Predicting 15N chemical shifts in proteins using the preceding residue-specific individual shielding surfaces from φ, ψi−1, and χ1torsion angles

Empirical shielding surfaces are most commonly used to predict chemical shifts in proteins from known backbone torsion angles, phi and psi. However, the prediction of (15)N chemical shifts using this technique is significantly poorer, compared to that for the other nuclei such as (1)H(alpha), (13)C(alpha), and (13)C(beta). In this study, we investigated the effects from the preceding residue and the side-chain geometry, chi(1), on (15)N chemical shifts by statistical methods. For an amino acid sequence XY, the (15)N chemical shift of Y is expressed as a function of the amino acid types of X and Y, as well as the backbone torsion angles, phi and psi(i-1). Accordingly, 380 empirical 'Preceding Residue Specific Individual (PRSI)' (15)N chemical shift shielding surfaces, representing all the combinations of X and Y (except for Y=Pro), were built and used to predict (15)N chemical shift from phi and psi(i-1). We further investigated the chi(1) effects, which were found to account for differences in (15)N chemical shifts by approximately 5 ppm for amino acids Val, Ile, Thr, Phe, His, Tyr, and Trp. Taking the chi(1) effects into account, the chi(1)-calibrated PRSI shielding surfaces (XPRSI) were built and used to predict (15)N chemical shifts for these amino acids. We demonstrated that (15)N chemical shift predictions are significantly improved by incorporating the preceding residue and chi(1) effects. The present PRSI and XPRSI shielding surfaces were extensively compared with three recently published programs, SHIFTX (Neal et al., 2003), SHIFTS (Xu and Case, 2001 and 2002), and PROSHIFT (Meiler, 2003) on a set of ten randomly selected proteins. A set of Java programs using XPRSI shielding surfaces to predict (15)N chemical shifts in proteins were developed and are freely available for academic users at http://www.pronmr.com or by sending email to one of the authors Yunjun Wang (yunjunwang@yahoo.com).     

Probing multiple effects on 15N, 13C alpha, 13C beta,and 13C' chemical shifts in peptides using density functional theory

We have used density functional calculations on model peptides to study conformational effects on (15)N, (13)C alpha, (13)C beta, and (13)C' chemical shifts, associated with hydrogen bonding, backbone conformation, and side-chain orientation. The results show a significant dependence on the backbone torsion angles of the nearest three residues. Contributions to (15)N chemical shifts from hydrogen bonding (up to 8 ppm), backbone conformation (up to 13 ppm), side-chain orientation and neighborhood residue effects (up to 22 ppm) are significant, and a unified theory will be required to account for their behavior in proteins. In contrast to this, the dependence on sequence and hydrogen bonding is much less for (13)C alpha and (13)C beta chemical shifts (<0.5 ppm), and moderate for carbonyl carbon shifts (<2 ppm). The effects of side-chain orientation are mainly limited to the residue itself for both nitrogen and carbon, but the chi(1) effect is also significant for the nitrogen shift of the following residue and for the (13)C' shift of the preceding residue. The calculated results are used, in conjunction with an additive model of chemical shift contributions, to create an algorithm for prediction of (15)N and (13)C shifts in proteins from their structure; this includes a model to extrapolate results to regions of torsion angle space that have not been explicitly studied by density functional theory (DFT) calculations. Crystal structures of 20 proteins with measured shifts have been used to test the prediction scheme. Root mean square deviations between calculated and experimental shifts 2.71, 1.22, 1.31, and 1.28 ppm for N, C alpha, C beta, and C', respectively. This prediction algorithm should be helpful in NMR assignment, crystal and solution structure comparison, and structure refinement.     

Assignment of protein backbone resonances using connectivity, torsion angles and 13Cα chemical shifts

A program is presented which will return the most probable sequence location for a short connected set of residues in a protein given just (13)C(alpha) chemical shifts (delta((13)C(alpha))) and data restricting the phi and psi backbone angles. Data taken from both the BioMagResBank and the Protein Data Bank were used to create a probability density function (PDF) using a multivariate normal distribution in delta((13)C(alpha)), phi, and psi space for each amino acid residue. Extracting and combining probabilities for particular amino acid residues in a short proposed sequence yields a score indicative of the correctness of the proposed assignment. The program is illustrated using several proteins for which structure and (13)C(alpha) chemical shift data are available.     

