NH Chemical Shifts Research Articles

Interactions of the products, 8-oxo-dGMP, dGMP, and pyrophosphate with the MutT nucleoside triphosphate pyrophosphohydrolase.

The MutT enzyme from E. coli, in the presence of a divalent cation, catalyzes the hydrolysis of nucleoside- and deoxynucleoside-triphosphate (NTP) substrates by nucleophilic substitution at Pbeta, to yield a nucleotide (NMP) and PPi. The best substrate of MutT is believed to be the mutagenic nucleotide 8-oxo-dGTP, on the basis of its 10(3.4)-fold lower K(m) than that of dGTP (Maki, H., and Sekiguchi, M. (1992) Nature 355, 273-275). To determine the true affinity of MutT for an 8-oxo-nucleotide and to elucidate the kinetic scheme, product inhibition by 8-oxo-dGMP and dGMP and direct binding of these nucleotides to MutT were studied. With Mg(2+)-activated dGTP hydrolysis, 8-oxo-dGMP is a noncompetitive inhibitor with K(I)(sl)(o)(pe) = 49 nM, which is 10(4.6)-fold lower than the K(I)(sl)(o)(pe)of dGMP (1.7 mM). Similarly, the K(I)(intercept) of 8-oxo-dGMP is 10(4.0)-fold lower than that of dGMP. PPi is a linear uncompetitive inhibitor, suggesting that it dissociates first from the product complex, followed by the nucleotide. Noncompetitive inhibition by dGMP and 8-oxo-dGMP indicates an "iso" mechanism in which the nucleotide product leaves an altered form of the enzyme which slowly reverts to the form which binds substrate. Consistent with this kinetic scheme, (1)H-(15)N HSQC titration of MutT with dGMP reveals weak binding and fast exchange from one site with a K(D) = 1.8 mM, in agreement with its K(I)(sl)(o)(pe). With 8-oxo-dGMP, tight binding and slow exchange (n = 1.0 +/- 0.1, K(D) < 0.25 mM) are found. Isothermal calorimetric titration of MutT with 8-oxo-dGMP yields a K(D) of 52 nM, in agreement with its K(I)(sl)(o)(pe). Changing the metal activator from Mg(2+) to Mn(2+) had little effect on the K(I)(sl)(o)(pe) of dGMP or of 8-oxo-dGMP, consistent with the second-sphere enzyme-M(2+)-H(2)O-NTP-M(2+) complex found by NMR (Lin, J., Abeygunawardana, C., Frick, D. N., Bessman, M. J., and Mildvan, A. S. (1997) Biochemistry 36, 1199-1211), but it decreased the K(I) of PPi 12-fold, suggesting direct coordination of the PPi product by the enzyme-bound divalent cation. The tight binding of 8-oxo-dGMP to MutT (DeltaG degrees = -9.8 kcal/mol) is driven by a highly favorable enthalpy (<DeltaH(binding)> = -32 +/- 7 kcal/mol), with an unfavorable entropy (<-TDeltaS(o)(binding)> = +22 +/- 7 kcal/mol), as determined by van't Hoff analysis of the effect of temperature on the K(I)(sl)(o)(pe) and by isothermal titration calorimetry in two buffer systems. The binding of 8-oxo-dGMP to MutT induces changes in backbone (15)N and NH chemical shifts of 62 residues widely distributed throughout the protein, while dGMP binding induces smaller changes in only 22 residues surrounding the nucleotide binding site, suggesting that the unusually high affinity of MutT for 8-oxo-nucleotides is due not only to interactions with the altered 8-oxo or 7-NH positions on guanine, but results primarily from diffuse structural changes which tighten the protein structure around the 8-oxo-nucleotide.

Solution structure of a D,L-alternating oligonorleucine as a model of double-stranded antiparallel beta-helix.

Conformational characteristics of alternating D,L linear peptides are of particular interest because of their capacity to form transmembrane channels with different transport properties, as some natural antibiotics do. Single- and double-stranded beta-helical structures are common for alternating D,L peptides. The stability of the beta-helix depends on several structural factors, such as the backbone peptide length, type and position of side chains, and nature of terminal groups. The NMR and molecular dynamics solution conformation of a synthetic alternating D,L-oligopeptide with 15 norleucines (XVMe) has been used as a model to get insight in to the conformational features of double-stranded beta-helix structures. The NH chemical shift values (delta(NH)) and long-range nuclear Overhauser effects (NOE) cross peaks, in particular interstrand connectivities, clearly point to an antiparallel double-stranded beta-helix for the XVMe major conformation in solution. An extensive set of distances (from NOE cross peaks) and H-bonds (from delta(NH)) has been included in the molecular dynamics calculations. The experimental NMR data and theoretical calculations clearly indicate that the most probable conformation of XVMe in solution is a double-strand antiparallel beta(5.6) increasing decreasing-helix structure.

