920 MHz ultra-high field NMR approaches to structural glycobiology

Although NMR spectroscopy has great potential to provide us with detailed structural information on oligosaccharides and glycoconjugates, the carbohydrate NMR analyses have been hampered by the severe spectral overlapping and the insufficiency of the conformational restraints. Recently, ultra-high field NMR spectrometers have become available for applications to structural analyses of biological macromolecules. Here we demonstrate that ultra-high fields offer not only increases in sensitivity and chemical shift dispersion but also potential benefits for providing unique information on chemical exchange and relaxation, by displaying NMR spectral data of oligosaccharide, glycoprotein, and glycolipid systems recorded at a 21.6 T magnetic field (corresponding to 920 MHz (1)H observation frequency). The ultra-high field NMR spectroscopy combined with sugar library and stable-isotope labeling approaches will open new horizons in structural glycobiology.     

Ultra-high field NMR studies of antibody binding and site-specific phosphorylation of alpha-synuclein

Although biological importance of intrinsically disordered proteins is becoming recognized, NMR analyses of this class of proteins remain as tasks with more challenge because of poor chemical shift dispersion. It is expected that ultra-high field NMR spectroscopy offers improved resolution to cope with this difficulty. Here, we report an ultra-high field NMR study of alpha-synuclein, an intrinsically disordered protein identified as the major component of the Lewy bodies. Based on NMR spectral data collected at a 920 MHz proton frequency, we performed epitope mapping of an anti-alpha-synuclein monoclonal antibody, and furthermore, characterized conformational effects of phosphorylation at Ser129 of alpha-synuclein.     

Analysis of the amide <Superscript>15</Superscript>N chemical shift tensor of the C<Subscript>α</Subscript> tetrasubstituted constituent of membrane-active peptaibols,the α-aminoisobutyric acid residue,compared to those of di- and tri-substituted proteinogenic amino acid residues

In protein NMR spectroscopy the chemical shift provides important information for the assignment of residues and a first structural evaluation of dihedral angles. Furthermore, angular restraints are obtained from oriented samples by solution and solid-state NMR spectroscopic approaches. Whereas the anisotropy of chemical shifts, quadrupolar couplings and dipolar interactions have been used to determine the structure, dynamics and topology of oriented membrane polypeptides using solid-state NMR spectroscopy similar concepts have been introduced to solution NMR through the measurements of residual dipolar couplings. The analysis of ¹⁵N chemical shift spectra depends on the accuracy of the chemical shift tensors. When investigating alamethicin and other peptaibols, i.e. polypeptides rich in α-aminoisobutyric acid (Aib), the ¹⁵N chemical shift tensor of this C_α-tetrasubstituted amino acid exhibits pronounced differences when compared to glycine, alanine and other proteinogenic residues. Here we present an experimental investigation on the ¹⁵N amide Aib tensor of N-acetyl-Aib-OH and for the Aib residues within peptaibols. Furthermore, a statistical analysis of the tensors published for di- (glycine) and tri-substituted residues has been performed, where for the first time the published data sets are compiled using a common reference. The size of the isotropic chemical shift and main tensor elements follows the order di- < tri- < tetra-substituted amino acids. A ¹⁵N chemical shift-¹H-¹⁵N dipolar coupling correlation NMR spectrum of alamethicin is used to evaluate the consequences of variations in the main tensor elements for the structural analysis of this membrane peptide.     

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.     

