Predicting <Superscript>13</Superscript>C<Superscript>α</Superscript> chemical shifts for validation of protein structures

The ¹³C^α chemical shifts for 16,299 residues from 213 conformations of four proteins (experimentally determined by X-ray crystallography and Nuclear Magnetic Resonance methods) were computed by using a combination of approaches that includes, but is not limited to, the use of density functional theory. Initially, a validation test of this methodology was carried out by a detailed examination of the correlation between computed and observed ¹³C^α chemical shifts of 10,564 (of the 16,299) residues from 139 conformations of the human protein ubiquitin. The results of this validation test on ubiquitin show agreement with conclusions derived from computation of the chemical shifts at the ab initio Hartree–Fock level. Further, application of this methodology to 5,735 residues from 74 conformations of the three remaining proteins that differ in their number of amino acid residues, sequence and three-dimensional structure, together with a new scoring function, namely the conformationally averaged root-mean-square-deviation, enables us to: (a) offer a criterion for an accurate assessment of the quality of NMR-derived protein conformations; (b) examine whether X-ray or NMR-solved structures are better representations of the observed ¹³C^α chemical shifts in solution; (c) provide evidence indicating that the proposed methodology is more accurate than automated predictors for validation of protein structures; (d) shed light as to whether the agreement between computed and observed ¹³C^α chemical shifts is influenced by the identity of an amino acid residue or its location in the sequence; and (e) provide evidence confirming the presence of dynamics for proteins in solution, and hence showing that an ensemble of conformations is a better representation of the structure in solution than any single conformation. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Rapid measurement of <Superscript>3</Superscript>J(H<Superscript>N</Superscript>–H<Superscript>α</Superscript>) and <Superscript>3</Superscript>J(N–H<Superscript>β</Superscript>) coupling constants in polypeptides

We present two NMR experiments, (3,2)D HNHA and (3,2)D HNHB, for rapid and accurate measurement of 3J(H N-H alpha) and 3J(N-H beta) coupling constants in polypeptides based on the principle of G-matrix Fourier transform NMR spectroscopy and quantitative J-correlation. These experiments, which facilitate fast acquisition of three-dimensional data with high spectral/digital resolution and chemical shift dispersion, will provide renewed opportunities to utilize them for sequence specific resonance assignments, estimation/characterization of secondary structure with/without prior knowledge of resonance assignments, stereospecific assignment of prochiral groups and 3D structure determination, refinement and validation. Taken together, these experiments have a wide range of applications from structural genomics projects to studying structure and folding in polypeptides.     

H<Superscript>N</Superscript>, N,C<Superscript>α</Superscript> and C<Superscript>β</Superscript> assignments of the two periplasmic domains of <Emphasis Type="Italic">Neisseria meningitidis</Emphasis> DsbD

DsbD is a disulfide bond reductase present in the inner membrane of many Gamma-Proteobacteria. In the human pathogen Neisseria meningitidis, DsbD is required for viability and represents a potential target for the development of antibiotics. Here we report the chemical shift assignments (H^N, N, C^α and C^β) for the reduced and oxidized forms of the two periplasmic domains of N. meningitidis DsbD, n-NmDsbD and c-NmDsbD. The backbone amide resonances in all four forms were completely assigned, and the secondary structures for the core regions of the proteins were calculated using ¹³C^αβ shifts. The reduced and oxidized forms of each domain have similar secondary shifts suggesting they retain the same fold. We anticipate that these data will provide an important basis for studying the interaction between n-NmDsbD and c-NmDsbD, which is required for electron transfer across the bacterial cytoplasmic membrane.     

Sequential nearest-neighbor effects on computed <Superscript>13</Superscript>C<Superscript>α</Superscript> chemical shifts

To evaluate sequential nearest-neighbor effects on quantum-chemical calculations of ¹³C^α chemical shifts, we selected the structure of the nucleic acid binding (NAB) protein from the SARS coronavirus determined by NMR in solution (PDB id 2K87). NAB is a 116-residue α/β protein, which contains 9 prolines and has 50% of its residues located in loops and turns. Overall, the results presented here show that sizeable nearest-neighbor effects are seen only for residues preceding proline, where Pro introduces an overestimation, on average, of 1.73 ppm in the computed ¹³C^α chemical shifts. A new ensemble of 20 conformers representing the NMR structure of the NAB, which was calculated with an input containing backbone torsion angle constraints derived from the theoretical ¹³C^α chemical shifts as supplementary data to the NOE distance constraints, exhibits very similar topology and comparable agreement with the NOE constraints as the published NMR structure. However, the two structures differ in the patterns of differences between observed and computed ¹³C^α chemical shifts, Δ _ca,i , for the individual residues along the sequence. This indicates that the Δ _ca,i -values for the NAB protein are primarily a consequence of the limited sampling by the bundles of 20 conformers used, as in common practice, to represent the two NMR structures, rather than of local flaws in the structures.     

