Dylan T. Murray

Solid state NMR strategy for characterizing native membrane protein structures.

Unlike water soluble proteins, the structures of helical transmembrane proteins depend on a very complex environment. These proteins sit in the midst of dramatic electrical and chemical gradients and are often subject to variations in the lateral pressure profile, order parameters, dielectric constant, and other properties. Solid state NMR is a collection of tools that can characterize high resolution membrane protein structure in this environment. Indeed, prior work has shown that this complex environment significantly influences transmembrane protein structure. Therefore, it is important to characterize such structures under conditions that closely resemble its native environment. Researchers have used two approaches to gain protein structural restraints via solid state NMR spectroscopy. The more traditional approach uses magic angle sample spinning to generate isotropic chemical shifts, much like solution NMR. As with solution NMR, researchers can analyze the backbone chemical shifts to obtain torsional restraints. They can also examine nuclear spin interactions between nearby atoms to obtain distances between atomic sites. Unfortunately, for membrane proteins in lipid preparations, the spectral resolution is not adequate to obtain complete resonance assignments. Researchers have developed another approach for gaining structural restraints from membrane proteins: the use of uniformly oriented lipid bilayers, which provides a method for obtaining high resolution orientational restraints. When the bilayers are aligned with respect to the magnetic field of the NMR spectrometer, researchers can obtain orientational restraints in which atomic sites in the protein are restrained relative to the alignment axis. However, this approach does not allow researchers to determine the relative packing between helices. By combining the two approaches, we can take advantage of the information acquired from each technique to minimize the challenges and maximize the quality of the structural results. By combining the distance, torsional, and orientational restraints, we can characterize high resolution membrane protein structure in native-like lipid bilayer environments.

Structure of FUS Protein Fibrils and Its Relevance to Self-Assembly and Phase Separation of Low-Complexity Domains

Polymerization and phase separation of proteins containing low-complexity (LC) domains are important factors in gene expression, mRNA processing and trafficking, and localization of translation. We have used solid-state nuclear magnetic resonance methods to characterize the molecular structure of self-assembling fibrils formed by the LC domain of the fused in sarcoma (FUS) RNA-binding protein. From the 214-residue LC domain of FUS (FUS-LC), a segment of only 57 residues forms the fibril core, while other segments remain dynamically disordered. Unlike pathogenic amyloid fibrils, FUS-LC fibrils lack hydrophobic interactions within the core and are not polymorphic at the molecular structural level. Phosphorylation of core-forming residues by DNA-dependent protein kinase blocks binding of soluble FUS-LC to FUS-LC hydrogels and dissolves phase-separated, liquid-like FUS-LC droplets. These studies offer a structural basis for understanding LC domain self-assembly, phase separation, and regulation by post-translational modification.

Helical membrane protein conformations and their environment

Evidence that membrane proteins respond conformationally and functionally to their environment is growing. Structural models, by necessity, have been characterized in preparations where the protein has been removed from its native environment. Different structural methods have used various membrane mimetics that have recently included lipid bilayers as a more native-like environment. Structural tools applied to lipid bilayer-embedded integral proteins are informing us about important generic characteristics of how membrane proteins respond to the lipid environment as compared with their response to other nonlipid environments. Here, we review the current status of the field, with specific reference to observations of some well-studied α-helical membrane proteins, as a starting point to aid the development of possible generic principles for model refinement.

Membrane Protein Structural Validation by Oriented Sample Solid-State NMR: Diacylglycerol Kinase

The validation of protein structures through functional assays has been the norm for many years. Functional assays perform this validation for water-soluble proteins very well, but they need to be performed in the same environment as that used for the structural analysis. This is difficult for membrane proteins that are often structurally characterized in detergent environments, although functional assays for these proteins are most frequently performed in lipid bilayers. Because the structure of membrane proteins is known to be sensitive to the membrane mimetic environment, such functional assays are appropriate for validating the protein construct, but not the membrane protein structure. Here, we compare oriented sample solid-state NMR spectral data of diacylglycerol kinase previously published with predictions of such data from recent structures of this protein. A solution NMR structure of diacylglycerol kinase has been obtained in detergent micelles and three crystal structures have been obtained in a monoolein cubic phase. All of the structures are trimeric with each monomer having three transmembrane and one amphipathic helices. However, the solution NMR structure shows typical perturbations induced by a micelle environment that is reflected in the predicted solid-state NMR resonances from the structural coordinates. The crystal structures show few such perturbations, especially for the wild-type structure and especially for the monomers that do not have significant crystal contacts. For these monomers the predicted and observed data are nearly identical. The thermostabilized constructs do show more perturbations, especially the A41C mutation that introduces a hydrophilic residue into what would be the middle of the lipid bilayer inducing additional hydrogen bonding between trimers. These results demonstrate a general technique for validating membrane protein structures with minimal data obtained from membrane proteins in liquid crystalline lipid bilayers by oriented sample solid-state NMR.

