Triosephosphate Isomerase: N and C Chemical Shift Assignments and Conformational Change upon Ligand Binding by Magic-Angle Spinning Solid-State NMR Spectroscopy

Microcrystalline uniformly ¹³C,¹⁵N-enriched yeast triosephosphate isomerase (TIM) is sequentially assigned by high-resolution solid-state NMR (SSNMR). Assignments are based on intraresidue and interresidue correlations, using dipolar polarization transfer methods, and guided by solution NMR assignments of the same protein. We obtained information on most of the active-site residues involved in chemistry, including some that were not reported in a previous solution NMR study, such as the side-chain carbons of His95. Chemical shift differences comparing the microcrystalline environment to the aqueous environment appear to be mainly due to crystal packing interactions. Site-specific perturbations of the enzyme's chemical shifts upon ligand binding are studied by SSNMR for the first time. These changes monitor proteinwide conformational adjustment upon ligand binding, including many of the sites probed by solution NMR and X-ray studies. Changes in Gln119, Ala163, and Gly210 were observed in our SSNMR studies, but were not reported in solution NMR studies (chicken or yeast). These studies identify a number of new sites with particularly clear markers for ligand binding, paving the way for future studies of triosephosphate isomerase dynamics and mechanism.     

Practical considerations over spectral quality in solid state NMR spectroscopy of soluble proteins

Great theoretical and methodological advances are pushing the limits of resolution and sensitivity in solid state NMR (SSNMR). However, sample preparation remains a critical issue for the success of an experiment. The factors affecting spectral quality in SSNMR samples are discussed, examining cases encountered in the literature and presenting new experimental data. A discussion on resolution and sensitivity in sedimented solutes is framed in this context.     

Separated local field NMR experiments on oriented samples rotating at the magic angle

Biophysical studies on membrane proteins by solid state NMR (SSNMR) can be carried out directly in a membrane environment. Samples are usually prepared in form of multi-lamellar dispersions for magic angle sample spinning or as aligned multi-layers for orientation dependent NMR experiments without sample rotation. A new development is the application of MAS NMR to aligned samples (MAOSS; Magic Angle Oriented Sample Spinning). In combination with separated local field (SLF) experiments, size and orientation of heteronuclear dipolar couplings may be extracted from two-dimensional experiments which correlate dipolar couplings with isotropic chemical shifts. The orientation of these ¹H–X dipolar couplings can be directly related to the orientation of molecular groups in the sample. Here, we demonstrate the feasibility of these experiments on highly ordered polyethylene fibers which serve as model compound. Based on these data, the experiment is also applied to ordered multi-layers of bacteriorhodopsin (purple membrane) which is used as a model for aligned membrane proteins. We present a detailed analysis of different experimental designs with respect to angular sensitivity and the influence of residual sample disorder (“mosaic spread”). The results of the MAOSS-SLF experiment are discussed within the context of established solid state NMR experiments which are usually performed without sample rotation and we compare the data to orientation information obtained from X-ray diffraction.     

Analysis of Structural Variability in Pharmaceutical Excipients Using Solid-State NMR Spectroscopy

Polysaccharide-based excipients comprise the majority of most solid dosage forms and can vary dramatically in terms of structural and functionally related properties. Analytical methods for characterizing these important formulation components are crucial. Solid-state NMR spectroscopy (SSNMR) can provide a wealth of information on these materials while offering the advantages of non-destructive sample preparation and selectivity. The overall objective of this work is to identify SSNMR parameters that can be used to detect differences among these excipients. Excipients were obtained from a wide range of suppliers and analyzed as received; ¹³C SSNMR spectra were acquired using a Chemagnetics CMX-300 spectrometer operating at approximately 75 MHz. The resolution of SSNMR signals of many excipients allows for positive identification of the major form present. Alginic acid and sodium alginate can be differentiated based on carbonyl peak position. Analysis of relative peak intensities provides insight into the purity of a carrageenan sample compared to known standards. The SSNMR spectrum of starch can be used to identify the source and to quantitate the amorphous and crystalline content. Relaxation values and peak areas of starch derivatives can be related to the degree of hydrolysis, providing an alternative method for determining dextrose equivalent. Differences in peak intensities and relaxation time values of HPMC samples can be correlated to the amount of methoxy subsituent groups. Important characteristics of excipients such as form identification, structural differences, crystalline and amorphous content, and water content variations can be detected using SSNMR spectroscopy.     