Computation of multicenter overlap integrals with Slater-type orbitals using Ψ<Superscript>α</Superscript>-ETOs

Multicenter overlap integrals appearing in the evaluation of multicenter–multielectron integrals of central and noncentral interaction potentials are calculated using complete orthonormal sets of -ETOs (=1, 0, –1, –2, ...). The final results are expressed in terms of two-center overlap integrals between STOs. The convergence of the series is tested by calculating concrete cases for arbitrary quantum numbers, screening constants and location of STOs.     

Study of Electric Explosion of Flat Micron-Thick Foils at Current Densities of (5−50)×10<Superscript>8</Superscript> A/cm<Superscript>2</Superscript>

Electric explosions of flat Al, Тi, Ni, Cu, and Та foils with thicknesses of 1?16 μm, widths of 1?8 mm, and lengths of 5?11 mm were studied experimentally on the BIN, XP, and COBRA high-current generators at currents of 40?1000 kA and current densities of (5–50) × 10⁸ A/cm². The images of the exploded foils were taken at different angles to the foil surface by using point projection radiography with an X-pinch hot spot as the radiation source, the spatial resolution and exposure time being 3 μm and 50 ps, respectively, as well by the laser probing method with a spatial resolution of 20 μm and an exposure time of 180 ps. In the course of foil explosion, rapidly expanding objects resembling the core and corona of an exploded wire were observed. It is shown that the core of the exploded foil has a complicated time-varying structure.     

Molecular modeling of the rabbit colonic (HKα2a) H<Superscript>+</Superscript>, K<Superscript>+</Superscript> ATPase

A model of the HK2a subunit of the rabbit colonic H⁺, K⁺ ATPase has been generated using the crystal structure of the Ca⁺² ATPase as a template. A pairwise sequence alignment of the deduced primary sequences of the two proteins demonstrated that they share 29% amino acid sequence identity and 47% similarity. Using O (version 7) the model of HK2a was constructed by interactively mutating, deleting, and inserting the amino acids that differed between the pairwise sequence alignment of the Ca⁺² ATPase and HK2a. Insertions and deletions in the HK2a sequence occur in apparent extra-membraneous loop regions. The HK2a model was energy minimized and globally refined to a level comparable to that of the Ca⁺² ATPase structure using CNS. The charge distribution over the surface of HK2a was evaluated in GRASP and possible secondary structure elements of HK2a were visualized in BOBSCRIPT. Conservation and placement of residues that may be involved in ouabain binding by the H⁺, K⁺ ATPase were considered and a putative location for the subunit was postulated within the structure.Figure Possible architecture of the HK2a subunit. The residue in green is the lysine (position 517, Fig. 1) that lies in the nucleotide binding pocket and the residue in red is the aspartic acid at the phosphorylation site (position 385). Based on an alignment with the Ca⁺² ATPase, ten transmembrane helices were modeled into HK2a. The ten transmembrane helices are drawn as rods and shown in different colors for clarity. From left to right, the transmembrane helix designations are M10 (blue), M7 (gray), M8 (purple), M9 (orange), M5 (pink), M6 (green), M3 (brown), M4 (cyan), M2 (teal), and M1 (almond).     

<Superscript>1</Superscript>H–<Superscript>15</Superscript>N correlation spectroscopy of nanocrystalline proteins

The limits of resolution that can be obtained in ¹H–¹⁵N 2D NMR spectroscopy of isotopically enriched nanocrystalline proteins are explored. Combinations of frequency switched Lee–Goldburg (FSLG) decoupling, fast magic angle sample spinning (MAS), and isotopic dilution via deuteration are investigated as methods for narrowing the amide ¹H resonances. Heteronuclear decoupling of ¹⁵N from the ¹H resonances is also studied. Using human ubiquitin as a model system, the best resolution is most easily obtained with uniformly ²H and ¹⁵N enriched protein where the amides have been exchanged in normal water, MAS at 20 kHz, and WALTZ-16 decoupling of the ¹⁵N nuclei. The combination of these techniques results in average ¹H lines of only 0.26 ppm full width at half maximum. Techniques for optimizing instrument stability and ¹⁵N decoupling are described for achieving the best possible performance in these experiments.     