NMR studies of aurein 1.2 analogs

Aurein 1.2 is an antimicrobial and anticancer peptide isolated from an Australian frog. To improve our understanding of the mechanism of action, two series of peptides were designed. The first series includes the N-terminal membrane anchor of bacterial glucose-specific enzyme IIA, aurein 1.2, and a newly identified aurein 1.2 analog from human LL-37 (LLAA). The order of antibacterial activity is LLAA > aurein 1.2 >> the membrane anchor (inactive). The structure of LLAA in detergent micelles was determined by ¹H NMR spectroscopy, including structural refinement by natural abundance ¹³C^α, ¹³C^β, and ¹⁵N chemical shifts. The hydrophobic surface area of the 3D structure is related to the retention time of the peptide on a reverse-phase HPLC column. The higher activity of LLAA compared to aurein 1.2 was attributed to additional cationic residues that enhance the membrane perturbation potential. The second peptide series was created by changing the C-terminal phenylalanine (F13) of aurein 1.2 to either phenylglycine or tryptophan. A closer or further location of the aromatic rings to the peptide backbone in the mutants relative to F13 is proposed to cause a drop in activity. Phenylglycine with unique chemical shifts may be a useful NMR probe for structure-activity relationship studies of antimicrobial peptides. To facilitate potential future use for NMR studies, random-coil chemical shifts for phenylglycine (X) were measured using the synthetic peptide GGXGG. Aromatic rings of phenylalanines in all the peptides penetrated 2-5 Å below the lipid head group and are essential for membrane targeting as illustrated by intermolecular peptide-lipid NOE patterns.     

Using P MAS NMR to monitor a gel phase thermal disorder transition in sphingomyelin/cholesterol bilayers

The impact of low cholesterol concentrations on an egg sphingomyelin bilayer is investigated using ³¹P magic angle spinning (MAS) NMR spectroscopy. The magnitude of the isotropic ³¹P MAS NMR line width is used to monitor the main gel to liquid crystalline phase transition, along with a unique gel phase pretransition. In addition, the ³¹P chemical shift anisotropy (CSA) and spin-spin relaxation times (T₂), along with the effects of spinning speed, proton decoupling and magnetic field strength, are reported. The variation of this unique gel phase thermal pretransition with the inclusion of 5 through 21 mol% cholesterol is presented and discussed.     

CSSI-PRO: a method for secondary structure type editing,assignment and estimation in proteins using linear combination of backbone chemical shifts

Estimation of secondary structure in polypeptides is important for studying their structure, folding and dynamics. In NMR spectroscopy, such information is generally obtained after sequence specific resonance assignments are completed. We present here a new methodology for assignment of secondary structure type to spin systems in proteins directly from NMR spectra, without prior knowledge of resonance assignments. The methodology, named Combination of Shifts for Secondary Structure Identification in Proteins (CSSI-PRO), involves detection of specific linear combination of backbone ¹H^α and ¹³C′ chemical shifts in a two-dimensional (2D) NMR experiment based on G-matrix Fourier transform (GFT) NMR spectroscopy. Such linear combinations of shifts facilitate editing of residues belonging to α-helical/β-strand regions into distinct spectral regions nearly independent of the amino acid type, thereby allowing the estimation of overall secondary structure content of the protein. Comparison of the predicted secondary structure content with those estimated based on their respective 3D structures and/or the method of Chemical Shift Index for 237 proteins gives a correlation of more than 90% and an overall rmsd of 7.0%, which is comparable to other biophysical techniques used for structural characterization of proteins. Taken together, this methodology has a wide range of applications in NMR spectroscopy such as rapid protein structure determination, monitoring conformational changes in protein-folding/ligand-binding studies and automated resonance assignment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Carboxyl pKa Values and Acid Denaturation of BBL

The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK_a values of all the carboxylic residues in this pH range. We employed ¹³C direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pK_a values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pK_a values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pK_a, and, using thermodynamic cycles, we could calculate their pK_as in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured ¹³C^α chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state.     

NMR crystallography: The effect of deuteration on high resolution C solid state NMR spectra of a 7-TM protein