Conformationally constrained macrocycles that mimic tripeptide beta-strands in water and aprotic solvents.

The beta-strand conformation is unknown for short peptides in aqueous solution, yet it is a fundamental building block in proteins and the crucial recognition motif for proteolytic enzymes that enable formation and turnover of all proteins. To create a generalized scaffold as a peptidomimetic that is pre-organized in a beta-strand, we individually synthesized a series of 15-22-membered macrocyclic analogues of tripeptides and analyzed their structures. Each cycle is highly constrained by two trans amide bonds and a planar aromatic ring with a short nonpeptidic linker between them. A measure of this ring strain is the restricted rotation of the component tyrosinyl aromatic ring (DeltaG(rot) 76.7 kJ mol(-1) (16-membered ring), 46.1 kJ mol(-1) (17-membered ring)) evidenced by variable temperature proton NMR spectra (DMF-d(7), 200-400 K). Unusually large amide coupling constants ((3)J(NH-CHalpha) 9-10 Hz) corresponding to large dihedral angles were detected in both protic and aprotic solvents for these macrocycles, consistent with a high degree of structure in solution. The temperature dependence of all amide NH chemical shifts (Deltadelta/T 7-12 ppb/deg) precluded the presence of transannular hydrogen bonds that define alternative turn structures. Whereas similar sized conventional cyclic peptides usually exist in solution as an equilibrium mixture of multiple conformers, these macrocycles adopt a well-defined beta-strand structure even in water as revealed by 2-D NMR spectral data and by a structure calculation for the smallest (15-membered) and most constrained macrocycle. Macrocycles that are sufficiently constrained to exclusively adopt a beta-strand-mimicking structure in water may be useful pre-organized and generic templates for the design of compounds that interfere with beta-strand recognition in biology.

A rare occurrence of a β‐turn in an amyloid βA4 peptide

AbstractThe fragment β(25–35) of the amyloid β‐peptide, like its parent βA4, has shown neurotrophic and late neurotoxic activities in cultured cells. The 3D structure of this important peptide was examined by 1H and 13C 2D‐NMR and MD simulations in DMSO‐d6 and water. The NMR parameters of chemical shift, 3J(N,Hα) coupling constants, temperature coefficients of NH chemical shifts and the pattern of intra and inter‐residue NOEs were used to deduce the structures. In DMSO‐d6, the peptide was found to take up a type I β‐turn around the C‐terminal residues Ile8–Gly9–Leu10–Met11, whereas in water at pH 5.5, it adopts a random coil conformation. This is only the second report of a β‐turn in the β‐amyloid class of peptides. The solution structures generated using restrained molecular dynamics were refined by MARDIGRAS to an R factor of 0.33 in the case of DMSO‐d6 and to 0.56 for water. Copyright © 2002 John Wiley &amp; Sons, Ltd.

Conformation of a model peptide of the tandem repeat decapeptide in mussel adhesive protein by NMR and MD simulations

The conformation of a model peptide (Ala–Lys–Pro–Ser–Tyr–Hyp–Hyp–Thr–Tyr–Lys) of the tandem repeat decapeptide sequence of Mytilus edulis foot protein-1 (mefp-1) has been studied by 1H and 13C 2D-NMR in three diverse media—DMSO-d 6, water (pH 3.5) and 40% hexafluoroacetone (HFA). Various NMR parameters that were used to deduce the secondary structure were chemical shift ( 1H and 13C), temperature coefficients of NH chemical shifts, 3 J NH α coupling constants and the pattern of intra and inter-residue NOEs. Molecular dynamics simulations making integral use of the NMR data shows that the conformation of the peptide is conserved in all the three media. The structure in the three solvents is best defined as a left-handed polyproline II helix (PPII).

The hairpin structure of the (6)F1(1)F2(2)F2 fragment from human fibronectin enhances gelatin binding.

