a homogeneous distribution. While the use of such a matrix protects the protein upon freezing, it also reduces the available sample volume (by ca.

Membrane proteins in their native habitat as seen by solid-state NMR spectroscopy.

Membrane proteins play many critical roles in cells, mediating flow of material and information across cell membranes. They have evolved to perform these functions in the environment of a cell membrane, whose physicochemical properties are often different from those of common cell membrane mimetics used for structure determination. As a result, membrane proteins are difficult to study by traditional methods of structural biology, and they are significantly underrepresented in the protein structure databank.

In situ structural studies of Anabaena sensory rhodopsin in the E. coli membrane.

Magic-angle spinning nuclear magnetic resonance is well suited for the study of membrane proteins in the nativelike lipid environment. However, the natural cellular membrane is invariably more complex than the proteoliposomes most often used for solid-state NMR (SSNMR) studies, and differences may affect the structure and dynamics of the proteins under examination. In this work we use SSNMR and other biochemical and biophysical methods to probe the structure of a seven-transmembrane helical photoreceptor, Anabaena sensory rhodopsin (ASR), prepared in the Escherichia coli inner membrane, and compare it to that in a bilayer formed by DMPC/DMPA lipids.

Advanced solid-state NMR techniques for characterization of membrane protein structure and dynamics: application to Anabaena Sensory Rhodopsin.

Studies of the structure, dynamics, and function of membrane proteins (MPs) have long been considered one of the main applications of solid-state NMR (SSNMR). Advances in instrumentation, and the plethora of new SSNMR methodologies developed over the past decade have resulted in a number of high-resolution structures and structural models of both bitopic and polytopic α-helical MPs. The necessity to retain lipids in the sample, the high proportion of one type of secondary structure, differential dynamics, and the possibility of local disorder in the loop regions all create challenges for structure determination.

Recent advances in magic angle spinning solid state NMR of membrane proteins.

Membrane proteins mediate many critical functions in cells. Determining their three-dimensional structures in the native lipid environment has been one of the main objectives in structural biology. There are two major NMR methodologies that allow this objective to be accomplished.

Conformational dynamics of a seven transmembrane helical protein Anabaena Sensory Rhodopsin probed by solid-state NMR.

The ability to detect and characterize molecular motions represents one of the unique strengths of nuclear magnetic resonance (NMR) spectroscopy. In this study, we report solid-state NMR site-specific measurements of the dipolar order parameters and (15)N rotating frame spin-lattice (R1ρ) relaxation rates in a seven transmembrane helical protein Anabaena Sensory Rhodopsin reconstituted in lipids. The magnitudes of the observed order parameters indicate that both the well-defined transmembrane regions and the less structured intramembrane loops undergo restricted submicrosecond time scale motions.