Improved accuracy in measuring one-bond and two-bond <Superscript>15</Superscript>N,<Superscript>13</Superscript>C<Superscript>α</Superscript> coupling constants in proteins by double-inphase/antiphase (DIPAP) spectroscopy

An extension to HN(CO-α/β-N,C^α-J)-TROSY (Permi and Annila in J Biomol NMR 16:221–227, 2000) is proposed that permits the simultaneous determination of the four coupling constants ¹ J _{N′(i)Cα(i)}, ² J _HN(i)Cα(i), ² J _{Cα(i−1)N′(i)}, and ³ J _{Cα(i−1)HN(i)} in ¹⁵N,¹³C-labeled proteins. Contrasting the original scheme, in which two separate subspectra exhibit the ² J _CαN′ coupling as inphase and antiphase splitting (IPAP), we here record four subspectra that exhibit all combinations of inphase and antiphase splittings possible with respect to both ² J _CαN′ and ¹ J _N′Cα (DIPAP). Complementary sign patterns in the different spectrum constituents overdetermine the coupling constants which can thus be extracted at higher accuracy than is possible with the original experiment. Fully exploiting data redundance, simultaneous 2D lineshape fitting of the E.COSY multiplet tilts in all four subspectra provides all coupling constants at ultimate precision. Cross-correlation and differential-relaxation effects were taken into account in the evaluation procedure. By applying a four-point Fourier transform, the set of spectra is reversibly interconverted between DIPAP and spin-state representations. Methods are exemplified using proteins of various size.     

Comprehensive determination of <Superscript>3</Superscript>J<Subscript>HNHα</Subscript> for unfolded proteins using <Superscript>13</Superscript>C′-resolved spin-echo difference spectroscopy

An experiment is presented to determine ³J_HNHα coupling constants, with significant advantages for applications to unfolded proteins. The determination of coupling constants for the peptide chain using 1D ¹H, or 2D and 3D ¹H-¹⁵N correlation spectroscopy is often hampered by extensive resonance overlap when dealing with flexible, disordered proteins. In the experiment detailed here, the overlap problem is largely circumvented by recording ¹H-¹³C′ correlation spectra, which demonstrate superior resolution for unfolded proteins. J-coupling constants are extracted from the peak intensities in a pair of 2D spin-echo difference experiments, affording rapid acquisition of the coupling data. In an application to the cytoplasmic domain of human neuroligin-3 (hNlg3cyt) data were obtained for 78 residues, compared to 54 coupling constants obtained from a 3D HNHA experiment. The coupling constants suggest that hNlg3cyt is intrinsically disordered, with little propensity for structure.     

Synthesis of the β-amyloid fragment <Superscript>5</Superscript>RHDSGY<Superscript>10</Superscript> and its isomers

The peptide RHDSGY, a fragment of the human β-amyloid Zn-binding site, and its isomers RH(D-Asp)SGY and RH(β-Asp)SGY have been obtained as amides by means of solid-phase synthesis and analyzed by HPLC and various mass spectrometric methods. The problem of low yield of the RHDSGY peptide and its isomers attributed to 9-fluorenylmethoxycarbonyl (Fmoc)-amino acids and/or formation of such side-products as RH(β-Asp)SGY (or RHDSGY during synthesis of RH(β-Asp)SGY) and RH(Asp-imide) SGY was solved via selection of individual reagents for removal of Fmoc groups from α-amino groups of the growing peptide chain.     