The solution structure of the (6)F1(1)F2(2)F2 fragment from the gelatin-binding region of fibronectin has been determined (Protein Data Bank entry codes 1e88 and 1e8b). The structure reveals an extensive hydrophobic interface between the non-contiguous (6)F1 and (2)F2 modules. The buried surface area between (6)F1 and (2)F2 ( approximately 870 A(2)) is the largest intermodule interface seen in fibronectin to date. The dissection of (6)F1(1)F2(2)F2 into the (6)F1(1)F2 pair and (2)F2 results in near-complete loss of gelatin-binding activity. The hairpin topology of (6)F1(1)F2(2)F2 may facilitate intramolecular contact between the matrix assembly regions flanking the gelatin-binding domain. This is the first high-resolution study to reveal a compact, globular arrangement of modules in fibronectin. This arrangement is not consistent with the view that fibronectin is simply a linear 'string of beads'.

Molecular carpentry: piecing together helices and hairpins in designed peptides.

The design of a peptide that contains two distinct elements of secondary structure, helix and beta-hairpin, is described. Two designed 17-residue peptides: Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Gly-Gly-Leu-Phe-Val-D-Pro-Gly-Leu-Phe-Val-OMe (I) and Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-Gly-Gly-Leu-Val-Val-D-Pro-Gly-Leu-Val-Val-OMe (II) have been conformationally characterized by NMR spectroscopy. Peptides I and II contain a seven-residue helical module at the N terminus and a eight-residue beta-hairpin module at the C terminus, which are connected by a conformationally flexible Gly-Gly segment. The choice of the secondary-structure modules is based upon prior crystallographic and spectroscopic analysis of the individual modules. Analysis of 500 MHz 1H NMR data, recorded as solutions in methanol, suggests that the observed pattern of chemical shifts, 3JHN CalphaH values, temperature coefficients of the NH chemical shifts, and backbone inter-residue nuclear Overhauser effects favor helical structures for residues 1-7 and beta-hairpin structures for residues 10-17. The spectroscopic data are compatible with termination of the helical segment by formation of a Schellman motif; this restricts Gly(8) to a left-handed alpha-helical conformation. Gly(9) is the only residue with multiple conformational possibilities in phi,psi space. Possible orientations of the two secondary-structure modules are considered. This study validates the use of stereochemically rigid peptide modules as prefabricated elements in the construction of synthetic protein mimics.

The structural basis for the perturbed pKa of the catalytic base in 4-oxalocrotonate tautomerase: kinetic and structural effects of mutations of Phe-50.

The amino-terminal proline of 4-oxalocrotonate tautomerase (4-OT) functions as the general base catalyst in the enzyme-catalyzed isomerization of beta,gamma-unsaturated enones to their alpha,beta-isomers because of its unusually low pK(a) of 6.4 +/- 0.2, which is 3 units lower than that of the model compound, proline amide. Recent studies show that this abnormally low pK(a) is not due to the electrostatic effects of nearby cationic residues (Arg-11, Arg-39, and Arg-61) [Czerwinski, R. M., Harris, T. K., Johnson, Jr., W. H., Legler, P. M., Stivers, J. T., Mildvan, A. S., and Whitman, C. P. (1999) Biochemistry 38, 12358-12366]. Hence, it may result solely from a low local dielectric constant of 14.7 +/- 0.8 at the otherwise hydrophobic active site. Support for this mechanism comes from the study of mutants of the active site Phe-50, which is 5.8 A from Pro-1 and is one of 12 apolar residues within 9 A of Pro-1. Replacing Phe-50 with Tyr does not significantly alter k(cat) or K(m) and results in a pK(a) of 6.0 +/- 0.1 for Pro-1 as determined by (15)N NMR spectroscopy, comparable to that observed for wild type. (1)H-(15)N HSQC and 3D (1)H-(15)N NOESY HSQC spectra of the F50Y mutant demonstrate its conformation to be very similar to that of the wild-type enzyme. In the F50Y mutant, the pK(a) of Tyr-50 is increased by two units from that of a model compound N-acetyl-tyrosine amide to 12.2 +/- 0.3, as determined by UV and (1)H NMR titrations, yielding a local dielectric constant of 13.4 +/- 1.7, in agreement with the value of 13.7 +/- 0.3 determined from the decreased pK(a) of Pro-1 in this mutant. In the F50A mutant, the pK(a) of Pro-1 is 7.3 +/- 0.1 by (15)N NMR titration, comparable to the pK(a) of 7.6 +/- 0.2 found in the pH vs k(cat)/K(m) rate profile, and is one unit greater than that of the wild-type enzyme, indicating an increase in the local dielectric constant to a value of 21.2 +/- 2.6. A loss of structure of the beta-hairpin from residues 50 to 57, which covers the active site, and is the site of the mutation, is indicated by the disappearance in the F50A mutant of four interstrand NOEs and one turn NOE found in wild-type 4-OT. (1)H-(15)N HSQC spectra of the F50A mutant reveal widespread and large changes in the backbone (15)N and NH chemical shifts including those of Gly residues 48, 51, 53, and 54 causing their loss of dispersion at 23 degrees C and their disappearance at 43 degrees C due to rapid exchange with solvent. These observations confirm that the active site of the F50A mutant is more accessible to the external aqueous environment, causing an increase in the local dielectric constant and in the pK(a) of Pro-1. In addition, the F50A mutation decreased k(cat) 167-fold and increased K(m) 11-fold from those of the wild-type enzyme, suggesting an important role for the hydrophobic environment in catalysis, beyond that of decreasing the pK(a) of Pro-1. The F50I and F50V mutations destabilize the protein and decrease k(cat) by factors of 58 and 1.6, and increase K(m) by 3.3- and 3.8-fold, respectively.