The effect of deuteration on the ¹³C linewidths of U-¹³C, ¹⁵N 2D crystalline bacteriorhodopsin (bR) from Halobacterium salinarium, a 248-amino acid protein with seven-transmembrane (7TM) spanning regions, has been studied in purple membranes as a prelude to potential structural studies. Spectral doubling of resonances was observed for receptor expressed in ²H medium (for both 50:50% 1H:2H, and a more highly deuterated form) with the resonances being of similar intensities and separated by < 0.3 ppm in the methyl spectral regions in which they were readily distinguished. Line-widths of the methyl side chains were not significantly altered when the protein was expressed in highly deuterated medium compared to growth in fully protonated medium (spectral line widths were about 0.5 ppm on average for receptor expressed both in the fully protonated and highly deuterated media from the Cδ, Cγ₁, and Cγ₂ Ile ¹³C signals observed in the direct, 21-39 ppm, and indirect, 9-17 ppm, dimensions). The measured ¹³C NMR line-widths observed for both protonated and deuterated form of the receptor are sufficiently narrow, indicating that this crystalline protein morphology is suitable for structural studies. ¹H decoupling comparison of the protonated and deuterated bR imply that deuteration may be advantageous for samples in which low power ¹H decoupling is required.     

Assignment Strategies for Large Proteins by Magic-Angle Spinning NMR: The 21-kDa Disulfide-Bond-Forming Enzyme DsbA

We present strategies for chemical shift assignments of large proteins by magic-angle spinning solid-state NMR, using the 21-kDa disulfide-bond-forming enzyme DsbA as prototype. Previous studies have demonstrated that complete de novo assignments are possible for proteins up to  ∼ 17 kDa, and partial assignments have been performed for several larger proteins. Here we show that combinations of isotopic labeling strategies, high field correlation spectroscopy, and three-dimensional (3D) and four-dimensional (4D) backbone correlation experiments yield highly confident assignments for more than 90% of backbone resonances in DsbA. Samples were prepared as nanocrystalline precipitates by a dialysis procedure, resulting in heterogeneous linewidths below 0.2 ppm. Thus, high magnetic fields, selective decoupling pulse sequences, and sparse isotopic labeling all improved spectral resolution. Assignments by amino acid type were facilitated by particular combinations of pulse sequences and isotopic labeling; for example, transferred echo double resonance experiments enhanced sensitivity for Pro and Gly residues; [2-¹³C]glycerol labeling clarified Val, Ile, and Leu assignments; in-phase anti-phase correlation spectra enabled interpretation of otherwise crowded Glx/Asx side-chain regions; and 3D NCACX experiments on [2-¹³C]glycerol samples provided unique sets of aromatic (Phe, Tyr, and Trp) correlations. Together with high-sensitivity CANCOCA 4D experiments and CANCOCX 3D experiments, unambiguous backbone walks could be performed throughout the majority of the sequence. At 189 residues, DsbA represents the largest monomeric unit for which essentially complete solid-state NMR assignments have so far been achieved. These results will facilitate studies of nanocrystalline DsbA structure and dynamics and will enable analysis of its 41-kDa covalent complex with the membrane protein DsbB, for which we demonstrate a high-resolution two-dimensional ¹³C-¹³C spectrum.     

Chemical shift assignment and structural plasticity of a HIV fusion peptide derivative in dodecylphosphocholine micelles

A “HFPK3” peptide containing the 23 residues of the human immunodeficiency virus (HIV) fusion peptide (HFP) plus three non-native C-terminal lysines was studied in dodecylphosphocholine (DPC) micelles with 2D ¹H NMR spectroscopy. The HFP is at the N-terminus of the gp41 fusion protein and plays an important role in fusing viral and target cell membranes which is a critical step in viral infection. Unlike HFP, HFPK3 is monomeric in detergent-free buffered aqueous solution which may be a useful property for functional and structural studies. H^α chemical shifts indicated that DPC-associated HFPK3 was predominantly helical from I4 to L12. In addition to the highest-intensity crosspeaks used for the first chemical shift assignment (denoted I), there were additional crosspeaks whose intensities were ∼ 10% of those used for assignment I. A second assignment (II) for residues G5 to L12 as well as a few other residues was derived from these lower-intensity crosspeaks. Relative to the I shifts, the II shifts were different by 0.01-0.23 ppm with the largest differences observed for H^N. Comparison of the shifts of DPC-associated HFPK3 with those of detergent-associated HFP and HFP derivatives provided information about peptide structures and locations in micelles.     