Application of cross-correlated NMR spin relaxation to the zinc-finger protein CRP2(LIM2): evidence for collective motions in LIM domains

The solution structure of quail CRP2(LIM2) was significantly improved by using an increased number of NOE constraints obtained from a 13C,15N-labeled protein sample and by applying a recently developed triple-resonance cross-correlated relaxation experiment for the determination of the backbone dihedral angle psi. Additionally, the relative orientation of the 15N(i)-1HN(i) dipole and the 13CO(i) CSA tensor, which is related to both backbone angles phi and psi, was probed by nitrogen-carbonyl multiple-quantum relaxation and used as an additional constraint for the refinement of the local geometry of the metal-coordination sites in CRP2(LIM2). The backbone dynamics of residues located in the folded part of CRP2(LIM2) have been characterized by proton-detected 13C'(i-1)-15N(i) and 15N(i)-1HN(i) multiple-quantum relaxation, respectively. We show that regions having cross-correlated time modulation of backbone isotropic chemical shifts on the millisecond to microsecond time scale correlate with residues that are structurally altered in the mutant protein CRP2(LIM2)R122A (disruption of the CCHC zinc-finger stabilizing side-chain hydrogen bond) and that these residues are part of an extended hydrogen-bonding network connecting the two zinc-binding sites. This indicates the presence of long-range collective motions in the two zinc-binding subdomains. The conformational plasticity of the LIM domain may be of functional relevance for this important protein recognition motif.     

Determination and comparative analysis of the conformation of bovine pancreatic trypsin inhibitor and trypsin inhibitors E and K from the data of two-dimensional 1H-NMR spectroscopy

On the basis of joint consideration of distance dependences between amide proton NH and protons C alpha H, NH, C beta H of the preceding in amino acid sequence residue from the torsion angles phi psi, chi 1, the correlation diagram of these proton-proton distances with the regions of sterically allowed conformational space (phi, psi) is presented and the method for the determination of the L-amino acid residues backbone conformations is proposed. The diagram was used for the determination of backbone conformations of bovine pancreatic trypsin inhibitor and trypsin inhibitors E and K from Dendroaspis polylepis using the data from two-dimensional 1H-NMR spectroscopy. The analysis of backbone conformations was carried out. The individual elements of these protein molecules secondary structure were characterized and their high conformational homology was shown. The inference about qualitative coincidence of three protein molecules conformation in solution, preservation of secondary structure basic elements and their similarity with bovine pancreatic trypsin inhibitor crystalline structure was made.     

Determination of solid-state NMR structures of proteins by means of three-dimensional 15N-13C-13C dipolar correlation spectroscopy and chemical shift analysis

In this paper, a three-dimensional (3D) NMR-based approach for the determination of the fold of moderately sized proteins by solid-state magic-angle spinning (MAS) NMR is presented and applied to the alpha-spectrin SH3 domain. This methodology includes the measurement of multiple (13)C-(13)C distance restraints on biosynthetically site-directed (13)C-enriched samples, obtained by growing bacteria on [2-(13)C]glycerol and [1,3-(13)C]glycerol. 3D (15)N-(13)C-(13)C dipolar correlation experiments were applied to resolve overlap of signals, in particular in the region where backbone carbon-carbon correlations of the C(alpha)-C(alpha), CO-CO, C(alpha)-CO, and CO-C(alpha) type appear. Additional restraints for confining the structure were obtained from phi and psi backbone torsion angles of 29 residues derived from C(alpha), C(beta), CO, NH, and H(alpha) chemical shifts. Using both distance and angular restraints, a refined structure was calculated with a backbone root-mean-square deviation of 0.7 A with respect to the average structure.     

Assignment of the natural abundance 13C spectrum of proteins using 13C 1H-detected heteronuclear multiple-bond correlation NMR spectroscopy: structural information and stereospecific assignments from two- and three-bond carbon-hydrogen coupling constants.