Automated protein resonance assignments of magic angle spinning solid-state NMR spectra of β1 immunoglobulin binding domain of protein G (GB1)

Magic-angle spinning solid-state NMR (MAS SSNMR) represents a fast developing experimental technique with great potential to provide structural and dynamics information for proteins not amenable to other methods. However, few automated analysis tools are currently available for MAS SSNMR. We present a methodology for automating protein resonance assignments of MAS SSNMR spectral data and its application to experimental peak lists of the β1 immunoglobulin binding domain of protein G (GB1) derived from a uniformly ¹³C- and ¹⁵N-labeled sample. This application to the 56 amino acid GB1 produced an overall 84.1% assignment of the N, CO, CA, and CB resonances with no errors using peak lists from NCACX 3D, CANcoCA 3D, and CANCOCX 4D experiments. This proof of concept demonstrates the tractability of this problem.     

Four-dimensional heteronuclear correlation experiments for chemical shift assignment of solid proteins

Chemical shift assignment is the first step in all established protocols for structure determination of uniformly labeled proteins by NMR. The explosive growth in recent years of magic-angle spinning (MAS) solid-state NMR (SSNMR) applications is largely attributable to improved methods for backbone and side-chain chemical shift correlation spectroscopy. However, the techniques developed so far have been applied primarily to proteins in the size range of 5–10 kDa, despite the fact that SSNMR has no inherent molecular weight limits. Rather, the degeneracy inherent to many 2D and 3D SSNMR spectra of larger proteins has prevented complete unambiguous chemical shift assignment. Here we demonstrate the implementation of 4D backbone chemical shift correlation experiments for assignment of solid proteins. The experiments greatly reduce spectral degeneracy at a modest cost in sensitivity, which is accurately described by theory. We consider several possible implementations and investigate the CANCOCX pulse sequence in detail. This experiment involves three cross polarization steps, from H to CA[i], CA[i] to N[i], and N[i] to C′[i−1], followed by a final homonuclear mixing period. With short homonuclear mixing times (<20 ms), backbone correlations are observed with high sensitivity; with longer mixing times (>200 ms), long-range correlations are revealed. For example, a single 4D experiment with 225 ms homonuclear mixing time reveals ∼200 uniquely resolved medium and long-range correlations in the 56-residue protein GB1. In addition to experimental demonstrations in the 56-residue protein GB1, we present a theoretical analysis of anticipated improvements in resolution for much larger proteins and compare these results in detail with the experiments, finding good agreement between experiment and theory under conditions of stable instrumental performance.     

Efficient solvent suppression with adiabatic inversion for 1H-detected solid-state NMR

This study introduces a conceptually new solvent suppression scheme with adiabatic inversion pulses for ¹H-detected multidimensional solid-state NMR (SSNMR) of biomolecules and other systems, which is termed “Solvent suppression of Liquid signal with Adiabatic Pulse” (SLAP). ¹H-detected 2D ¹³C/¹H SSNMR data of uniformly ¹³C- and ¹⁵N-labeled GB1 sample using ultra-fast magic angle spinning at a spinning rate of 60 kHz demonstrated that the SLAP scheme showed up to 3.5-fold better solvent suppression performance over a traditional solvent-suppression scheme for SSNMR, MISSISSIPPI (Zhou and Rienstra, J Magn Reson 192:167–172, 2008) with 2/3 of the average RF power.