High-Resolution Two-Dimensional J-Resolved NMR Spectroscopy for Biological Systems

NMR spectroscopy is a principal tool in metabolomic studies and can, in theory, yield atom-level information critical for understanding biological systems. Nevertheless, NMR investigations on biological tissues generally have to contend with field inhomogeneities originating from variations in macroscopic magnetic susceptibility; these field inhomogeneities broaden spectral lines and thereby obscure metabolite signals. The congestion in one-dimensional NMR spectra of biological tissues often leads to ambiguities in metabolite identification and quantification. We propose an NMR approach based on intermolecular double-quantum coherences to recover high-resolution two-dimensional (2D) J-resolved spectra from inhomogeneous magnetic fields, such as those created by susceptibility variations in intact biological tissues. The proposed method makes it possible to acquire high-resolution 2D J-resolved spectra on intact biological samples without recourse to time-consuming shimming procedures or the use of specialized hardware, such as magic-angle-spinning probes. Separation of chemical shifts and J couplings along two distinct dimensions is achieved, which reduces spectral crowding and increases metabolite specificity. Moreover, the apparent J coupling constants observed are magnified by a factor of 3, facilitating the accurate measurement of small J couplings, which is useful in metabolic analyses. Dramatically improved spectral resolution is demonstrated in our applications of the technique on pig brain tissues. The resulting spectra contain a wealth of chemical shift and J-coupling information that is invaluable for metabolite analyses. A spatially localized experiment applied on an intact fish (Crossocheilus siamensis) reveals the promise of the proposed method in in vivo metabolite studies. Moreover, the proposed method makes few demands on spectrometer hardware and therefore constitutes a convenient and effective manner for metabonomics study of biological systems.     

Structure, dynamics and mapping of membrane-binding residues of micelle-bound antimicrobial peptides by natural abundance C NMR spectroscopy

Worldwide bacterial resistance to traditional antibiotics has drawn much research attention to naturally occurring antimicrobial peptides (AMPs) owing to their potential as alternative antimicrobials. Structural studies of AMPs are essential for an in-depth understanding of their activity, mechanism of action, and in guiding peptide design. Two-dimensional solution proton NMR spectroscopy has been the major tool. In this article, we describe the applications of natural abundance ¹³C NMR spectroscopy that provides complementary information to 2D ¹H NMR. The correlation of ¹³Cα secondary shifts with both 3D structure and heteronuclear ¹⁵N NOE values indicates that natural abundance carbon chemical shifts are useful probes for backbone structure and dynamics of membrane peptides. Using human LL-37-derived peptides (GF-17, KR-12, and RI-10), as well as amphibian antimicrobial and anticancer peptide aurein 1.2 and its analog LLAA, as models, we show that the cross peak intensity plots of 2D ¹H-¹³Cα HSQC spectra versus residue number present a wave-like pattern (HSQC wave) where key hydrophobic residues of micelle-bound peptides are located in the troughs with weaker intensities, probably due to fast exchange between the free and bound forms. In all the cases, the identification of aromatic phenylalanines as a key membrane-binding residue is consistent with previous intermolecular Phe-lipid NOE observations. Furthermore, mutation of one of the key hydrophobic residues of KR-12 to Ala significantly reduced the antibacterial activity of the peptide mutants. These results illustrate that natural abundance heteronuclear-correlated NMR spectroscopy can be utilized to probe backbone structure and dynamics, and perhaps to map key membrane-binding residues of peptides in complex with micelles. ¹H-¹³Cα HSQC wave, along with other NMR waves such as dipolar wave and chemical shift wave, offers novel insights into peptide-membrane interactions from different angles.     

Structural heterogeneity in microcrystalline ubiquitin studied by solid-state NMR

By applying [1-¹³C]- and [2-¹³C]-glucose labeling schemes to the folded globular protein ubiquitin, a strong reduction of spectral crowding and increase in resolution in solid-state NMR (ssNMR) spectra could be achieved. This allowed spectral resonance assignment in a straightforward manner and the collection of a wealth of long-range distance information. A high precision solid-state NMR structure of microcrystalline ubiquitin was calculated with a backbone rmsd of 1.57 to the X-ray structure and 1.32 Å to the solution NMR structure. Interestingly, we can resolve structural heterogeneity as the presence of three slightly different conformations. Structural heterogeneity is most significant for the loop region β1-β2 but also for β-strands β1, β2, β3, and β5 as well as for the loop connecting α1 and β3. This structural polymorphism observed in the solid-state NMR spectra coincides with regions that showed dynamics in solution NMR experiments on different timescales.     