Proton-detected heteronuclear multiple-bond 1H-13C correlations (HMBC) previously have been used for assignment purposes in a variety of isotopically enriched proteins. In the present study it is demonstrated that the technique yields an almost complete assignment of the natural abundance 13C spectrum of the protein basic pancreatic trypsin inhibitor (BPTI). In addition, the technique permits additional 1H assignments to be made for this well-studied protein. The intensities of observed correlations permit rough estimates to be made of 2J(C,H) and 3J(C,H) coupling constants. These couplings can be used for conformational studies of both the side chains and the backbone. Intra- and interresidue coupling between C alpha H and the carbonyl carbon provides information about the backbone angles psi and phi. Side-chain conformations can be determined from both two- and three-bond carbon-hydrogen coupling constants. The present study of BPTI together with its known high-precision solution structure yields an experimental correlation between resonance intensities and secondary structure. The spectra show the potential of the method in analyzing 13C NMR spectra of nonenriched proteins. The method yields 13C NMR chemical shifts, which are versatile parameters to be used to monitor structural changes, titrations, etc.     

Determination of the local structure of the protein insectotoxin I5A from the scorpion Buthus eupeus from 1H-NMR spectroscopy data

The local structure (torsion angles phi, psi and chi 1 of amino acid residues) of insectotoxin I5A (35 residues) of scorpion Buthus eupeus has been determined from cross-peak integral intensities in two-dimensional nuclear Overhauser enhancement (NOESY) spectra and spin coupling constants of vicinal H--NC alpha--H and H--C alpha C beta--H protons. The local structure determination was carried out by fitting complete relaxation matrix of peptide unit protons (protons of a given residue and NH proton of the next residue in the amino acid sequence) with experimental NOESY cross-peak intensities. The obtained intervals of backbone torsional angles phi and psi consistent with NMR data were determined for all but Gly residues. The predominant C alpha--C beta rotamer of the side chain has been unambiguously determined for 42% of the insectotoxin amino acid residues whereas for another 46% residues experimental data are fitted equally well with two rotamers. Stereospecific assignments were obtained for 38% of beta-methylene groups. The determined torsional angles phi, psi and chi 1 correspond to the sterically allowed conformations of the amino acid residues and agree with the insectotoxin secondary structure established earlier by 1H NMR spectroscopy.     

Empirical isotropic chemical shift surfaces

A list of proteins is given for which spatial structures, with a resolution better than 2.5 A, are known from entries in the Protein Data Bank (PDB) and isotropic chemical shift (ICS) values are known from the RefDB database related to the Biological Magnetic Resonance Bank (BMRB) database. The structures chosen provide, with unknown uncertainties, dihedral angles phi and psi characterizing the backbone structure of the residues. The joint use of experimental ICSs of the same residues within the proteins, again with mostly unknown uncertainties, and ab initio ICS(phi,psi) surfaces obtained for the model peptides For-(L-Ala)(n)-NH(2), with n = 1, 3, and 5, resulted in so-called empirical ICS(phi,psi) surfaces for all major nuclei of the 20 naturally occurring alpha-amino acids. Out of the many empirical surfaces determined, it is the 13C(alpha)-1H(alpha) ICS(phi,psi) surface which seems to be most promising for identifying major secondary structure types, alpha-helix, beta-strand, left-handed helix (alpha(D)), and polyproline-II. Detailed tests suggest that Ala is a good model for many naturally occurring alpha-amino acids. Two-dimensional empirical 13C(alpha)-1H(alpha) ICS(phi,psi) correlation plots, obtained so far only from computations on small peptide models, suggest the utility of the experimental information contained therein and thus they should provide useful constraints for structure determinations of proteins.     

