Solid-state NMR Structural Studies Research Articles

Akron Section Award to Christopher Jaroniec

Christopher Jaroniec, Professor of Chemistry and Evans Scholar at Ohio State University, is the recipient of the 2017 ACS Akron Section Award, given by the ACS Akron Section to promising young industrial or academic scientists. The award consists of $1,000 and a plaque. Jaroniec’s research is focused on the development of multidimensional magic-angle spinning solid-state NMR techniques and their application to the detailed analysis of the molecular structure, conformational dynamics, and intermolecular interactions of biological macromolecules. He will receive the award on Nov. 14 at the University of Akron. He will give two presentations, titled “Solid-State NMR Structural Studies of Proteins Using Paramagnetic Probes” and “Molecular Mechanisms of Prion & Amyloid Propagation Explored by Solid-State NMR Spectroscopy.”

Solid-state NMR of the Yersinia pestis outer membrane protein Ail in lipid bilayer nanodiscs sedimented by ultracentrifugation.

Solid-state NMR studies of sedimented soluble proteins has been developed recently as an attractive approach for overcoming the size limitations of solution NMR spectroscopy while bypassing the need for sample crystallization or precipitation (Bertini et al. Proc Natl Acad Sci USA 108(26):10396-10399, 2011). Inspired by the potential benefits of this method, we have investigated the ability to sediment lipid bilayer nanodiscs reconstituted with a membrane protein. In this study, we show that nanodiscs containing the outer membrane protein Ail from Yersinia pestis can be sedimented for solid-state NMR structural studies, without the need for precipitation or lyophilization. Optimized preparations of Ail in phospholipid nanodiscs support both the structure and the fibronectin binding activity of the protein. The same sample can be used for solution NMR, solid-state NMR and activity assays, facilitating structure-activity correlation experiments across a wide range of timescales.

Solid State NMR Structural Studies of Antimicrobial Peptides LPcin Analogs with Enhanced Activities

The availability of antibiotics has allowed for the successful treatment of many bacterial infections as well as the ability to perform invasive medical procedures including surgery and chemotherapy. However, their wide use has led to pathogens’ increased drug resistance and the need to find novel classes of antimicrobial peptides as alternatives to antibiotics. Lactophoricin (LPcin), a cationic amphipathic peptide consists of 23-mer peptide, corresponds to the carboxy terminal 113-135 region of component-3 of proteose-peptone. LPcin is a good candidate as a peptide antibiotic because it has an antibacterial activity but no hemolytic activity. Three different analogs of LPcin, LPcin-yk2 which has mutant amino acids, LPcin-yk1 and LPcin-yk3 that has shorter mutant amino acids are recently developed by using peptide engineering in our laboratory. These three LPcin analogs show better antibioitic activities than wild-type LPcin and no toxicity at all.In order to understand the structural correlation between LPcin analogs structure and antimicrobial activity under the membrane environments, we tried to express and purify as large as amounts of LPcin and three different LPcin analogs. We finally optimized and succeed to overexpress in the form of fusion protein in Escherichia coli and purified with biophysical techniques like Ni-affinity chromatography, dialysis, centrifuge, chemical cleavage, and reversed-phase semiprep HPLC. In here, we will present the optimizing processes with high-yield expression and purification of three LPcin analogs and solid- state NMR structural studies to figure out antibacterial killing mechanisms.

ChemInform Abstract: Elucidating Silk Structure Using Solid‐State NMR

AbstractReview: 104 refs.

