Estimates of ϕ and ψ torsion angles in proteins from one-, two- and three-bond nuclear spin-spin couplings: Application to staphylococcal nuclease

Summary Calculated coupling constants (³J_H ^N _H , ¹J_{C H} , ²J_CH , ¹J_{C N} and ²J_{C N}) from our accompanying paper [Edison, A.S. et al. (1994) J. Biomol. NMR, 4, 519–542] have been used to generate error surfaces that can provide estimates of the and angles in proteins. We have used experimental coupling data [³J_H ^N _H : Kay, L.E. et al. (1989) J. Am. Chem. Soc., 111, 5488–5490; ¹J_{C H} : Vuister, G.W. et al. (1993) J. Biomol. NMR, 3, 67–80; ²J_CH : Vuister, G.W. and Bax, A. (1992) J. Biomol. NMR, 2, 401–405; ¹J_{C N} and ²J_{C N}: Delaglio, F. et al. (1991) J. Biomol. NMR, 1, 439–446] to create error surfaces for selected residues of the protein staphylococcal nuclease. The residues were chosen to include all those with five experimental couplings, as well as some with four experimental couplings, to demonstrate the relative importance of ³J_H ^N _H and ¹J_{C H} . For most of the cases, we obtained good agreement between the X-ray structure [Loll, P.J. and Lattman, E.E. (1989) Protein Struct. Funct. Genet., 5, 183–201] and the NMR data.Abbreviations CUPID Contin Uous ProbabIlity Distribution analysis of rotamers - ⁿJ_AB single-bond (n=1), geminal (n=2), or vicinal (n=3) coupling constant between nuclei A and B - NOE nuclear Overhauser enhancement - r² correlation coefficient     

Differential deuterium isotope shifts and one-bond 1H−13C scalar couplings in the conformational analysis of protein glycine residues

Summary The one-bond deuterium isotope shift effect for glycine C resonances exhibits a conformational dependence comparable to that of the corresponding ¹J_HC scalar coupling in both magnitude (11 Hz at 14.1 T) and dihedral angle dependence. The similarity in the conformational dependence of the ¹J_HC and deuterium isotope shift values suggests a common physical basis. Given the known distribution of (,) main-chain dihedral angles for glycine residues, the deuterium isotope shifts and the ¹J_HC scalar couplings can determine conformations in the left-and right-handed helical-to-bridge regions of the (,) plane to an accuracy of approximately 13°. In the absence of stereochemical assignments, the differential deuterium isotope shifts and the ¹J_HC scalar couplings can be combined with limited independent structural information (e.g., the sign of ) to determine the chirality of the deuterium substitution.     

Measurement of one-bond 1Hα-13Cα couplings in backbone-labelled proteins

NMR dipole-dipole couplings between protein backbone nuclei (¹H, ¹³C, ¹⁵N, ¹H^N,¹³C) offer enormous scope for the rapid determination of protein global folds. Here, we show that measurement of one-bond splittings in the protein backbone is facilitated by use of protein that is selectively isotopically enriched only in the backbone atoms. In particular, ¹H-¹³C couplings can be measured simply and with high sensitivity by use of conventional heteronuclear single quantum correlation (HSQC) techniques.     

Measurement of 1H�C15N and 1H�C13C residual dipolar couplings in nucleic acids from TROSY intensities

Analogous to the recently introduced ARTSY method for measurement of one-bond (1)H-(15)N residual dipolar couplings (RDCs) in large perdeuterated proteins, we introduce methods for measurement of base (13)C-(1)H and (15)N-(1)H RDCs in protonated nucleic acids. Measurements are based on quantitative analysis of intensities in (1)H-(15)N and (13)C-(1)H TROSY-HSQC spectra, and are illustrated for a 71-nucleotide adenine riboswitch. Results compare favorably with those of conventional frequency-based measurements in terms of completeness and convenience of use. The ARTSY method derives the size of the coupling from the ratio of intensities observed in two TROSY-HSQC spectra recorded with different dephasing delays, thereby minimizing potential resonance overlap problems. Precision of the RDC measurements is limited by the signal-to-noise ratio, S/N, achievable in the 2D TROSY-HSQC reference spectrum, and is approximately given by 30/(S/N) Hz for (15)N-(1)H and 65/(S/N) Hz for (13)C-(1)H. The signal-to-noise ratio of both (1)H-(15)N and (1)H-(13)C spectra greatly benefits when water magnetization during the experiments is not perturbed, such that rapid magnetization transfer from bulk water to the nucleic acid, mediated by rapid amino and hydroxyl hydrogen exchange coupled with (1)H-(1)H NOE transfer, allows for fast repetition of the experiment. RDCs in the mutated helix 1 of the riboswitch are compatible with nucleotide-specifically modeled, idealized A-form geometry and a static orientation relative to the helix 2/3 pair, which differs by ca 6° relative to the X-ray structure of the native riboswitch.     