Probability-based protein secondary structure identification using combined NMR chemical-shift data

For a long time, NMR chemical shifts have been used to identify protein secondary structures. Currently, this is accomplished through comparing the observed (1)H(alpha), (13)C(alpha), (13)C(beta), or (13)C' chemical shifts with the random coil values. Here, we present a new protocol, which is based on the joint probability of each of the three secondary structural types (beta-strand, alpha-helix, and random coil) derived from chemical-shift data, to identify the secondary structure. In combination with empirical smooth filters/functions, this protocol shows significant improvements in the accuracy and the confidence of identification. Updated chemical-shift statistics are reported, on the basis of which the reliability of using chemical shift to identify protein secondary structure is evaluated for each nucleus. The reliability varies greatly among the 20 amino acids, but, on average, is in the order of: (13)C(alpha)>(13)C'>(1)H(alpha)>(13)C(beta)>(15)N>(1)H(N) to distinguish an alpha-helix from a random coil; and (1)H(alpha)>(13)C(beta) >(1)H(N) approximately (13)C(alpha) approximately (13)C' approximately (15)N for a beta-strand from a random coil. Amide (15)N and (1)H(N) chemical shifts, which are generally excluded from the application, in fact, were found to be helpful in distinguishing a beta-strand from a random coil. In addition, the chemical-shift statistical data are compared with those reported previously, and the results are discussed. A JAVA User Interface program has been developed to make the entire procedure fully automated and is available via http://ccsr3150-p3.stanford.edu.     

Solution structure of the soluble domain of the NfeD protein YuaF from <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

The transmembrane protein YuaF from B. subtilis is a member of the NfeD-like clan with a potential role in maintaining membrane integrity during conditions of cellular stress. nfeD-genes are primarily found in highly conserved operon structures together with the gene of another membrane protein belonging to the SPFH superfamily, in this case YuaG. This strongly suggests a functional if not physical interaction between YuaF and YuaG. Secondary structure predictions of NfeD proteins that accompany SPFH proteins all indicate a high content of beta-sheets in the C-terminal domains indicating a conserved core structure despite very low homology at the level of primary structure. Here we report the high-resolution solution structure of YuaF's soluble C-terminal domain derived from NMR data (sYuaF, residues 97-174 of full-length YuaF). Full backbone and side chain assignments of sYuaF were obtained from triple-resonance spectra. The structure was determined from distance restraints derived from 3D NOESY spectra collected at 600 MHz and 800 MHz, together with phi, psi, and chi(1) torsion angle restraints based on the analysis of (1)H(N), (15)N, (1)H(alpha), (13)C(alpha), (13)CO, and (13)C(beta) chemical shifts, and HNHA, HNHB and HACAHB-COSY spectra. Structures were calculated using CYANA 2.0 and refined in AMBER 8. sYuaF is composed of an extended N-terminal alpha-helix and a beta-barrel formed by five beta-strands. This beta-sheet core structure is well known from the diverse class of OB-fold proteins and can also be found in the distantly related NfeD protein Ph0471 from the archaeon P. horikoshii. Despite significant differences of their amino acid sequences the structural homology of these proteins suggests a conserved function of SPFH-associated NfeD proteins.     

Structure and conformation of linear peptides. VIII. Structure of t-Boc-glycyl-L-phenylalanine

The crystal structure of t-Boc-glycyl-L-phenylalanine (C14H22N2O5, molecular weight = 298) has been determined. Crystals are monoclinic, space group P2(1), with a = 7.599(1) A, b = 9.576(2), c = 12.841(2), beta = 97.21(1) degrees, Z = 2, Dm = 1.149, Dc = 1.168 g X cm-3. Trial structure was obtained by direct methods and refined to a final R-index of 0.064 for 1465 reflections with I greater than 1 sigma. The peptide unit is trans planar and is nearly perpendicular to the plane containing the urethane moiety. The plane of the carboxyl group makes a dihedral angle of 16.0 degrees with the peptide unit. The backbone torsion angles are omega 0 = -176.9 degrees, phi 1 = -88.0 degrees, psi 1 = -14.5 degrees, omega 1 = 176.4 degrees, phi 2 = -164.7 degrees and psi 2 = 170.3 degrees. The phenylalanine side chain conformation is represented by the torsion angles chi 1 = 52.0 degrees, chi 2 = 85.8 degrees.     

Backbone assignments and secondary structure of the Escherichia coli enzyme-II mannitol A domain determined by heteronuclear three-dimensional NMR spectroscopy.