     

Transmembrane helix orientation and dynamics: insights from ensemble dynamics with solid-state NMR observables

As the major component of membrane proteins, transmembrane helices embedded in anisotropic bilayer environments adopt preferential orientations that are characteristic or related to their functional states. Recent developments in solid-state nuclear magnetic resonance (SSNMR) spectroscopy have made it possible to measure NMR observables that can be used to determine such orientations in a native bilayer environment. A quasistatic single conformer model is frequently used to interpret the SSNMR observables, but important motional information can be missing or misinterpreted in the model. In this work, we have investigated the orientation of the single-pass transmembrane domain of viral protein ”u“ (VpuTM) from HIV-1 by determining an ensemble of structures using multiple conformer models based on the SSNMR ensemble dynamics technique. The resulting structure ensemble shows significantly larger orientational fluctuations while the ensemble-averaged orientation is compatible with the orientation based on the quasistatic model. This observation is further corroborated by comparison with the VpuTM orientation from comparative molecular dynamics simulations in explicit bilayer membranes. SSNMR ensemble dynamics not only reveals the importance of transmembrane helix dynamics in interpretation of SSNMR observables, but also provides a means to simultaneously extract both transmembrane helix orientation and dynamics information from the SSNMR measurements.     

Cationic membrane peptides: atomic-level insight of structure–activity relationships from solid-state NMR

Many membrane-active peptides, such as cationic cell-penetrating peptides (CPPs) and antimicrobial peptides (AMPs), conduct their biological functions by interacting with the cell membrane. The interactions of charged residues with lipids and water facilitate membrane insertion, translocation or disruption of these highly hydrophobic species. In this review, we will summarize high-resolution structural and dynamic findings towards the understanding of the structure–activity relationship of lipid membrane-bound CPPs and AMPs, as examples of the current development of solid-state NMR (SSNMR) techniques for studying membrane peptides. We will present the most recent atomic-resolution structure of the guanidinium-phosphate complex, as constrained from experimentally measured site-specific distances. These SSNMR results will be valuable specifically for understanding the intracellular translocation pathway of CPPs and antimicrobial mechanism of AMPs, and more generally broaden our insight into how cationic macromolecules interact with and cross the lipid membrane.     

Nano-Mole Scale Side-Chain Signal Assignment by 1H-Detected Protein Solid-State NMR by Ultra-Fast Magic-Angle Spinning and Stereo-Array Isotope Labeling

We present a general approach in ¹H-detected ¹³C solid-state NMR (SSNMR) for side-chain signal assignments of 10-50 nmol quantities of proteins using a combination of a high magnetic field, ultra-fast magic-angle spinning (MAS) at ~80 kHz, and stereo-array-isotope-labeled (SAIL) proteins [Kainosho M. et al., Nature 440, 52–57, 2006]. First, we demonstrate that ¹H indirect detection improves the sensitivity and resolution of ¹³C SSNMR of SAIL proteins for side-chain assignments in the ultra-fast MAS condition. ¹H-detected SSNMR was performed for micro-crystalline ubiquitin (~55 nmol or ~0.5mg) that was SAIL-labeled at seven isoleucine (Ile) residues. Sensitivity was dramatically improved by ¹H-detected 2D ¹H/¹³C SSNMR by factors of 5.4-9.7 and 2.1-5.0, respectively, over ¹³C-detected 2D ¹H/¹³C SSNMR and 1D ¹³C CPMAS, demonstrating that 2D ¹H-detected SSNMR offers not only additional resolution but also sensitivity advantage over 1D ¹³C detection for the first time. High ¹H resolution for the SAIL-labeled side-chain residues offered reasonable resolution even in the 2D data. A ¹H-detected 3D ¹³C/¹³C/¹H experiment on SAIL-ubiquitin provided nearly complete ¹H and ¹³C assignments for seven Ile residues only within ~2.5 h. The results demonstrate the feasibility of side-chain signal assignment in this approach for as little as 10 nmol of a protein sample within ~3 days. The approach is likely applicable to a variety of proteins of biological interest without any requirements of highly efficient protein expression systems.     