Fractional <Superscript>13</Superscript>C enrichment of isolated carbons using [1-<Superscript>13</Superscript>C]- or [2-<Superscript>13</Superscript>C]-glucose facilitates the accurate measurement of dynamics at backbone C<Superscript>α</Superscript> and side-chain methyl positions in proteins

A simple labeling approach is presented based on protein expression in [1-¹³C]- or [2-¹³C]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C^α sites, respectively. All of the methyl groups, with the exception of Thr and Ile(δ1) are produced with isolated ¹³C spins (i.e., no ¹³C–¹³C one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-α sites are labeled without concomitant labeling at C^β positions for 17 of the common 20 amino acids and there are no cases for which ¹³C^α−¹³CO spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone ¹⁵N studies. The utility of the labeling is established by recording ¹³C R _1ρ and CPMG-based experiments on a number of different protein systems.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N resonance assignments for the pro-inflammatory cytokine interleukin-36α

Interleukin-36α (IL-36α) is a recently characterised member of the interleukin-1 superfamily. It is involved in the pathogenesis of inflammatory arthritis in one third of psoriasis patients. By binding of IL-36α to its receptor IL-36R via the NF-κB pathway other cytokines involved in inflammatory and apoptotic cascade are activated. The efficacy of complex formation is controlled by N-terminal processing. To obtain a more detailed view on the structure function relationship we performed a heteronuclear multidimensional NMR investigation and here report the ¹H, ¹³C, and ¹⁵N resonance assignments for the backbone and side chain nuclei of the pro-inflammatory cytokine interleukin-36α.     

<Emphasis Type="Italic">N</Emphasis>-Methylation of <Emphasis Type="Italic">N</Emphasis><Superscript>α</Superscript>-Acetylated,Fully C<Superscript>α</Superscript>-Ethylated,Linear Peptides

“Mono-N-methyl scan” is a rational approach for the optimization of the peptide biological properties. N-Methylation of the –CONH– functionality is also a useful tool for discriminating solvent exposed from intramolecularly H-bonded secondary amide groups in peptides. We are currently extending this reaction to linear peptides based on C^α-tetrasubstituted α-amino acids. Following our study on the synthesis and conformation of the mono-N-methylated peptides from C^α-methylated residues, in this work we investigated the N-methylation reaction on homo-peptides to the pentamer level from the C^α-ethylated residue C^α,α-diethylglycine. Under the classical experimental conditions used, exclusively mono-N-methylation (on the N-terminal, acetylated residue) takes place, as unambiguously shown by mass spectrometry, 2D-NMR, and X-ray diffraction techniques. This backbone modification does not seem to involve any significant change in the peptide conformation in the crystalline state. Dedicated to the memory of Prof. Miroslav T. Leplawy (Technical University of Łodz, Poland), who performed the first synthesis of the extremely sterically demanding C^α,α-diethylglycine peptides.     

Impact of two-bond <Superscript>15</Superscript>N–<Superscript>15</Superscript>N scalar couplings on <Superscript>15</Superscript>N transverse relaxation measurements for arginine side chains of proteins

NMR relaxation of arginine (Arg) ¹⁵N_ε nuclei is useful for studying side-chain dynamics of proteins. In this work, we studied the impact of two geminal ¹⁵N–¹⁵N scalar couplings on measurements of transverse relaxation rates (R ₂ ) for Arg side-chain ¹⁵N_ε nuclei. For 12 Arg side chains of the DNA-binding domain of the Antp protein, we measured the geminal ¹⁵N–¹⁵N couplings ( ² J _NN ) of the ¹⁵N_ε nuclei and found that the magnitudes of the ² J _NN coupling constants were virtually uniform with an average of 1.2 Hz. Our simulations, assuming ideal 180° rotations for all ¹⁵N nuclei, suggested that the two ² J _NN couplings of this magnitude could in principle cause significant modulation in signal intensities during the Carr–Purcell-Meiboom–Gill (CPMG) scheme for Arg ¹⁵N_ε R ₂ measurements. However, our experimental data show that the expected modulation via two ² J _NN couplings vanishes during the ¹⁵N CPMG scheme. This quenching of J modulation can be explained by the mechanism described in Dittmer and Bodenhausen (Chemphyschem 7:831–836, 2006). This effect allows for accurate measurements of R ₂ relaxation rates for Arg side-chain ¹⁵N_ε nuclei despite the presence of two ² J _NN couplings. Although the so-called recoupling conditions may cause overestimate of R ₂ rates for very mobile Arg side chains, such conditions can readily be avoided through appropriate experimental settings.     