Solid state NMR structural studies of the lithiation of nano-silicon:

Abstract The electrochemical lithiation of undoped, P-doped and B-doped nano-silicon particles (100–200 nm diameter) has been studied during the first cycle by ex-situ 6 Li and 7 Li magic angle spinning nuclear magnetic resonance (MAS-NMR) spectroscopy. Samples were charged within pouch cells up to capacities of 4000 mAh/g, at C/40 followed by NMR analysis. The spectra reveal important quantitative information on the local lithium environments during the various stages of the intercalation process. Approximate Li/Si ratios of the lithium silicides present in the nanoparticles can be deduced, based on the initial formation of the SEI layer, which accounts for an irreversible capacity of up to 500 mAh/g. Surface lithium silicide environments with high Li concentrations (corresponding to the composition Li 15 Si 4 ) are preferentially formed at charging capacities near 1000 mAh/g. At higher charging capacities, irreversible capacity losses are lower and a wide distribution of lithium silicide environments is found, resembling those present in the crystalline phases Li 12 Si 7 , Li 7 Si 3 , and Li 13 Si 4 . At a charging capacity of and above 2000 mAh/g the large majority of silicon is converted to lithiated silicide particles. Boron-doped nano-Si materials behave generally similar, while phosphorus-doping reveals clear beneficial effects, in particular concerning the initial lithiation stages. Both irreversible capacity losses and surface “over-lithiation” are significantly diminished in these samples. Exposure of lithiated nano-Si samples to elevated temperatures (400–440 K) results in the crystallization of Li 7 Si 3 in all of those nano-Si samples charged with at least 1500 mAh/g.

Importance of lipid–pore loop interface for potassium channel structure and function

Potassium (i.e., K(+)) channels allow for the controlled and selective passage of potassium ions across the plasma membrane via a conserved pore domain. In voltage-gated K(+) channels, gating is the result of the coordinated action of two coupled gates: an activation gate at the intracellular entrance of the pore and an inactivation gate at the selectivity filter. By using solid-state NMR structural studies, in combination with electrophysiological experiments and molecular dynamics simulations, we show that the turret region connecting the outer transmembrane helix (transmembrane helix 1) and the pore helix behind the selectivity filter contributes to K(+) channel inactivation and exhibits a remarkable structural plasticity that correlates to K(+) channel inactivation. The transmembrane helix 1 unwinds when the K(+) channel enters the inactivated state and rewinds during the transition to the closed state. In addition to well-characterized changes at the K(+) ion coordination sites, this process is accompanied by conformational changes within the turret region and the pore helix. Further spectroscopic and computational results show that the same channel domain is critically involved in establishing functional contacts between pore domain and the cellular membrane. Taken together, our results suggest that the interaction between the K(+) channel turret region and the lipid bilayer exerts an important influence on the selective passage of potassium ions via the K(+) channel pore.

The Discreet Charm of the Curve

The Discreet Charm of the Curve

Elucidating silk structure using solid-state NMR

An overview of solid-state NMR structural studies on various silk forms and analogs conducted by the authors' research groups is presented. The well-studied silkworm and spider silks together with related silk peptides have a mixture of secondary structures including β-sheet, β-turn, helix and random coil that are difficult to analyze by X-ray diffraction and electron microscopy but conveniently investigated by solid-state NMR. Several newly developed solid-state NMR techniques and stable isotope labeling approaches of the silks were effectively used to characterize silk structure. The techniques discussed provide not only information on the secondary structure, but also on the hydrogen-bonding interactions present in the silks. Structural studies on other types of silk, silk peptide mimics and recombinant silk proteins are also discussed.

Improved chemical shift prediction by Rosetta conformational sampling

Chemical shift frequencies represent a time-average of all the conformational states populated by a protein. Thus, chemical shift prediction programs based on sequence and database analysis yield higher accuracy for rigid rather than flexible protein segments. Here we show that the prediction accuracy can be significantly improved by averaging over an ensemble of structures, predicted solely from amino acid sequence with the Rosetta program. This approach to chemical shift and structure prediction has the potential to be useful for guiding resonance assignments, especially in solid-state NMR structural studies of membrane proteins in proteoliposomes.