Measurement of residual dipolar couplings from 1Hα to 13Cα and 15N using a simple HNCA-based experiment

Novel NMR pulse schemes for simultaneous measurement of ¹ D _CHand ² D _NHresidual dipolar couplings in proteins is presented. We show that ² D _NHcoupling can be very useful for protein structure determination. The ² D _NHcoupling can be measured from ¹⁵N dimension with good accuracy on a slowly relaxing TROSY resonance, utilizing HNCA-TROSY-based experiments, which concomitantly supply large ¹ D _CHcoupling. The dynamic range of ² D _NHcoupling is comparable to ¹ D _NC coupling, but instead, it also serves non-redundant information on the course of protein backbone, thanks to rotational degree of freedom with respect to peptide bond. The HNCA-TROSY-based experiments are optimal for measuring residual dipolar couplings at high magnetic fields owing to absence of rapid transverse relaxation of carbonyl carbon. The reliability of the proposed approach was tested on ¹⁵N/¹³C human ubiquitin. A very good correlation with ubiquitin solution as well as crystal structure, for both ¹ D _CHand ² D _NHcouplings, was obtained.     

An HNCO-based Pulse Scheme for the Measurement of 13Cα-1Hα One-bond Dipolar couplings in 15N, 13C Labeled Proteins

A triple resonance pulse scheme is presented for recording 13C-1H one-bond dipolar couplings in 15N, 13C labeled proteins. HNCO correlation maps are generated where the carbonyl chemical shift is modulated by the 13C-1H coupling, with the two doublet components separated into individual data sets. The experiment makes use of recently described methodology whereby the protein of interest is dissolved in a dilute solution of bicelles which orient above a critical temperature, thus permitting measurement of significant couplings (Tjandra and Bax, 1997a). An application to the protein ubiquitin is described.     

Measurement of one-bond 15N-13C′ dipolar couplings in medium sized proteins

A simple and accurate method is described for measurement of ¹ J _CN splittings in isotopically enriched proteins. The method is of the quantitative J correlation type, and the ¹ J _CN splitting is derived from the relative intensity in two 3D TROSY-HNCO spectra with ¹ J _CN dephasing intervals of 1/(2¹ J _CN) (reference intensity) and 1/¹ J _CN (residual intensity). If the two spectra are recorded under identical conditions and with the same number of scans, the random error in the ¹ J _CN value extracted in this manner is inversely related to the signal-to-noise (S/N) in the reference spectrum. A S/N of 30:1 in the reference spectrum yields random errors of less than 0.2 Hz in the extracted ¹ J _CN value. Dipolar couplings obtained from the difference in ¹ J _CN splitting in the isotropic and liquid crystalline phase for the C-terminal domain of calmodulin are in excellent agreement with its 1.68-Å crystal structure, but agree considerably less with the 2.2-Å structure.     

Quantitative J correlation methods for the accurate measurement of 13C′-13Cαdipolar couplings in proteins

Methods are described for the precise and accurate measurement of one-bond dipolar (13)C'-(13)C(alpha) couplings in weakly aligned proteins. The experiments are based on the principle of quantitative J correlation, where (1)J(C'C(alpha)) (or (1)J(C'C(alpha)) + 1D(C'C(alpha)) is measured from the relative intensity of two interleaved 3D TROSY-HN(CO)CA or 3DTROSY-HNCO spectra recorded with dephasing intervals of zero (reference spectrum) and approximately 3/(2(1)J(C'C(alpha)) (attenuated spectrum). In analogy to other quantitative J correlation techniques, the random error in the measured (1)J(C'C(alpha)) value is inversely proportional to the signal-to-noise ratio in the reference spectrum. It is shown that for weakly aligned proteins, with the magnitude of the alignment tensor of D(a)(NH) < or = 10-15 Hz, the systematic errors are typically negligible. The methods are demonstrated for the third IgG-binding domain of protein G (GB3) and a-synuclein in complex with a detergent micelle, where errors in (1)D(C'C(alpha)) of less than 0.1 Hz and ca. 0.2 Hz,respectively, are estimated. Remarkably, the dipolar couplings determined for GB3 are in even better agreement with the recently refined 1.1-angstroms X-ray structure than the input (13)C'-(13)C(alpha) couplings used for the refinement.     