High resolution NMR spectroscopy of nanocrystalline proteins at ultra-high magnetic field

Magic-angle spinning (MAS) solid-state NMR (SSNMR) spectroscopy of uniformly-¹³C,¹⁵N labeled protein samples provides insight into atomic-resolution chemistry and structure. Data collection efficiency has advanced remarkably in the last decade; however, the study of larger proteins is still challenged by relatively low resolution in comparison to solution NMR. In this study, we present a systematic analysis of SSNMR protein spectra acquired at 11.7, 17.6 and 21.1 Tesla (¹H frequencies of 500, 750, and 900 MHz). For two protein systems—GB1, a 6 kDa nanocrystalline protein and DsbA, a 21 kDa nanocrystalline protein—line narrowing is demonstrated in all spectral regions with increasing field. Resolution enhancement is greatest in the aliphatic region, including methine, methylene and methyl sites. The resolution for GB1 increases markedly as a function of field, and for DsbA, resolution in the C–C region increases by 42%, according to the number of peaks that can be uniquely picked and integrated in the 900 MHz spectra when compared to the 500 MHz spectra. Additionally, chemical exchange is uniquely observed in the highest field spectra for at least two isoleucine Cδ1 sites in DsbA. These results further illustrate the benefits of high-field MAS SSNMR spectroscopy for protein structural studies.     

Magic-angle spinning solid-state NMR of a 144 kDa membrane protein complex: E. coli cytochrome bo3 oxidase

Recent progress in magic-angle spinning (MAS) solid-state NMR (SSNMR) has enabled multidimensional studies of large, macroscopically unoriented membrane proteins with associated lipids, without the requirement of solubility that limits other structural techniques. Here we present initial sample preparation and SSNMR studies of a 144 kDa integral membrane protein, E. coli cytochrome bo₃ oxidase. The optimized protocol for expression and purification yields ∼5 mg of the enzymatically active, uniformly ¹³C,¹⁵N-enriched membrane protein complex from each liter of growth medium. The preparation retains endogenous lipids and yields spectra of high sensitivity and resolution, consistent with a folded, homogenous protein. Line widths of isolated signals are less than 0.5 ppm, with a large number of individual resonances resolved in the 2D and 3D spectra. The ¹³C chemical shifts, assigned by amino acid type, are consistent with the secondary structure previously observed by diffraction methods. Although the structure is predominantly helical, the percentage of non-helical signals varies among residue types; these percentages agree well between the NMR and diffraction data. Samples show minimal evidence of degradation after several weeks of NMR data acquisition. Use of a triple resonance scroll resonator probe further improves sample stability and enables higher power decoupling, higher duty cycles and more advanced 3D experiments to be performed. These initial results in cytochrome bo₃ oxidase demonstrate that multidimensional MAS SSNMR techniques have sufficient sensitivity and resolution to interrogate selected parts of a very large uniformly ¹³C,¹⁵N-labeled membrane protein. Heather L. Frericks and Lai Lai Yap contributed equally to this work.     

Investigating Gabapentin Polymorphism Using Solid-State NMR Spectroscopy

Solid-state NMR spectroscopy (SSNMR), coupled with powder X-ray diffraction (PXRD), was used to identify the physical forms of gabapentin in samples prepared by recrystallization, spray drying, dehydration, and milling. Four different crystalline forms of gabapentin were observed: form I, a monohydrate, form II, the most stable at ambient conditions, form III, produced by either recrystallization or milling, and an isomorphous desolvate produced from desolvating the monohydrate. As-received gabapentin (form II) was ball-milled for 45 min in both the presence and absence of hydroxypropylcellulose (HPC). The samples were then stored for 2 days at 50°C under 0% relative humidity and analyzed by ¹³C SSNMR and PXRD. High-performance liquid chromatography was run on the samples to determine the amount of degradation product formed before and after storage. The ¹H T ₁ values measured for the sample varied from 130 s for the as-received unstressed material without HPC to 11 s for the material that had been ball-milled in the presence of HPC. Samples with longer ¹H T ₁ values were substantially more stable than samples that had shorter T ₁ values. Samples milled with HPC had detectable form III crystals as well. These results suggest that SSNMR can be used to predict gabapentin stability in formulated products.     

