The Dynamics of Lysozyme from Bacteriophage Lambda in Solution Probed by NMR and MD Simulations

¹⁵N NMR relaxation studies, analyses of NMR data to include chemical shifts, residual dipolar couplings (RDC), NOEs and H^N–H^α coupling constants, and molecular dynamics (MD) simulations have been used to characterise the behaviour of lysozyme from bacteriophage lambda (λ lysozyme) in solution. The lower and upper lip regions in λ lysozyme (residues 51–60 and 128–141, respectively) show reduced ¹H–¹⁵N order parameters indicating mobility on a picosecond timescale. In addition, residues in the lower and upper lips also show exchange contributions to T₂ indicative of slower timescale motions. The chemical shift, RDC, coupling constant and NOE data for λ lysozyme indicate that two fluctuating β‐strands (β3 and β4) are populated in the lower lip region while the N terminus of helix α6 (residues 136–139) forms dynamic helical turns in the upper lip region. This behaviour is confirmed by MD simulations that show hydrogen bonds, indicative of the β‐sheet and helical secondary structure in the lip regions, with populations of 40–60 %. Thus in solution λ lysozyme adopts a conformational ensemble that will contain both the open and closed forms observed in the crystal structures of the protein.     

Substrate Binding Drives Active‐Site Closing of Human Blood Group B Galactosyltransferase as Revealed by Hot‐Spot Labeling and NMR Spectroscopy Experiments

Crystallography has shown that human blood group A (GTA) and B (GTB) glycosyltransferases undergo transitions between “open”, “semiclosed”, and “closed” conformations upon substrate binding. However, the timescales of the corresponding conformational reorientations are unknown. Crystal structures show that the Trp and Met residues are located at “conformational hot spots” of the enzymes. Therefore, we utilized ¹⁵N side‐chain labeling of Trp residues and ¹³C‐methyl labeling of Met residues to study substrate‐induced conformational transitions of GTB. Chemical‐shift perturbations (CSPs) of Met and Trp residues in direct contact with substrate ligands reflect binding kinetics, whereas the CSPs of Met and Trp residues at remote sites reflect conformational changes of the enzyme upon substrate binding. Acceptor binding is fast on the chemical‐shift timescale with rather small CSPs in the range of less than approximately 20 Hz. Donor binding matches the intermediate exchange regime to yield an estimate for exchange rate constants of approximately 200–300 Hz. Donor or acceptor binding to GTB saturated with acceptor or donor substrate, respectively, is slow (<10 Hz), as are coupled protein motions, reflecting mutual allosteric control of donor and acceptor binding. Remote CSPs suggest that substrate binding drives the enzyme into the closed state required for catalysis. These findings should contribute to better understanding of the mechanism of glycosyl transfer of GTA and GTB.     

Fluorine-protein interactions and ¹⁹F NMR isotropic chemical shifts: An empirical correlation with implications for drug design

An empirical correlation between the fluorine isotropic chemical shifts, measured by ¹⁹F NMR spectroscopy, and the type of fluorine–protein interactions observed in crystal structures is presented. The CF, CF₂, and CF₃ groups present in fluorinated ligands found in the Protein Data Bank were classified according to their ¹⁹F NMR chemical shifts and their close intermolecular contacts with the protein atoms. Shielded fluorine atoms, i.e., those with increased electron density, are observed primarily in close contact to hydrogen bond donors within the protein structure, suggesting the possibility of intermolecular hydrogen bond formation. Deshielded fluorines are predominantly found in close contact with hydrophobic side chains and with the carbon of carbonyl groups of the protein backbone. Correlation between the ¹⁹F NMR chemical shift and hydrogen bond distance, both derived experimentally and computed through quantum chemical methods, is also presented. The proposed “rule of shielding” provides some insight into and guidelines for the judicious selection of appropriate fluorinated moieties to be inserted into a molecule for making the most favorable interactions with the receptor.     

pH-Dependent Protonation of Surface Carboxylate Groups in PsbO Enables Local Buffering and Triggers Structural Changes

Photosystem II (PSII) catalyzes the splitting of water, releasing protons and dioxygen. Its highly conserved subunit PsbO extends from the oxygen-evolving center (OEC) into the thylakoid lumen and stabilizes the catalytic Mn₄CaO₅ cluster. The high degree of conservation of accessible negatively charged surface residues in PsbO suggests additional functions, as local pH buffer or by affecting the flow of protons. For this discussion, we provide an experimental basis, through the determination of pK_a values of water-accessible aspartate and glutamate side-chain carboxylate groups by means of NMR. Their distribution is strikingly uneven, with high pK_a values around 4.9 clustered on the luminal PsbO side and values below 3.5 on the side facing PSII. pH-dependent changes in backbone chemical shifts in the area of the lumen-exposed loops are observed, indicating conformational changes. In conclusion, we present a site-specific analysis of carboxylate group proton affinities in PsbO, providing a basis for further understanding of proton transport in photosynthesis.     