Heteronuclear relayed E.COSY revisited: Determination of 3J(Hα,Cγ) couplings in Asx and aromatic residues in proteins

Constant-time 3D heteronuclear relayed E.COSY [Schmidt et al. (1996) J. Biomol. NMR, 7, 142–152], as based on generic 2D small-flip-angle HMQC-COSY [Schmidt et al. (1995) J. Biomol. NMR, 6, 95–105], has been modified to allow for quantitative determination of heteronuclear three-bond ³ J(H,C) couplings. The method is applicable to amino acid spin topologies with carbons in the position which lack attached protons, i.e. to asparagine, aspartate, and aromatic residues in uniformly ¹³C-enriched proteins. The pulse sequence critically exploits heteronuclear triple-quantum coherence (HTQC) of CH₂ moieties involving geminal H proton pairs, taking advantage of improved multiple-quantum relaxation properties, at the same time avoiding scalar couplings between those spins involved in multiple-quantum coherence, thus yielding E.COSY-type multiplets with a splitting structure that is simpler than with the original scheme. Numerical least-squares 2D line-shape simulation is used to extract ³ J(H,C) coupling constants which are of relevance to side-chain ₁ dihedral-angle conformations in polypeptides. Methods are demonstrated with recombinant ¹⁵N,¹³C-enriched ribonuclease T1 and Desulfovibrio vulgaris flavodoxin with bound oxidized FMN.     

A conformational study of the tetrapeptide CH3CO-Ala-Asp-Gly-Lys-NHCH3 corresponding to a β-bend in staphylococcal nuclease

The tetrapeptide sequence Ala-Asp-Gly-Lys occurs as a type I′ β-bend at residues 94–97 in staphylococcal nuclease. We have synthesized theN-acetyl,N′-methylamide derivative of this tetrapeptide and studied its conformation in solution, using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. In the synthesis, special attention was paid to the possibility of cyclic aspartimide formation giving rise to mixtures of α- and β-Asp-Gly products. The presence of such a mixture was excluded by infrared, NMR, and other analytical procedures applied to the products and to models for α- and β-linked aspartyl residues. The CD spectra of the protected tetrapeptide in water, methanol, and trifluoroethanol show no evidence of preferred chain conformations. In dimethylsulfoxide-d ₆ , however, the NMR spectra are consistent with the presence of a population of conformers in which the Lys and C-terminal NHCH₃ amide protons are shielded from solvent. Taken together with the observed³J_NH-C ^α _H coupling constants for all residues, this permitted the construction and energetic evaluation of possible conformations in solution. Only one such conformation was fully compatible with the NMR data; this is a type II β-bend in which the Lys and C-terminal NHCH₃ amide protons are close to the Ala C=O group and may form bifurcated hydrogen bonds with it. This conformation can be converted into the conformation existing in staphylococcal nuclease by rotating the plane of the Ala-Asp peptide group by about 120° around a line connecting the Ala and Asp C_α atoms and by making small shifts in dihedral angles elsewhere in the peptide.     

Backbone 1H, 13C and 15N resonance assignments of the 39?kDa staphylococcal hemoglobin receptor IsdH

During infections Stahpylococcus aureus preferentially uses heme as an iron source, which it captures from human hemoglobin using the Iron regulated surface determinant (Isd) system. On the cell surface two related staphylococcal surface receptors called IsdH and IsdB bind to hemoglobin and extract its heme. Both receptors contain multiple NEAr iron Transporter (NEAT) domains that either bind to hemoglobin, or to heme. All previous structural studies have investigated individual NEAT domains and have not explored how the domains might interact with one another to synergistically extract heme from hemoglobin. Here, we report the near complete (1)H, (13)C and (15)N backbone resonance assignments of a bi-domain unit from IsdH that contains the N2 and N3 NEAT domains, which bind to hemoglobin and heme, respectively (IsdH(N2N3), residues 326-660, 39 kDa). The assigned backbone resonances lay the foundation for future NMR studies that will explore the molecular basis of IsdH function.     