A New Structural Model of Aβ40 Fibrils

The amyloid fibrils of beta-amyloid (Aβ) peptides play important roles in the pathology of Alzheimer's disease. Comprehensive solid-state NMR (SSNMR) structural studies on uniformly isotope-labeled Aβ assemblies have been hampered for a long time by sample heterogeneity and low spectral resolution. In this work, SSNMR studies on well-ordered fibril samples of Aβ(40) with an additional N-terminal methionine provide high-resolution spectra which lead to an accurate structural model. The fibrils studied here carry distinct structural features compared to previous reports. The inter-β-strand contacts within the U-shaped β-strand-turn-β-strand motif are shifted, the N-terminal region adopts a β-conformation, and new inter-monomer contacts occur at the protofilament interface. The revealed structural diversity in Aβ fibrils points to a complex picture of Aβ fibrillation.

Expression, refolding, and initial structural characterization of the Y. pestis Ail outer membrane protein in lipids

Ail is an outer membrane protein and virulence factor of Yersinia pestis, an extremely pathogenic, category A biothreat agent, responsible for precipitating massive human plague pandemics throughout history. Due to its key role in bacterial adhesion to host cells and bacterial resistance to host defense, Ail is a key target for anti-plague therapy. However, little information is available about the molecular aspects of its function and interactions with the human host, and the structure of Ail is not known. Here we describe the recombinant expression, purification, refolding, and sample preparation of Ail for solution and solid-state NMR structural studies in lipid micelles and lipid bilayers. The initial NMR and CD spectra show that Ail adopts a well-defined transmembrane β-sheet conformation in lipids.

Wheat-germ cell-free production of prion proteins for solid-state NMR structural studies

The expression of soluble, functional protein on a preparative scale poses a central challenge for structural studies. Cell-free protein expression has become a valuable alternative to cell-based methods, and allows today the expression of milligram quantities of protein. Its use is particularly attractive for NMR studies as it allows a multitude of isotopic labeling schemes. We have implemented and further developed protocols to prepare cell-free extracts from wheat germ to produce recombinant protein for solid-state NMR studies. Furthermore, we established the Renilla luciferase model to optimise and evaluate extract quality, and report first productions of the prions Ure2p and HET-s devoted to structural studies currently ongoing in our laboratories.

High-level expression and purification of the second transmembrane domain of wild-type and mutant human melanocortin-4 receptor for solid-state NMR structural studies

It has been demonstrated that human melanocortin-4 receptor (hMC4R) plays an important role in the control of energy homeostasis, and heterozygous mutations in the hMC4R gene are the most frequent genetic cause of severe human obesity. In order to obtain additional insight into the structure and function, we cloned, expressed, and purified the second transmembrane domain of the wild-type hMC4R (wt-TM2) and D90N mutant hMC4R (m-TM2). To facilitate structural studies of these hMC4R by solid-state NMR, efficient methods for the production of milligram quantities of isotopically labeled protein are necessary. However, large-scale production of most transmembrane proteins has been limited by experimental adversities due to insufficient yields and low solubility of protein. Nevertheless, through the optimization of the expression and purification approach, we could obtain uniformly or selectively labeled fusion proteins in yields as high as 200–250 mg per liter M9 minimal medium. These proteins were overexpressed in inclusion bodies as a fusion protein with ketosteroid isomerase (KSI) in Escherichia coli, and the fusion protein was purified using immobilized metal affinity chromatography under denaturing conditions. wt-/m-TM2 peptides were released from the fusion by cyanogen bromide cleavage at the Met residue and separated from the carrier KSI by size exclusion chromatography. Initial structural data obtained by solution NMR measurements of wt-/m-TM2 is also presented. The successful application to the production of the second transmembrane domain of human MC4R indicates that the method can be applied to other transmembrane proteins as well and also enable its structural and functional studies using solid-state NMR spectroscopy.