This report presents the backbone assignments and the secondary structure determination of the A domain of the Escherichia coli mannitol transport protein, enzyme-IImtl. The backbone resonances were partially assigned using three-dimensional heteronuclear 1H NOE 1H-15N single-quantum coherence (15N NOESY-HSQC) spectroscopy and three-dimensional heteronuclear 1H total correlation 1H-15N single-quantum coherence (15N TOCSY-HSQC) spectroscopy on uniformly 15N enriched protein. Triple-resonance experiments on uniformly 15N/13C enriched protein were necessary to complete the backbone assignments, due to overlapping 1H and 15N frequencies. Data obtained from three-dimensional 1H-15N-13C alpha correlation experiments (HNCA and HN(CO)CA), a three-dimensional 1H-15N-13CO correlation experiment (HNCO), and a three-dimensional 1H alpha-13C alpha-13CO correlation experiment (COCAH) were combined using SNARF software, and yielded the assignments of virtually all observed backbone resonances. Determination of the secondary structure of IIAmtl is based upon NOE information from the 15N NOESY-HSQC and the 1H alpha and 13C alpha secondary chemical shifts. The resulting secondary structure is considerably different from that reported for IIAglc of E. coli and Bacillus subtilis determined by NMR and X-ray.     

Spectral fitting for signal assignment and structural analysis of uniformly <Superscript>13</Superscript>C-labeled solid proteins by simulated annealing based on chemical shifts and spin dynamics

We describe an approach for the signal assignment and structural analysis with a suite of two-dimensional (13)C-(13)C magic-angle-spinning solid-state NMR spectra of uniformly (13)C-labeled peptides and proteins. We directly fit the calculated spectra to experimental ones by simulated annealing in restrained molecular dynamics program CNS as a function of atomic coordinates. The spectra are calculated from the conformation dependent chemical shift obtained with SHIFTX and the cross-peak intensities computed for recoupled dipolar interactions. This method was applied to a membrane-bound 14-residue peptide, mastoparan-X. The obtained C', C(alpha) and C(beta) chemical shifts agreed with those reported previously at the precisions of 0.2, 0.7 and 0.4 ppm, respectively. This spectral fitting program also provides backbone dihedral angles with a precision of about 50 degrees from the spectra even with resonance overlaps. The restraints on the angles were improved by applying protein database program TALOS to the obtained chemical shifts. The peptide structure provided by these restraints was consistent with the reported structure at the backbone RMSD of about 1 A.     

Structure and conformation of linear peptides. XII. Structure of tryptophanyl-glycyl-glycine dihydrate

The crystal structure of a tripeptide, tryptophanyl-glycyl-glycine dihydrate (C15H18N4O4.2H2O, molecular weight = 354) has been determined. The crystals are orthorhombic, space group P2(1)2(1)2(1), with a = 7.875 (1) A, b = 9.009(1), c = 24.307(1) and Z = 4. The final R-index is 0.058 for 1488 reflections [sin theta)/lambda less than or equal to 0.6 A-1) with I greater than 2 sigma (I). The molecule exists as a zwitterion, with terminal NH3+ and COO- groups. The peptide units are trans and nearly perpendicular to the plane of the carboxyl group. The backbone torsion angles are: psi 1 = 132.7 degrees, omega 1 = 174.2 degrees, phi 2 = 88.2 degrees, psi 2 = 8.6 degrees, omega 2 = -179.8 degrees, phi 3 = -85.2 degrees, psi 31 = -178.1 degrees, psi 32 = 5.0 degrees. For the sidechain of tryptophan, chi 1 = -171.6 degrees, chi 2 = 101.0 degrees.     