Simultaneous measurement of 1H�C15N and Methyl 1Hm�C13Cm residual dipolar couplings in large proteins

A two-dimensional TROSY-based SIM-(13)C(m)-(1)H(m)/(1)H-(15)N NMR experiment for simultaneous measurements of methyl (1) D (CH) and backbone amide (1) D (NH) residual dipolar couplings (RDC) in {U-[(15)N,(2)H]; Ileδ1-[(13)CH(3)]; Leu,Val-[(13)CH(3)/(12)CD(3)]}-labeled samples of large proteins is described. Significant variation in the alignment tensor of the 82-kDa enzyme Malate synthase G is observed as a function of only slight changes in experimental conditions. The SIM-(13)C(m)-(1)H(m)/(1)H-(15)N data sets provide convenient means of establishing the alignment tensor characteristics via the measurement of (1) D (NH) RDCs in the same protein sample.     

Improved three-dimensional1H−13C−1H correlation spectroscopy of a13C-labeled protein using constant-time evolution

Summary An improved version of the three-dimensional HCCH-COSY NMR experiment is described that correlates the resonances of geminal and vicinal proton pairs with the chemical shift of the¹³C nucleus attached to one of the protons. The experiment uses constant-time evolution of transverse¹³C magnetization which optimizes transfer of magnetization and thus improves the sensitivity of the experiment over the original scheme. The experiment is demonstrated for calmodulin complexed with a 26-residue peptide comprising the binding site of skeletal muscle myosin light chain kinase.     

Application of H(N)CA,CO-E.COSY experiments for calibrating the φ angular dependences of vicinal couplings J(C′i−1,Hi α), J(C′i−1,Ci β) and J(C′i−1,C′i) in proteins

A triple-resonance NMR technique suitable for the determination ofcarbonyl-related couplings in polypeptide systems is introduced. Theapplication of three novel pulse sequences to uniformly¹³C/¹⁵N-enriched proteins yields E.COSY-likemultiplet patterns exhibiting either one of the³J(C_i–1,H_i ), ³J(C_i–1,C_i ) and³J(C_i–1,C_i)coupling constants in the indirectly detected ¹³Cdimension, depending on the passive spin selected. The experiments aredemonstrated with oxidized flavodoxin from Desulfovibrio vulgaris. On thebasis of the J-values measured and the backbone -angles derived from ahigh-resolution X-ray structure of the protein, the three associated Karplusequations were reparametrized. The root-mean-square differences between theexperimental coupling constants and those predicted by the optimized Karpluscurves are 0.41, 0.33 and 0.32 Hz for³J(C_i–1,H_i ),³J(C_i–1,C_i ) and³J(C_i–1,C_i),respectively. The results are compared with the Karplus parameters previouslypublished for the same couplings.     

Measurement and application of 1H-19F dipolar couplings in the structure determination of 2′-fluorolabeled RNA

Residual dipolar couplings can provide powerful restraints for determination and refinement of the solution structure of macromolecules. The application of these couplings in nucleic acid structure elucidation can have an especially dramatic impact, since they provide long-range restraints, typically absent in NOE and J-coupling measurements. Here we describe sensitive X-filtered-E.COSY-type methods designed to measure both the sign and magnitude of long-range ¹H-¹⁹F dipolar couplings in selectively fluorine labeled RNA oligonucleotides oriented in solution by a liquid crystalline medium. The techniques for measuring ¹H-¹⁹F dipolar couplings are demonstrated on a 21-mer RNA hairpin, which has been specifically labeled with fluorine at the 2-hydroxyl position of three ribose sugars. Experimentally measured ¹H-¹⁹F dipolar couplings for the 2-deoxy-2-fluoro-sugars located in the helical region of the RNA hairpin were found to be in excellent agreement with values predicted using canonical A-form helical geometry, demonstrating that these couplings can provide accurate restraints for the refinement of RNA structures determined by NMR.     