Detergent Optimized Membrane Protein Reconstitution in Liposomes for Solid State NMR

For small helical membrane proteins, their structures are highly sensitive to their environment, and solid state NMR is a structural technique that can characterize these membrane proteins in native-like lipid bilayers and proteoliposomes. To date, a systematic method by which to evaluate the effect of the solubilizing detergent on proteoliposome preparations for solid state NMR of membrane proteins has not been presented in the literature. A set of experiments are presented aimed at determining the conditions most amenable to dialysis mediated reconstitution sample preparation. A membrane protein from M. tuberculosis is used to illustrate the method. The results show that a detergent that stabilizes the most protein is not always ideal and sometimes cannot be removed by dialysis. By focusing on the lipid and protein binding properties of the detergent, proteoliposome preparations can be readily produced, which provide double the signal-to-noise ratios for both the oriented sample and magic angle spinning solid state NMR. The method will allow more membrane protein drug targets to be structurally characterized in lipid bilayer environments.

Assignment of oriented sample NMR resonances from a three transmembrane helix protein

Oriented sample solid state NMR techniques have been routinely employed to determine the structures of membrane proteins with one or two transmembrane helices. For larger proteins the technique has been limited by spectral resolution and lack of assignment strategies. Here, a strategy for resonance assignment is devised and applied to a three transmembrane helix protein. Sequence specific assignments for all labeled transmembrane amino acid sites are obtained, which provide a set of orientational restraints and helix orientations in the bilayer. Our experiments expand the utility of solid state NMR in membrane protein structure characterization to three transmembrane helix proteins and represent a straightforward strategy for routinely characterizing multiple transmembrane helix protein structures.

Geometry of kinked protein helices from NMR data

Mathematical questions related to determining the structure of a protein from NMR orientational restraints are discussed. The protein segment is a kinked alpha helix modeled as a regular alpha helix in which two adjacent torsion angles have been varied from their ideal values. Varying these torsion angles breaks the helix into two regular helical segments joined at a kink. The problem is to find the torsion angles at the kink from the relationship of the helical segments to the direction of the magnetic field.

Chaperonin GroEL accelerates protofibril formation and decorates fibrils of the Het-s prion protein

Significance The interaction of amyloids with chaperones, a large group of proteins responsible for proteostasis, is thought to play a significant role in the etiology of amyloidosis. Here, we study the interaction of the model chaperonin GroEL with a model amyloid protein, the prion domain of Het-s, by using NMR and EPR spectroscopies and electron and atomic force microscopies. We show that GroEL accelerates protofibril formation, eventually leading to the formation of fibrils densely decorated by GroEL. This type of chaperone–amyloid interaction may serve to reduce the toxicity of amyloidogenic oligomers and target the fully formed fibrils for rapid elimination by facilitating in vivo clearance. We have studied the interaction of the prototypical chaperonin GroEL with the prion domain of the Het-s protein using solution and solid-state NMR, electron and atomic force microscopies, and EPR. While GroEL accelerates Het-s protofibril formation by several orders of magnitude, the rate of appearance of fibrils is reduced. GroEL remains bound to Het-s throughout the aggregation process and densely decorates the fibrils at a regular spacing of ∼200 Å. GroEL binds to the Het-s fibrils via its apical domain located at the top of the large open ring. Thus, apo GroEL and bullet-shaped GroEL/GroES complexes in which only a single ring is capped by GroES interact with the Het-s fibrils; no evidence is seen for any interaction with football-shaped GroEL/GroES complexes in which both rings are capped by GroES. EPR spectroscopy shows that rotational motion of a nitroxide spin label, placed at the N-terminal end of the first β-strand of Het-s fibrils, is significantly reduced in both Het-s/GroEL aggregates and Het-s fibrils, but virtually completely eliminated in Het-s/GroEL fibrils, suggesting that in the latter, GroEL may come into close proximity to the nitroxide label. Solid-state NMR measurements indicate that GroEL binds to the mobile regions of the Het-s fibril comprising the N-terminal tail and a loop connecting β-strands 4 and 5, consistent with interactions involving GroEL binding consensus sequences located therein.