Structure and conformation of linear peptides. XIII. Structure of L-phenylalanyl-glycyl-glycine

The crystal structure of a tripeptide, L-phenylalanyl-glycyl-glycine (C13H17N3O4), molecular weight = 279.3, has been determined. The crystals are orthorhombic, space group P2(1)2(1)2(1), with a = 5.462(1) A, b = 15.285(5), c = 16.056(4), Z = 4, and P (calc) = 1.384 g.cm-3. The final R-index is 0.052 for 866 reflections with sin theta/lambda less than or equal to 0.55 A-1 and I greater than 1 sigma. The molecule exists as a zwitterion, with the N-terminus protonated and the C-terminus in an ionized form. Both the peptide units are in the trans configuration and planar, though one of them shows significant deviations from planarity ([delta w[ = 5.1 degrees). The peptide backbone is folded, with the torsion angles of: psi 1 = 116.2(5) degrees, omega 1 = 178.8(4), phi 2 = -89.7(5). psi 2 = -28.9(6), omega 2 = -174.9(4), phi 3 = 134.9(5), psi 31 = 7.8(6), psi 32 = -172.6(4). The terminal glycine adopts a "D-residue" conformation. For the sidechain of phenylalanine, chi 1 = 175.5(4), chi 2 = -127.0(6).     

Structural and dynamic characterization of an unfolded state of poplar apo-plastocyanin formed under nondenaturing conditions

Plastocyanin is a predominantly beta-sheet protein containing a type I copper center. The conformational ensemble of a denatured state of apo-plastocyanin formed in solution under conditions of low salt and neutral pH has been investigated by multidimensional heteronuclear NMR spectroscopy. Chemical shift assignments were obtained by using three-dimensional triple-resonance NMR experiments to trace through-bond heteronuclear connectivities along the backbone and side chains. The (3)J(HN,Halpha) coupling constants, (15)N-edited proton-proton nuclear Overhauser effects (NOEs), and (15)N relaxation parameters were also measured for the purpose of structural and dynamic characterization. Most of the residues corresponding to beta-strands in the folded protein exhibit small upfield shifts of the (13)C(alpha) and (13)CO resonances relative to random coil values, suggesting a slight preference for backbone dihedral angles in the beta region of (phi,psi) space. This is further supported by the presence of strong sequential d(alphaN)(i, i + 1) NOEs throughout the sequence. The few d(NN)(i, i + 1) proton NOEs that are observed are mostly in regions that form loops in the native plastocyanin structure. No medium or long-range NOEs were observed. A short sequence, between residues 59 and 63, was found to populate a nonnative helical conformation in the unfolded state, as indicated by the shift of the (13)C(alpha), (13)CO, and (1)H(alpha) resonances relative to random coil values and by the decreased values of the (3)J(HN,Halpha) coupling constants. The (15)N relaxation parameters indicate restriction of motions on a nanosecond timescale in this region. Intriguingly, this helical conformation is present in a sequence that is close to but not in the same location as the single short helix in the native folded protein. The results are consistent with earlier NMR studies of peptide fragments of plastocyanin and confirm that the regions of the sequence that form beta-strands in the native protein spontaneously populate the beta-region of (phi,psi) space under folding conditions, even in the absence of stabilizing tertiary interactions. We conclude that the state of apo-plastocyanin present under nondenaturing conditions is a noncompact unfolded state with some evidence of nativelike and nonnative local structuring that may be initiation sites for folding of the protein.     

3d Trosy-HncaCodedcb and Trosy-HncaCodedco Experiments: Triple Resonance nmr Experiments With two Sequential Connectivity Pathways and High Sensitivity

The concept of chemical shift-coding monitors chemical shifts in multi-dimensional NMR experiments without additional polarization transfer elements and evolution periods. The chemical shifts are coded in the line-shape of the cross-peak through an apparent scalar coupling dependent upon the chemical shift. This concept is applied to the three-dimensional triple-resonance experiment HNCA adding the information of (13)C(beta) or (13)C' chemical shifts. On average, the proposed TROSY-HNCA(coded)CB experiment is a factor of 2 less sensitive than the HNCA experiment. However, it contains correlations via the chemical shifts of both (13)C(alpha) and (13)C(beta), and provides up to three times higher resolution along the (13)C(alpha) chemical shift axis. Thus, it dramatically reduces ambiguities in linking the spin systems of adjacent residues in the protein sequence during the sequential assignment. The TROSY-HNCA(coded)CO experiment which is conceptually similar contains correlations via the chemical shifts of (13)C(alpha) and (13)C' without major signal losses. The proposed triple resonance experiments are applied to a approximately 70% (2)H, approximately 85% (13)C,(15)N labeled protein with a molecular weight of 25 kDa.     