Sequence specific <Superscript>1</Superscript>H, <Superscript>13</Superscript>C and <Superscript>15</Superscript>N resonance assignments of a cataract-related variant G57W of human γS-crystallin

γS-crystallin is a major structural component of the human eye lens, which maintains its stability over the lifetime of an organism with negligible turnover. The G57W mutant of human γS-crystallin (abbreviated hereafter as γS-G57W) is associated with dominant congenital cataracts. In order to provide a structural basis for the ability of γS-G57W causing cataract, we have cloned, overexpressed, isolated and purified the protein. The 2D [¹⁵N–¹H]-HSQC spectrum recorded with uniformly ¹³C/¹⁵N-labelled γS-G57W was highly dispersed indicating the protein to adopt an ordered conformation. In this paper, we report almost complete sequence-specific ¹H, ¹³C and ¹⁵N resonance assignments of γS-G57W using a suite of heteronuclear 3D NMR experiments.     

How uniform is the peptide plane geometry? A high-accuracy NMR study of dipolar C<Superscript>α</Superscript>–C′/H<Superscript>N</Superscript>–N cross-correlated relaxation

Highly precise and accurate measurements of very small NMR cross-correlated relaxation rates, namely those between protein H_i^N–N_i and C_i−1^α–C_i−1′ dipoles, are demonstrated with an error of 0.03 s⁻¹ for GB3. Because the projection angles between the two dipole vectors are very close to the magic angle the rates range only from −0.2 to +0.2 s⁻¹. Small changes of the average vector orientations have a dramatic impact on the relative values. The rates suggest deviation from idealized peptide plane geometry caused by twists around the C′–N bonds and/or pyramidalization of the nitrogen atoms. A clear alternating pattern along the sequence is observed in β strands 1, 3 and 4 of GB3, where the side chains of almost all residues with large positive rates are solvent exposed. In the α helix all rates are relatively large and positive. Some of the currently most accurate structures of GB3 determined by both high resolution X-ray crystallography and NMR are in satisfactory agreement with the experimental rates in the helix and β strand 3, but not in the loops and the two central strands of the sheet for which no alternating pattern is predicted.     

The role of Ca<Superscript>2+</Superscript>, H<Superscript>+</Superscript>, and Cl<Superscript>−</Superscript> ions in generation of variation potential in pumpkin plants

The contributions of Ca²⁺, H⁺, and Cl⁻ in generation of variation potentials (VP) in 3- to 4-week-old pumpkin (Cucurbita pepo L., cv. Mozoleevskaya) plants were assessed. During VP generation, transient alkalinization of the medium around the stem was recorded with a potentiometric method. The pH changes were kinetically similar to the electric potential changes and were apparently due to temporal suppression of the plasma-membrane electrogenic H⁺ pump. These data and the observed inhibition of VP in the stem zone treated locally with a metabolic inhibitor (NaN₃) indicate that the VP generation is related to the reversible suppression of the H⁺-pump. The anion channel blocker (ethacrynic acid) decelerated significantly the front slope of VP and reduced the VP amplitude. A short-term increase in external Cl⁻ concentration around the stem was observed during potential transients representing the VP front slope and the pulses integrated into VP. The removal of Ca²⁺ from extracellular medium inhibited the VP generation. It is proposed that Ca²⁺ plays a role in activation of anion channels and in the H⁺-pump inactivation. The VP generation is probably determined by a complex mechanism, with contributions from passive ion fluxes (Ca²⁺, Cl⁻) moving along the electrochemical gradients and from changes in the electrogenic pump activity.     

Detection of C′,C<Superscript>α</Superscript> correlations in proteins using a new time- and sensitivity-optimal experiment

Sensitivity- and time-optimal experiment, called COCAINE (CO-CA In- and aNtiphase spectra with sensitivity Enhancement), is proposed to correlate chemical shifts of ¹³C and ¹³C spins in proteins. A comparison of the sensitivity and duration of the experiment with the corresponding theoretical unitary bounds shows that the COCAINE experiment achieves maximum possible transfer efficiency in the shortest possible time, and in this sense the sequence is optimal. Compared to the standard HSQC, the COCAINE experiment delivers a 2.7-fold gain in sensitivity. This newly proposed experiment can be used for assignment of backbone resonances in large deuterated proteins effectively bridging ¹³C and ¹³C resonances in adjacent amino acids. Due to the spin-state selection employed, the COCAINE experiment can also be used for efficient measurements of one-bond couplings (e.g. scalar and residual dipolar couplings) in any two-spin system (e.g. the N/H in the backbone of protein).