Solution structure of the c-terminal dimerization domain of SARS coronavirus nucleocapsid protein solved by the SAIL-NMR method

The C-terminal domain (CTD) of the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP) contains a potential RNA-binding region in its N-terminal portion and also serves as a dimerization domain by forming a homodimer with a molecular mass of 28 kDa. So far, the structure determination of the SARS-CoV NP CTD in solution has been impeded by the poor quality of NMR spectra, especially for aromatic resonances. We have recently developed the stereo-array isotope labeling (SAIL) method to overcome the size problem of NMR structure determination by utilizing a protein exclusively composed of stereo- and regio-specifically isotope-labeled amino acids. Here, we employed the SAIL method to determine the high-quality solution structure of the SARS-CoV NP CTD by NMR. The SAIL protein yielded less crowded and better resolved spectra than uniform ¹³C and ¹⁵N labeling, and enabled the homodimeric solution structure of this protein to be determined. The NMR structure is almost identical with the previously solved crystal structure, except for a disordered putative RNA-binding domain at the N-terminus. Studies of the chemical shift perturbations caused by the binding of single-stranded DNA and mutational analyses have identified the disordered region at the N-termini as the prime site for nucleic acid binding. In addition, residues in the β-sheet region also showed significant perturbations. Mapping of the locations of these residues onto the helical model observed in the crystal revealed that these two regions are parts of the interior lining of the positively charged helical groove, supporting the hypothesis that the helical oligomer may form in solution.     

Differences in lysine pKa values may be used to improve NMR signal dispersion in reductively methylated proteins

Reductive methylation of lysine residues in proteins offers a way to introduce ¹³C methyl groups into otherwise unlabeled molecules. The ¹³C methyl groups on lysines possess favorable relaxation properties that allow highly sensitive NMR signal detection. One of the major limitations in the use of reductive methylation in NMR is the signal overlap of ¹³C methyl groups in NMR spectra. Here we show that the uniform influence of the solvent on chemical shifts of exposed lysine methyl groups could be overcome by adjusting the pH of the buffering solution closer to the pKa of lysine side chains. Under these conditions, due to variable pKa values of individual lysine side chains in the protein of interest different levels of lysine protonation are observed. These differences are reflected in the chemical shift differences of methyl groups in reductively methylated lysines. We show that this approach is successful in four different proteins including Ca²⁺-bound Calmodulin, Lysozyme, Ca²⁺-bound Troponin C, and Glutathione S-Transferase. In all cases significant improvement in NMR spectral resolution of methyl signals in reductively methylated proteins was obtained. The increased spectral resolution helps with more precise characterization of protein structural rearrangements caused by ligand binding as shown by studying binding of Calmodulin antagonist trifluoperazine to Calmodulin. Thus, this approach may be used to increase resolution in NMR spectra of ¹³C methyl groups on lysine residues in reductively methylated proteins that enhances the accuracy of protein structural assessment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High-Resolution Two-Dimensional J-Resolved NMR Spectroscopy for Biological Systems