Leucine Side‐Chain Conformation and Dynamics in Proteins from 13C NMR Chemical Shifts

Look to the left : The carbon nucleus of a substituent in the gauche position about a subtending dihedral angle experiences an NMR chemical shift of about 5 ppm relative to the same chemical group present in the trans position. We demonstrate that this “γ‐gauche effect” can be utilized to determine the conformation and extent of rotameric averaging for leucine amino acid side chains in the protein calbindin D_9k. The success of this approach suggests that rules can be established to define the orientation of other side chains in proteins as well, offering an easy gauge of protein side‐chain flexibility, as well as avenues to advance protein structure determination by using side‐chain chemical shifts.

     

Impact of Hydrostatic Pressure on an Intrinsically Disordered Protein: A High‐Pressure NMR Study of α‐Synuclein

The impact of pressure on the backbone ¹⁵N, ¹H and ¹³C chemical shifts in N‐terminally acetylated α‐synuclein has been evaluated over a pressure range 1–2500 bar. Even while the chemical shifts fall very close to random coil values, as expected for an intrinsically disordered protein, substantial deviations in the pressure dependence of the chemical shifts are seen relative to those in short model peptides. In particular, the nonlinear pressure response of the ¹H^N chemical shifts, which commonly is associated with the presence of low‐lying “excited states”, is much larger in α‐synuclein than in model peptides. The linear pressure response of ¹H^N chemical shift, commonly linked to H‐bond length change, correlates well with those in short model peptides, and is found to be anticorrelated with its temperature dependence. The pressure dependence of ¹³C chemical shifts shows remarkably large variations, even when accounting for residue type, and do not point to a clear shift in population between different regions of the Ramachandran map. However, a nearly universal decrease in ³J_HN–Hα by 0.22±0.05 Hz suggests a slight increase in population of the polyproline II region at 2500 bar. The first six residues of N‐terminally acetylated synuclein show a transient of approximately 15 % population of α‐helix, which slightly diminishes at 2500 bar. The backbone dynamics of the protein is not visibly affected beyond the effect of slight increase in water viscosity at 2500 bar.     

Structural Characterization of Frustrated Lewis Pairs and Their Reaction Products Using Modern Solid-State NMR Spectroscopy Techniques

Frustrated Lewis pair (FLP) chemistry has provided a new strategy for small-molecule binding and/or catalytic activation. The most prominent FLPs are based on intramolecular phosphane borane adducts, the catalytic properties of which can be tailored over wide ranges of reactivity and selectivity. Advanced solid-state NMR spectroscopic techniques, together with DFT calculations, can provide new structural insights in these systems. This review illustrates the utility of ³¹P and ¹¹B NMR chemical shifts, ¹¹B electric field gradient tensors, and ³¹P ¹¹B indirect and direct dipole dipole interactions for characterizing intramolecular borane phosphane FLPs. We demonstrate the potential of this method to 1) quantify the extent of boron phosphorus bonding interactions (and hence, the “degree of frustration”); 2) reveal specific structural details (i.e., boron phosphorus distances and other local geometric aspects) related to their catalytic activities; and (3) characterize products of FLP reactions with regard to molecular structure, stereochemistry, and aggregation properties in terms of internuclear distances, bonding connectivities, and orientational parameters.     

Structural Mechanism of Barriers to Interspecies Seeding Transmissibility of Full-Length Prion Protein Amyloid

A puzzling feature of prion diseases is the cross-species barriers. The detailed molecular mechanisms underlying these interspecies barriers remain poorly understood because of a lack of high-resolution structural information on the scrapie isoform of the prion protein (PrP^Sc). In this study we identified the critical role of the residues 165/167 in the barrier to seeding mouse PrP (mPrP) fibril seeds to human cellular prion protein (PrP^C). Solid-state NMR revealed a C-terminal β-sheet core spanning residues 165–230 and the packing arrangement of mPrP fibrils. Residues 165/167 are located on one end of the fibril core. Molecular dynamics simulations demonstrated that the stabilities of the seeding-induced β-strand structures are significantly impacted by hydrogen bonds involving the side chain of residue 167 and steric resistance involving residue 165. These findings suggest that the α2–β2 loop containing residues 165/167 could be the initial site of seed–template conformational conversion.     