Solid-state NMR ensemble dynamics as a mediator between experiment and simulation

Solid-state NMR (SSNMR) is a powerful technique to describe the orientations of membrane proteins and peptides in their native membrane bilayer environments. The deuterium (²H) quadrupolar splitting (DQS), one of the SSNMR observables, has been used to characterize the orientations of various single-pass transmembrane (TM) helices using a semistatic rigid-body model such as the geometric analysis of labeled alanine (GALA) method. However, dynamic information of these TM helices, which could be related to important biological function, can be missing or misinterpreted with the semistatic model. We have investigated the orientation of WALP23 in an implicit membrane of dimyristoylglycerophosphocholine by determining an ensemble of structures using multiple conformer models with a DQS restraint potential. When a single conformer is used, the resulting helix orientation (tilt angle (τ) of 5.6 ± 3.2° and rotation angle (ρ) of 141.8 ± 40.6°) is similar to that determined by the GALA method. However, as the number of conformers is increased, the tilt angles of WALP23 ensemble structures become larger (26.9 ± 6.7°), which agrees well with previous molecular dynamics simulation results. In addition, the ensemble structure distribution shows excellent agreement with the two-dimensional free energy surface as a function of WALP23's τ and ρ. These results demonstrate that SSNMR ensemble dynamics provides a means to extract orientational and dynamic information of TM helices from their SSNMR observables and to explain the discrepancy between molecular dynamics simulation and GALA-based interpretation of DQS data.     

The interactions of the N-terminal fusogenic peptide of HIV-1 gp41 with neutral phospholipids

We have studied the interactions with neutral phospholipid bilayers of FPI, the 23-residue fusogenic N-terminal peptide of the HIV-1_LAI transmembrane glycoprotein gp41, by CD, EPR, NMR, and solid state NMR (SSNMR) with the objective of understanding how it lyses and fuses cells. Using small unilamellar vesicles made from egg yolk phoshatidylcholine which were not fused or permeabilised by the peptide we obtained results suggesting that it was capable of inserting as an α-helix into neutral phospholipid bilayers but was only completely monomeric at peptide/lipid (P/L) ratios of 1/2000 or lower. Above this value, mixed populations of monomeric and multimeric forms were found with the proportion of multimer increasing proportionally to P/L, as calculated from studies on the interaction between the peptide and spin-labelled phospholipid. The CD data indicated that, at P/L between 1/200 and 1/100, approximately 68% of the peptide appeared to be in α-helical form. When P/L=1/25 the α-helical content had decreased to 41%. Measurement at a P/L of 1/100 of the spin lattice relaxation effect on the ¹³C nuclei of the phospholipid acyl chains of an N-terminal spin label attached to the peptide showed that most of the peptide N-termini were located in the interior hydrocarbon region of the membrane. SSNMR on multilayers of ditetradecylphosphatidyl choline at P/Ls of 1/10, 1/20 and 1/30 showed that the peptide formed multimers that affected the motion of the lipid chains and disrupted the lipid alignment. We suggest that these aggregates may be relevant to the membrane-fusing and lytic activities of FPI and that they are worthy of further study. Received: 8 June 1998 / Revised version: 18 November 1998 / Accepted: 28 December 1998     

Solid state NMR: new tools for insight into enzyme function

NMR has had considerable impact in enzymology, probing evidence for ionization states, conformational 'strain', compressed interactions, electronically unusual species, and conformational dynamics of enzymes. Solid-state NMR is becoming increasingly important in studying enzymes because of a number of recent tools for analysis of proteins by SSNMR, and because of the growing ability to isolate the species of interest for analysis. Here, we review recent studies of a Michaelis complex, of the dynamic functioning of membrane-associated enzymes, and initial studies of several enzymes with redox-active and paramagnetic centers.     