Structural characterization of the D290V mutation site in hnRNPA2 low-complexity–domain polymers

Significance Genetic studies have shown that mutations of conserved Asp residues in three analogous heterogeneous ribonucleoproteins are causative of three neurological diseases. All three Asp residues map to domains of low complexity (LC) or intrinsic disorder. These domains form labile self-associated polymers as normal functional states, and the mutations abnormally enhance the stability of the polymers via heretofore unknown mechanisms. The present study gives evidence that the charged Asp residues are closely aligned in the polymer core and removal of electrostatic repulsion enhances polymer stability. These results may provide insight into other neurodegenerative diseases also caused by mutations in LC domains. Human genetic studies have given evidence of familial, disease-causing mutations in the analogous amino acid residue shared by three related RNA binding proteins causative of three neurological diseases. Alteration of aspartic acid residue 290 of hnRNPA2 to valine is believed to predispose patients to multisystem proteinopathy. Mutation of aspartic acid 262 of hnRNPA1 to either valine or asparagine has been linked to either amyotrophic lateral sclerosis or multisystem proteinopathy. Mutation of aspartic acid 378 of hnRNPDL to either asparagine or histidine has been associated with limb girdle muscular dystrophy. All three of these aspartic acid residues map to evolutionarily conserved regions of low-complexity (LC) sequence that may function in states of either intrinsic disorder or labile self-association. Here, we present a combination of solid-state NMR spectroscopy with segmental isotope labeling and electron microscopy on the LC domain of the hnRNPA2 protein. We show that, for both the wild-type protein and the aspartic acid 290-to-valine mutant, labile polymers are formed in which the LC domain associates into an in-register cross-β conformation. Aspartic acid 290 is shown to be charged at physiological pH and immobilized within the polymer core. Polymers of the aspartic acid 290-to-valine mutant are thermodynamically more stable than wild-type polymers. These observations give evidence that removal of destabilizing electrostatic interactions may be responsible for the increased propensity of the mutated LC domains to self-associate in disease-causing conformations.

Mycobacterium Tuberculosis Protein Rv1861: Structural Insights into GTP Hydrolysis in a Native-Like Membrane Environment

1/3 of the worlds population is infected with Mycobacterium tuberculosis 10% of whom will become sick from the bacilli. Multidrug resistant strains resistant to the leading tuberculosis antibiotics, isoniazid and rifampicin have emerged necessitating new treatments. Integral membrane proteins are an excellent source of novel drug targets. Structural biology can provide rich information regarding the influence of lead compounds on protein function. Solid state nuclear magnetic resonance is increasingly being used to understand membrane protein structure and dynamics in lipid membrane environments. The structural information can be accompanied with other biophysical techniques to make conclusions regarding membrane protein function and the influence of regulatory compounds. Rv1861 is an integral membrane protein from Mycobacterium tuberculosis. It contains the signature GTP binding domain motif AXXXXGKT near the N-terminus of the protein and is predicted to contain three transmembrane alpha helices from hydrophobicity analysis.Oriented sample solid state magnetic resonance accurately measures peptide plane orientations through PISEMA experiments on protein in liquid crystalline lipid bilayers aligned between glass slides. The structural data can be correlated to functional information from experiments such as isothermal titration calorimetry on the protein in liposomes allowing protein structure and function to be understood in a native-like environment. Here we present the initial structural characterization of the Rv1861 protein in lipid bilayers. PISEMA experiments on uniformly and amino acid type specifically labeled samples are used to determine helix orientation. Light scattering, size exclusion chromotography and electrophoresis experiments on the protein in micellar environments will provide the oligomeric state and compliment the secondary structure characterization. The data provide a firm foundation for further structure determination and functional characterization of the protein in a native-like environment.