NMR spectroscopy is a principal tool in metabolomic studies and can, in theory, yield atom-level information critical for understanding biological systems. Nevertheless, NMR investigations on biological tissues generally have to contend with field inhomogeneities originating from variations in macroscopic magnetic susceptibility; these field inhomogeneities broaden spectral lines and thereby obscure metabolite signals. The congestion in one-dimensional NMR spectra of biological tissues often leads to ambiguities in metabolite identification and quantification. We propose an NMR approach based on intermolecular double-quantum coherences to recover high-resolution two-dimensional (2D) J-resolved spectra from inhomogeneous magnetic fields, such as those created by susceptibility variations in intact biological tissues. The proposed method makes it possible to acquire high-resolution 2D J-resolved spectra on intact biological samples without recourse to time-consuming shimming procedures or the use of specialized hardware, such as magic-angle-spinning probes. Separation of chemical shifts and J couplings along two distinct dimensions is achieved, which reduces spectral crowding and increases metabolite specificity. Moreover, the apparent J coupling constants observed are magnified by a factor of 3, facilitating the accurate measurement of small J couplings, which is useful in metabolic analyses. Dramatically improved spectral resolution is demonstrated in our applications of the technique on pig brain tissues. The resulting spectra contain a wealth of chemical shift and J-coupling information that is invaluable for metabolite analyses. A spatially localized experiment applied on an intact fish (Crossocheilus siamensis) reveals the promise of the proposed method in in vivo metabolite studies. Moreover, the proposed method makes few demands on spectrometer hardware and therefore constitutes a convenient and effective manner for metabonomics study of biological systems.     

αB-Crystallin: A Hybrid Solid-State/Solution-State NMR Investigation Reveals Structural Aspects of the Heterogeneous Oligomer

Atomic-level structural information on αB-Crystallin (αB), a prominent member of the small heat-shock protein family, has been a challenge to obtain due its polydisperse oligomeric nature. We show that magic-angle spinning solid-state NMR can be used to obtain high-resolution information on an ∼ 580-kDa human αB assembled from 175-residue 20-kDa subunits. An ∼ 100-residue α-crystallin domain is common to all small heat-shock proteins, and solution-state NMR was performed on two different α-crystallin domain constructs isolated from αB. In vitro, the chaperone-like activities of full-length αB and the isolated α-crystallin domain are identical. Chemical shifts of the backbone and C^β resonances have been obtained for residues 64-162 (α-crystallin domain plus part of the C-terminus) in αB and the isolated α-crystallin domain by solid-state and solution-state NMR, respectively. Both sets of data strongly predict six β-strands in the α-crystallin domain. A majority of residues in the α-crystallin domain have similar chemical shifts in both solid-state and solution-state, indicating similar structures for the domain in its isolated and oligomeric forms. Sites of intersubunit interaction are identified from chemical shift differences that cluster to specific regions of the α-crystallin domain. Multiple signals are observed for the resonances of M68 in the oligomer, identifying the region containing this residue as existing in heterogeneous environments within αB. Evidence for a novel dimerization motif in the human α-crystallin domain is obtained by a comparison of (i) solid-state and solution-state chemical shift data and (ii) ¹H-¹⁵N heteronuclear single quantum coherence spectra as a function of pH. The isolated α-crystallin domain undergoes a dimer-monomer transition over the pH range 7.5-6.8. This steep pH-dependent switch may be important for αB to function optimally (e.g., to preserve the filament integrity of cardiac muscle proteins such as actin and desmin during cardiac ischemia, which is accompanied by acidosis).     

Structure-based prediction of methyl chemical shifts in proteins

Protein methyl groups have recently been the subject of much attention in NMR spectroscopy because of the opportunities that they provide to obtain information about the structure and dynamics of proteins and protein complexes. With the advent of selective labeling schemes, methyl groups are particularly interesting in the context of chemical shift based protein structure determination, an approach that to date has exploited primarily the mapping between protein structures and backbone chemical shifts. In order to extend the scope of chemical shifts for structure determination, we present here the CH3Shift method of performing structure-based predictions of methyl chemical shifts. The terms considered in the predictions take account of ring current, magnetic anisotropy, electric field, rotameric type, and dihedral angle effects, which are considered in conjunction with polynomial functions of interatomic distances. We show that the CH3Shift method achieves an accuracy in the predictions that ranges from 0.133 to 0.198 ppm for ¹H chemical shifts for Ala, Thr, Val, Leu and Ile methyl groups. We illustrate the use of the method by assessing the accuracy of side-chain structures in structural ensembles representing the dynamics of proteins.