Efficient Screening of Target-Specific Selected Compounds in Mixtures by 19F NMR Binding Assay with Predicted 19F NMR Chemical Shifts

Ligand-based ¹⁹F NMR screening is a highly effective and well-established hit-finding approach. The high sensitivity to protein binding makes it particularly suitable for fragment screening. Different criteria can be considered for generating fluorinated fragment libraries. One common strategy is to assemble a large, diverse, well-designed and characterized fragment library which is screened in mixtures, generated based on experimental ¹⁹F NMR chemical shifts. Here, we introduce a complementary knowledge-based ¹⁹F NMR screening approach, named ¹⁹Focused screening, enabling the efficient screening of putative active molecules selected by computational hit finding methodologies, in mixtures assembled and on-the-fly deconvoluted based on predicted ¹⁹F NMR chemical shifts. In this study, we developed a novel approach, named LEFshift, for ¹⁹F NMR chemical shift prediction using rooted topological fluorine torsion fingerprints in combination with a random forest machine learning method. A demonstration of this approach to a real test case is reported.     

Structural analysis of cellulose triacetate polymorphs by two-dimensional solid-state C-C and H-C correlation NMR spectroscopies

For the elucidation of the crystal structures of the two crystalline allomorphs of cellulose triacetate (CTA), namely CTA I and CTA II, two-dimensional (2D) solid-state through-bond ¹³C-¹³C and ¹H-¹³C correlations NMR techniques applied to the two crystalline allomorphs of CTA. As a result, the ¹³C and ¹H chemical shifts of the glucopyranose ring of CTA I and CTA II were completely assigned by the 2D NMR spectra of these allomorphs. On the 2D ¹³C-¹³C correlation spectrum of CTA II, two sets of the ¹³C-¹³C correlations from C1 to C6 were observed. This indicated that the CP/MAS ¹³C NMR spectrum of CTA II can be characterized by its overlapping of the ¹³C subspectra of two kinds of 2,3,6-triacetyl-anhydroglucopyranose units and that there are two magnetically non-equivalent sites in the unit cell of CTA II. In the case of CTA I, the numbers of respective ¹³C and ¹H shifts of CTA I agreed with the those of the glucopyranose residue in the allomorph, strongly suggesting that the asymmetric unit of CTA I is only one glucose residue. In addition, conformational differences in the exocyclic C5-C6 bonds between CTA I and CTA II were strongly suggested by the notable differences in the ¹H and ¹³C chemical shifts at the C6 sites of these allomorphs.     

Grow to Fit Molecular Dynamics (G2FMD): an ab initio method for protein side-chain assignment and refinement

The rough energy landscapes and tight packing of protein interiors are two of the critical factors that have prevented the wide application of physics-based models in protein side-chain assignment and protein structure prediction in general. Complementing the rotamer-based methods, we propose an ab initio method that utilizes molecular mechanics simulations for protein side-chain assignment and refinement. By reducing the side-chain size, a smooth energy landscape was obtained owing to the increased distances between the side chains. The side chains then gradually grow back during molecular dynamics simulations while adjusting to their surrounding driven by the interaction energies. The method overcomes the barriers due to tight packing that limit conformational sampling of physics-based models. A key feature of this approach is that the resulting structures are free from steric collisions and allow the application of all-atom models in the subsequent refinement. Tests on a small set of proteins showed nearly 100% accuracy on both chi1 and chi2 of buried residues and 94% of them were within 20 degrees from the native conformation, 79% were within 10 degrees and 42% were within 5 degrees . However, the accuracy decreased when exposed side chains were involved. Further improvement and application of the method and the possible reasons that affect the accuracy on the exposed side chains are discussed.     