Leucine side-chain rotamers in a glycophorin A transmembrane peptide as revealed by three-bond carbon—carbon couplings and 13C chemical shifts

Summary We have used a spin-echo difference NMR pulse sequence to measure three-bond J couplings between - and -carbons of the leucine residues in a micelle-associated helical peptide dimer that corresponds to residues 62–101 of the transmembrane erythrocyte protein glycophorin A. The observed ³J couplings correlate strongly with the ¹³C chemical shift of the -methyl groups, and within experimental error both the shift distribution of the methyl carbons and the variations in ³J can be accounted for by variations in side-chain rotamer populations. We infer that all leucine side chains in this peptide dimer are in fast exchange among X ² rotamers and sample two of the three possible rotameric states, even when the side chain forms part of the dimer interface. The observed correlation of chemical shift with couplings can be traced to a -gauche interaction of methyl and -carbons. This correlation may provide an alternate route to rotamer analysis in some protein systems.     

Measurement of HN-Hα J couplings in calcium-free calmodulin using new 2D and 3D water-flip-back methods

Summary Two new methods are described for the measurement of three-bond J_H ^NHcouplings in proteins isotopically enriched with ¹⁵N. Both methods leave the water magnetization in an unsaturated state, parallel to the z-axis, and therefore offer significant enhancements in sensitivity for rapidly exchanging backbone amide protons. The J couplings can be measured either from a set of constant-time 2D ¹H-¹⁵N HMQC spectra, which are modulated in intensity by J_H ^NH, or from a water-flip-back version of the 3D HNHA experiment. The method is demonstrated for a sample of calcium-free calmodulin. Residues Lys⁷⁵-Asp⁸⁰ have J_H ^NHvalues in the 6–7 Hz range, suggesting that a break in the central helix occurs at the same position as previously observed in solution NMR studies of Ca²⁺-ligated calmodulin.     

Study of the residues involved in the binding of β1 to β3 subunits in the sodium channel

The voltage-gated sodium channel (VGSC) is a complex, which is composed of one pore-forming α subunit and at least one β subunit. Up to now, five β subunits are known: β1/β1A, β1B, β2, β3, and β4, encoded by four genes (SCN1B∼SCN4B). It is critical to have a deep understanding of the interaction between β1 and β3 subunits, two subunits which frequently appear in many diseases concurrently. In this study, we had screened out the new template of β1 subunit for homology modelling, which shares higher similarity to β3. Docking studies of the β1 and β3 homology model were conducted, and likely β1 and β3 binding loci were investigated. The results revealed that β1–β3 is more likely to form a di-polymer than β1–β1 based on molecular interaction analysis, including potential energy analysis, Van der Waals (VDW) energy analysis and electrostatic energy analysis, and in addition, consideration of the hydrogen bonds and hydrophobic contacts that are involved. Based on these analyses, the residues His122 and Lys140 of β1 and Glu 66, Asn 131, Asp 118, Glu 120, Glu133, Asn135, Ser 137 of β3 were predicted to play a functional role.     

Combined use of 13C chemical shift and 1Hα−13Cα heteronuclear NOE data in monitoring a protein NMR structure refinement

Summary A large portion of the ¹³C resonance assignments for murine epidermal growth factor (mEGF) at pH 3.1 and 28°C has been determined at natural isotope abundance. Sequence-specific ¹³C assignments are reported for 100% of the assignable C, 96% of the C, 86% of the aromatic and 70% of the remaining peripheral aliphatic resonances of mEGF. A good correlation was observed between experimental and back-calculated C chemical shifts for regions of regular -sheet structure. These assignments also provide the basis for interpreting ¹H–¹³C heteronuclear NOE (HNOE) values in mEGF at natural isotope abundance. Some of the backbone polypeptide segments with high internal mobility, indicated by these ¹H–¹³C HNOE measurements, correlate with locations of residues involved in the putative mEGF-receptor binding site. Using four families of mEGF structures obtained over the last few years, we demonstrate that standard deviations between experimental and back-calculated C values can be used to monitor the refinement of this protein's structure, particularly for -sheet regions. Improved agreement between calculated and observed values of C is correlated with other measures of structure quality, including lowered values of residual constraint violations and more negative values of conformational energy. These results support the view that experimental conformation-dependent chemical shifts, C, can provide a reliable source of information for monitoring the process of protein structure refinement and are potentially useful restraints for driving the refinement.Abbreviations HSQC heteronuclear single-quantum coherence spectroscopy - PFG pulsed-field gradient - TOCSY ¹H-¹H total correlation spectroscopy - EGF epidermal growth factor - mEGF murine EGF - hEGF human EGF - hTGF human type- transforming growth factor - DIPSI spm-locking pulse sequence - NOE nuclear Overhauser effect - HNOE heteronuclear Overhauser effect