Optimal control based NCO and NCA experiments for spectral assignment in biological solid-state NMR spectroscopy

We present novel pulse sequences for magic-angle-spinning solid-state NMR structural studies of 13C, 15N-isotope labeled proteins. The pulse sequences have been designed numerically using optimal control procedures and demonstrate superior performance relative to previous methods with respect to sensitivity, robustness to instrumental errors, and band-selective excitation profiles for typical biological solid-state NMR applications. Our study addresses specifically 15N to 13C coherence transfers being important elements in spectral assignment protocols for solid-state NMR structural characterization of uniformly 13C, 15N-labeled proteins. The pulse sequences are analyzed in detail and their robustness towards spin system and external experimental parameters are illustrated numerically for typical 15N– 13C spin systems under high-field solid-state NMR conditions. Experimentally the methods are demonstrated by 1D 15N → 13C coherence transfer experiments, as well as 2D and 3D 15N, 13C and 15N, 13C, 13C chemical shift correlation experiments on uniformly 13C, 15N-labeled ubiquitin.

NMR Structural Studies of Interactions of a Small, Nonpeptidyl Tpo Mimic with the Thrombopoietin Receptor Extracellular Juxtamembrane and Transmembrane Domains

Thrombopoietin (Tpo) is a glycoprotein growth factor that supports hematopoietic stem cell survival and expansion and is the principal regulator of megakaryocyte growth and differentiation. Several small, nonpeptidyl molecules have been identified as selective human Tpo receptor (hTpoR) agonists. To understand how the small molecule Tpo mimic SB394725 interacts and activates hTpoR, we performed receptor domain swap and mutagenesis studies. The results suggest that SB394725 interacts specifically with the extracellular juxtamembrane region (JMR) and the transmembrane (TM) domain of hTpoR. Solution and solid-state NMR structural studies using a peptide containing the JMR-TM sequences showed that this region of hTpoR, unexpectedly, consists of two alpha-helices separated by a few nonhelical residues. SB394725 interacts specifically with His-499 in the TM domain and a few distinct residues in the JMR-TM region and affects several specific C-terminal TM domain residues. The unique structural information provided by these studies both sheds light on the distinctive mechanism of action of SB394725 and provides valuable insight into the mechanism of ligand-induced cytokine receptor activation.

Structure, dynamics and topology of membrane polypeptides by oriented 2H solid-state NMR spectroscopy

Knowledge of the structure, dynamics and interactions of polypeptides when associated with phospholipid bilayers is key to understanding the functional mechanisms of channels, antibiotics, signal- or translocation peptides. Solid-state NMR spectroscopy on samples uniaxially aligned relative to the magnetic field direction offers means to determine the alignment of polypeptide bonds and domains relative to the bilayer normal. Using this approach the (15)N chemical shift of amide bonds provides a direct indicator of the approximate helical tilt, whereas the (2)H solid-state NMR spectra acquired from peptides labelled with 3,3,3-(2)H(3)-alanines contain valuable complimentary information for a more accurate analysis of tilt and rotation pitch angles. The deuterium NMR line shapes are highly sensitive to small variations in the alignment of the C(alpha)-C(beta) bond relative to the magnetic field direction and, therefore, also the orientational distribution of helices relative to the membrane normal. When the oriented membrane samples are investigated with their normal perpendicular to the magnetic field direction, the rate of rotational diffusion can be determined in a semi-quantitative manner and thereby the aggregation state of the peptides can be analysed. Here the deuterium NMR approach is first introduced showing results from model amphipathic helices. Thereafter investigations of the viral channel peptides Vpu(1-27) and Influenza A M2(22-46) are shown. Whereas the (15)N chemical shift data confirm the transmembrane helix alignments of these hydrophobic sequences, the deuterium spectra indicate considerable mosaic spread in the helix orientations. At least two peptide populations with differing rotational correlation times are apparent in the deuterium spectra of the viral channels suggesting an equilibrium between monomeric peptides and oligomeric channel configurations under conditions where solid-state NMR structural studies of these peptides have previously been performed.