Solution secondary structure of calcium-saturated troponin C monomer determined by multidimensional heteronuclear NMR spectroscopy.

The solution secondary structure of calcium-saturated skeletal troponin C (TnC) in the presence of 15% (v/v) trifluoroethanol (TFE), which has been shown to exist predominantly as a monomer (Slupsky CM, Kay CM, Reinach FC, Smillie LB, Sykes BD, 1995, Biochemistry 34, forthcoming), has been investigated using multidimensional heteronuclear nuclear magnetic resonance spectroscopy. The 1H, 15N, and 13C NMR chemical shift values for TnC in the presence of TFE are very similar to values obtained for calcium-saturated NTnC (residues 1-90 of skeletal TnC), calmodulin, and synthetic peptide homodimers. Moreover, the secondary structure elements of TnC are virtually identical to those obtained for calcium-saturated NTnC, calmodulin, and the synthetic peptide homodimers, suggesting that 15% (v/v) TFE minimally perturbs the secondary and tertiary structure of this stably folded protein. Comparison of the solution structure of calcium-saturated TnC with the X-ray crystal structure of half-saturated TnC reveals differences in the phi/psi angles of residue Glu 41 and in the linker between the two domains. Glu 41 has irregular phi/psi angles in the crystal structure, producing a kink in the B helix, whereas in calcium-saturated TnC, Glu 41 has helical phi/psi angles, resulting in a straight B helix. The linker between the N and C domains of calcium-saturated TnC is flexible in the solution structure.     

A sequence preference for nucleation of alpha-helix--crystal structure of Gly-L-Ala-L-Val and Gly-L-Ala-L-Leu: some comments on the geometry of leucine zippers

The synthetic peptide Gly-L-Ala-L-Val (C10H19N3O4.3H2O; GAV) crystallizes in the monoclinic space group P21, with a = 8.052(2), b = 6.032(2), c = 15.779(7) A, beta = 98.520(1) degree, V = 757.8 A3, Dx = 1.312 g cm-3, and Z = 2. The peptide Gly-L-Ala-L-Leu (C11H21N3O4.3H2O; GAL) crystallizes in the orthorhombic space group P212121, with a = 6.024(1), b = 8.171(1), c = 32.791(1) A, V = 1614 A3, Dx = 1.289 g cm-3, and Z = 4. Their crystal structures were solved by direct methods using the program SHELXS-86, and refined to an R index of 0.05 for 1489 reflections for GAV and to an R index of 0.05 for 1563 reflections for GAL. The tripeptides exist as a zwitterion in the crystal and assume a near alpha-helical backbone conformation with the following torsion angles: psi 1 = -150.7 degrees; phi 2, psi 2 = -68.7 degrees, -38.1 degrees; phi 3, psi 32 = -74.8 degrees, -44.9 degrees, 135.9 degrees for GAV; psi 1 = -150.3 degrees; phi 2, psi 2 = -67.7 degrees, -38.9 degrees; phi 3, psi 31, psi 32 = -72.2 degrees, -45.3 degrees, 137.5 degrees for GAL. Both the peptide units in both of the tripeptides show significant deviation from planarity [omega 1 = -171.3(6) degrees and omega 2 = -172.0(6) degrees for GAV; omega 1 = -171.9(5) degrees and omega 2 = -173.2(6) degrees for GAL]. The side-chain conformational angles chi 21 and chi 22 are -61.7(5) degrees and 175.7(5) degrees, respectively, for valine, and the side-chain conformations chi 12 and chi 23's are -68.5(5) degrees and (-78.4(6) degrees, 159.10(5) degrees) respectively, for leucine. Each of the tripeptide molecule is held in a near helical conformation by a water molecule that bridges the NH3+ and COO- groups, and acts as the fourth residue needed to complete the turn by forming two hydrogen bonds. Two other water molecules form intermolecular hydrogen bonds in stabilizing the helical structure so that the end result is a column of molecules that looks like an alpha-helix.