Prediction of protein side-chain conformations by principal component analysis for fixed main-chain atoms

A method of side-chain prediction without calculating the potential function is introduced. It is based on the assumption that similar side- chain conformations have a similar structural environment around the side chains. The environment information is represented by vectors that were obtained from principle component analysis and represented by the variance of positions of main-chain atoms around side chains. This information was added to the side-chain library (rotamer library) made from X-ray structures. Side-chain conformations were constructed using this side-chain library without using potential functions. An optimal solution was determined by comparing environmental information with the backbone conformation around the side chain to be predicted and native ones in the library. The method was performed for 15 proteins whose structures were known. The result for the root-mean-square deviation between the predicted and X-ray side-chain conformations was approximately 1.5 A (the value for core residues was approximately 1.1 A) and the percentage of predicted chi 1 angles correct within 40 degrees was approximately 65% (75% for the core). The computational time was short (approximately 60 s for the prediction of proteins with 200 amino acid residues). About 70% of the side-chain conformations were constructed by location of the main-chain atoms around the central C beta atom and the average of r.m.s.d. was approximately 1.4 A (for core residues the average was approximately 1.0 A).      

Errors in the Calculation of 27Al Nuclear Magnetic Resonance Chemical Shifts

Computational chemistry is an important tool for signal assignment of ²⁷Al nuclear magnetic resonance spectra in order to elucidate the species of aluminum(III) in aqueous solutions. The accuracy of the popular theoretical models for computing the ²⁷Al chemical shifts was evaluated by comparing the calculated and experimental chemical shifts in more than one hundred aluminum(III) complexes. In order to differentiate the error due to the chemical shielding tensor calculation from that due to the inadequacy of the molecular geometry prediction, single-crystal X-ray diffraction determined structures were used to build the isolated molecule models for calculating the chemical shifts. The results were compared with those obtained using the calculated geometries at the B3LYP/6-31G(d) level. The isotropic chemical shielding constants computed at different levels have strong linear correlations even though the absolute values differ in tens of ppm. The root-mean-square difference between the experimental chemical shifts and the calculated values is approximately 5 ppm for the calculations based on the X-ray structures, but more than 10 ppm for the calculations based on the computed geometries. The result indicates that the popular theoretical models are adequate in calculating the chemical shifts while an accurate molecular geometry is more critical.     

Characterisation of the supramolecular structure of chemically and physically modified regenerated cellulosic fibres by means of high-resolution Carbon-13 solid-state NMR

Carbon-13 high-resolution solid-state NMR techniques have been invaluable in elucidating the structure of regenerated cellulosic materials. Studies of a range of fibres have shown systematic changes in chemical shifts, which can be related to the influences of physical processing or chemical modification. A constrained curve fitting method has been applied, where the C4 spectral envelope is represented as the sum of contributions from polymer in ordered, partially-ordered and disordered environments, associated with differing conformational arrangements of the cellulose hydroxymethyl and glycocidic bonds. The empirical gamma-gauche effect seems likely to provide the best rationalization for the relationship between C4 shifts and conformational order, taking into account the increased range of bond angles in disordered environments. The quantification of proportions of polymer units within different conformational groupings will provide new insights into the development of supramolecular texture. This will allow better appreciation of the relationships between fibre processing and ultimate fibre performance.     

Systematic Approach to Find the Global Minimum of Relaxation Dispersion Data for Protein-Induced B–Z Transition of DNA

Carr–Purcell–Meiboom–Gill (CPMG) relaxation dispersion spectroscopy is commonly used for quantifying conformational changes of protein in μs-to-ms timescale transitions. To elucidate the dynamics and mechanism of protein binding, parameters implementing CPMG relaxation dispersion results must be appropriately determined. Building an analytical model for multi-state transitions is particularly complex. In this study, we developed a new global search algorithm that incorporates a random search approach combined with a field-dependent global parameterization method. The robust inter-dependence of the parameters carrying out the global search for individual residues (GSIR) or the global search for total residues (GSTR) provides information on the global minimum of the conformational transition process of the Zα domain of human ADAR1 (hZα_ADAR1)–DNA complex. The global search results indicated that a α-helical segment of hZα_ADAR1 provided the main contribution to the three-state conformational changes of a hZα_ADAR1—DNA complex with a slow B–Z exchange process. The two global exchange rate constants, k_ex and k_ZB, were found to be 844 and 9.8 s⁻¹, respectively, in agreement with two regimes of residue-dependent chemical shift differences—the “dominant oscillatory regime” and “semi-oscillatory regime”. We anticipate that our global search approach will lead to the development of quantification methods for conformational changes not only in Z-DNA binding protein (ZBP) binding interactions but also in various protein binding processes.     