Magic-angle spinning solid-state NMR spectroscopy of nanodisc-embedded human CYP3A4

Cytochrome P450 (CYP) 3A4 contributes to the metabolism of approximately 50% of commercial drugs by oxidizing a large number of structurally diverse substrates. Like other endoplasmic reticulum-localized P450s, CYP3A4 contains a membrane-anchoring N-terminal helix and a significant number of hydrophobic domains, important for the interaction between CYP3A4 and the membrane. Although the membrane affects specificity of CYP3A4 ligand binding, the structural details of the interaction have not been revealed so far because X-ray crystallography studies are available only for the soluble domain of CYP3A4. Here we report sample preparation and initial magic-angle spinning (MAS) solid-state NMR (SSNMR) of CYP3A4 (Delta3-12) embedded in a nanoscale membrane bilayer, or Nanodisc. The growth protocol yields approximately 2.5 mg of the enzymatically active, uniformly 13C,15N-enriched CYP3A4 from 1 L of growth medium. Polyethylene glycol 3350-precipitated CYP3A4 in Nanodiscs yields spectra of high resolution and sensitivity, consistent with a folded, homogeneous protein. CYP3A4 in Nanodiscs remains enzymatically active throughout the precipitation protocol as monitored by bromocriptine binding. The 13C line widths measured from 13C-13C 2D chemical shift correlation spectra are approximately 0.5 ppm. The secondary structure distribution within several amino acid types determined from 13C chemical shifts is consistent with the ligand-free X-ray structures. These results demonstrate that MAS SSNMR can be performed on Nanodisc-embedded membrane proteins in a folded, active state. The combination of SSNMR and Nanodisc methodologies opens up new possibilities for obtaining structural information on CYP3A4 and other integral membrane proteins with full retention of functionality.     

VITAL NMR: using chemical shift derived secondary structure information for a limited set of amino acids to assess homology model accuracy

Homology modeling is a powerful tool for predicting protein structures, whose success depends on obtaining a reasonable alignment between a given structural template and the protein sequence being analyzed. In order to leverage greater predictive power for proteins with few structural templates, we have developed a method to rank homology models based upon their compliance to secondary structure derived from experimental solid-state NMR (SSNMR) data. Such data is obtainable in a rapid manner by simple SSNMR experiments (e.g., ¹³C–¹³C 2D correlation spectra). To test our homology model scoring procedure for various amino acid labeling schemes, we generated a library of 7,474 homology models for 22 protein targets culled from the TALOS+/SPARTA+ training set of protein structures. Using subsets of amino acids that are plausibly assigned by SSNMR, we discovered that pairs of the residues Val, Ile, Thr, Ala and Leu (VITAL) emulate an ideal dataset where all residues are site specifically assigned. Scoring the models with a predicted VITAL site-specific dataset and calculating secondary structure with the Chemical Shift Index resulted in a Pearson correlation coefficient (−0.75) commensurate to the control (−0.77), where secondary structure was scored site specifically for all amino acids (ALL 20) using STRIDE. This method promises to accelerate structure procurement by SSNMR for proteins with unknown folds through guiding the selection of remotely homologous protein templates and assessing model quality.     

Nucleotide-type chemical shift assignment of the encapsulated 40 kbp dsDNA in intact bacteriophage T7 by MAS solid-state NMR

The icosahedral bacteriophage T7 is a 50 MDa double-stranded DNA (dsDNA) virus that infects Escherichia coli. Although there is substantial information on the physical and morphological properties of T7, structural information, based mostly on Raman spectroscopy and cryo-electron microscopy, is limited. Here, we apply the magic-angle spinning (MAS) solid-state NMR (SSNMR) technique to study a uniformly ¹³C and ¹⁵N labeled wild-type T7 phage. We describe the details of the large-scale preparation and purification of an isotopically enriched phage sample under fully hydrated conditions, and show a complete ¹³C and a near-complete ¹⁵N nucleotide-type specific assignment of the sugar and base moieties in the 40 kbp dsDNA of T7 using two-dimensional ¹³C–¹³C and ¹⁵N–¹³C correlation experiments. The chemical shifts are interpreted as reporters of a B-form conformation of the encapsulated dsDNA. While MAS SSNMR was found to be extremely useful in determining the structures of proteins in native-like environments, its application to nucleic acids has lagged behind, leaving a missing ¹³C and ¹⁵N chemical shift database. This work therefore expands the ¹³C and ¹⁵N database of real B-form DNA systems, and opens routes to characterize more complex nucleic acid systems by SSNMR.