Preparation of α-synuclein fibrils for solid-state NMR: Expression, purification, and incubation of wild-type and mutant forms

We report the expression and purification of α-synuclein, a protein implicated in Parkinson’s disease, from isotopically ( 13C, 15N) labeled bacterial growth media, as required for solid-state NMR structural studies. Expression from Escherichia coli (BL21(DE3)) was performed with a protocol optimized for time efficiency and yield. Chemical lysis, crude purification by ammonium sulfate precipitation, and two chromatography steps (hydrophobic interaction and size exclusion) yield 30–35 mg/L of growth medium. Purity is confirmed by gel electrophoresis and mass spectrometry. Furthermore, we demonstrate reproducible fibril growth by control of environmental incubation conditions. Highly resolved multidimensional solid-state NMR spectra indicate microscopic order throughout the majority of the AS fibril structure. The number of signals and intensities of well-resolved residue types (Thr, Ser, Ala, Gly, Val, and Ile) are consistent with a single conformation, which is reproducibly prepared by seeding consecutive preparations. Variations in the fibril growth rates and structural polymorphisms exhibited in the solid-state NMR spectra are minimized by careful control of incubation conditions.

Solid-state NMR structural studies of the fibril form of a mutant mouse prion peptide PrP 89–143(P101L)

The peptide fragment 89–143 of the prion protein (carrying a P101L mutation) is biologically active in transgenic mice when in a fibrillar form. Injection of these fibrils into transgenic mice (expressing full length PrP with the P101L mutation) induces a neurodegenerative prion disease (Kaneko et al., J. Mol. Biol. 295 (2000) 997). Here we present solid-state NMR studies of PrP 89–143(P101L) fibrils, probing the conformation of residues in the hydrophobic segment 112–124 with chemical shifts. The conformations of glycine residues were analyzed using doubly 13C=O labeled peptides by two-dimensional (2D) double-quantum correlation, and double-quantum filtered dephasing distance measurements. MQ-NMR experiments were carried out to probe the relative alignment of the individual peptides fibrils. These NMR studies indicate that the 112–124 segment adopts an extended β-sheet conformation, though not in a parallel, in register alignment. There is evidence for conformational variability at Gly 113. DQ correlation experiments provide useful information in regions with conformational heterogeneity.

Hydration-optimized oriented phospholipid bilayer samples for solid-state NMR structural studies of membrane proteins

The preparation of oriented, hydration-optimized lipid bilayer samples, for NMR structure determination of membrane proteins, is described. The samples consist of planar phospholipid bilayers, containing membrane proteins, that are oriented on single pairs of glass slides, and are placed in the coil of the NMR probe with the bilayer plane perpendicular to the direction of the magnetic field. Lipid bilayers provide a medium that closely resembles the biological membrane, and sample orientation both preserves the intrinsic membrane-defined directional quality of membrane proteins, and provides the mechanism for resonance line narrowing. The hydration-optimized samples overcome some of the difficulties associated with multi-dimensional, high-resolution, solid-state NMR spectroscopy of membrane proteins. These samples have greater stability over the course of multi-dimensional NMR experiments, they have lower sample conductance for greater rf power efficiency, and enable greater rf coil filling factors to be obtained for improved experimental sensitivity. Sample preparation is illustrated for the membrane protein CHIF (channel inducing factor), a member of the FXYD family of ion transport regulators.

Myristoylation as a general method for immobilization and alignment of soluble proteins for solid-state NMR structural studies.

N-terminal myristoylation of the immunoglobulin-binding domain of protein G (GB1) from group G Streptococcus provides the means to bind the protein to aligned phospholipid bilayers for solid-state NMR structural studies. The myristoylated protein is immobilized by its interactions with bilayers, and the sample alignment enables orientationally dependent 15N chemical shifts and 1H-15N-dipolar couplings to be measured. Spectra calculated for the average solution NMR structure of the protein at various orientations with respect to the magnetic field direction were compared to the experimental spectrum. The best fit identified the orientation of the myristoylated protein on the lipid bilayers, and demonstrated that the protein adopts a similar structure in both its myristoylated and non-myristoylated forms, and that the structure is not grossly distorted by its interaction with the phosholipid bilayer surface or by its location in the restricted aqueous space between bilayer leaflets. The protein is oriented such that its charged sides face the phosphatidylcholine headgroups of the lipids with the single amphiphilic helix running parallel to the bilayer surface.