Structural Study of the Partially Disordered Full‐Length δ Subunit of RNA Polymerase from Bacillus subtilis

The partially disordered δ subunit of RNA polymerase was studied by various NMR techniques. The structure of the well‐folded N‐terminal domain was determined based on inter‐proton distances in NOESY spectra. The obtained structural model was compared to the previously determined structure of a truncated construct (lacking the C‐terminal domain). Only marginal differences were identified, thus indicating that the first structural model was not significantly compromised by the absence of the C‐terminal domain. Various ¹⁵N relaxation experiments were employed to describe the flexibility of both domains. The relaxation data revealed that the C‐terminal domain is more flexible, but its flexibility is not uniform. By using paramagnetic labels, transient contacts of the C‐terminal tail with the N‐terminal domain and with itself were identified. A propensity of the C‐terminal domain to form β‐type structures was obtained by chemical shift analysis. Comparison with the paramagnetic relaxation enhancement indicated a well‐balanced interplay of repulsive and attractive electrostatic interactions governing the conformational behavior of the C‐terminal domain. The results showed that the δ subunit consists of a well‐ordered N‐terminal domain and a flexible C‐terminal domain that exhibits a complex hierarchy of partial ordering.     

MAS NMR study of surface-modified calcined kaolin

Calcined kaolins, modified by a silane coupling agent, were analyzed by magic angle spinning nuclear magnetic resonance (MAS NMR) and compared to unmodified samples. The results show no chemical shift for ²⁹Si, whereas there is a marked change for ²⁷Al. The chemical shifts of 5.44 and 65.69 ppm of ²⁷Al in unmodified samples are respectively moved to 3.8–4.4 and 54.6–59.9 ppm after modification. This is attributed to changes in the chemical environment around the surface Al ions on the calcined kaolin resulting from chemical bonding of the silane coupling agent molecules to the Al. The results demonstrate that MAS NMR is a useful technique for characterizing the surface modification mechanisms of minerals.     

13C n.m.r. relaxation study of poly(aspartic acid)

¹³C n.m.r. relaxation parameters (T₁, line widths and NOE factors) and chemical shifts were measured in dependence on pH for several samples of poly(α-l-Asp) differing in molecular mass and polydispersity, and for samples of poly(α,β-d,l-Asp) differing in molecular mass. Poly(α-l-Asp) dissolved at pH 9 and acidified to pH 6-4 can appear in two different forms, A and B. In n.m.r. spectra these forms differ mainly by the width of the bands of side chain carbons which is larger in form A. Conditions for a reproducible generation of either of the two forms were not found although the effect of molecular mass (3–6 × 10⁴), polydispersity, sample concentration (2.0-0.2 mol l^?1), temperature of dissolution (4°C–37°C), concentration of NaCl (0–4 mol l^?1) or rate of acidification (1 pH unit s^?1 week^?1) and rate of mixing were investigated. The dynamics of poly(α,β-d,l-Asp) is almost unaffected by change of pH. The relaxation parameters of poly(α-l-Asp) at pH 9 and 4 differ more in form A than in form B. Analysis of relaxation data for the methine carbon of poly(α-l-Asp) in form A by means of the isotropic model with a log x² distribution of correlation times yields a correlation time of 1 ns at pH 4. This indicates that the dynamics of the backbone is dominated by rapid segmental motions even at pH 4. The dependence of chemical shifts on pH indicates that the chemical shift values are determined mainly by the ionization of the carboxyl group in the side chain rather than by a conformational transition. The evaluated relaxation parameters suggest, when compared with those of polypeptides with known conformational behaviour, that the two forms of poly(α-l-Asp) differ in conformation (α-helix, β-structure).     

Mesomorphic behavior of polydiethylphosphazenes as described by13C,14N,and2H NMR

The existence of mesomorphism in polydiethylphosphazene was recently established by MAS NMR and X-ray diffraction characterization. In the present work the mechanism of motion of the ethyl side groups in the high-temperature polymorph tabove 45 C) is identified and compared to the arrangement of side groups in the low-temperature polymorph. For this purpose a few NMR active nuclei (¹³C,¹⁴N, and²H) were exploited to define the side-chain motions occurring at transition. Experiments performed at varying temperatures close to the onset of solid transition suggest the presence of jumps between two conformations in the pretransition state. Rotor-synchronized triple-resonance NMR of the high-temperature phase determined the average distances between the carbons and the nitrogens in the polymorphs. The theoretical prediction of the dipolar interaction between the nuclei supports the hypothesis that ethyl groups can undergo a complete rotation about the P CH₂ bond by jumping across a conformational barrier. The mechanism of motion of the ethyl groups must be cooperative and the collapse of the rigid shell around the main chain is described at the transition.