Nanoconstruction of Microspheres and Microcapsules Using Proton-Induced Phase Transitions: Molecular Self-Recognition by Diamide Diacids in Water

Bis(N α -amido-L-phenylalanine)-1,1-cyclobutane dicarboxylate (5) was studied by Fourier transform infrared (FTIR) spectroscopy, variable-temperature NMR (VT-NMR), transmission electron microscopy, X-ray crystallography, Raman microscopy, and a novel imaging technique known as soft X-ray microscopy (XRM). Diamide diacid 5 was shown to self-associate into solid microspheres during a proton-induced phase transition from the solvated state to the desolvated assembled state. These diverse techniques allowed for the delineation of the molecular recognition events involved in the assembly process. X-ray crystallography revealed that 5 packs in a bundled helical array comprised of two types of intermolecular hydrogen bonds (i.e., OC=O…HN and COOH…O=CN). VT-NMR and IR measurements of 5 (1 mM in CDCl 3 ) revealed the small temperature dependence of the amide NH chemical shift (Δδ/ΔT = -1.1 ppb/K) and the availability of the free amide NH of 5 to form intermolecular hydrogen bonds. Supramolecular rodlike structures were observed during the aqueous assembly of 5 into microspheres by XRM. Raman microscopy confirmed that nearly identical bonding patterns are present in the assembled microsphere and the crystal architecture of 5. Collectively, these observations provide compelling evidence that the assembly of 5 occurs via crystalline supramolecular intermediates, which are similar in shape and have complementary bonding motifs for proper self-recognition. Competition experiments involving varying concentrations of 5 and its microcapsule-forming cyclopropane analogue 3 revealed that molecular fidelity was less important to the microsphere-forming process than the related capsule-forming process.

Hydrogen-Bonded Double Strands: Crystal Structure and Spectroscopic Propertiesof a 2,2′-Dipyrryl Ketone

The synthesis, crystal structure determination, conformational analysis, and spectroscopic properties of 3,3′-diethyl-4,4′-dimethyl-2,2′-dipyrryl ketone (1) are reported. The dipyrryl ketone is a model for the dipyrrole core of 10-oxobilirubin, a presumed metabolite in alternate pathways of excretion of the yellow pigment of jaundice, bilirubin. In the crystal, 1 adopts a helical conformation, with a molecule of one helicity being hydrogen-bonded to two molecules of the opposite helicity. Thus, 1 self-assembles via hydrogen bonding into supramolecular double-stranded arrays, where molecules of the same helicity comprise one strand and are paired through hydrogen bonding to molecules of opposite helicity in the second strand. In the observed molecular conformation each pyrrole ring and adjacent carbonyl group are rotated into an sc conformation (torsion angle ∼29 °), with each N-H pointing in the same direction as the C*O. Molecular mechanics/dynamics calculations predict the sc,sc conformation, absent hydrogen bonding, to be the most stable, but only by a few tenths of a kj/mol. In CHCl3, 1 is monomeric according to vapor pressure osmometry studies (\(\)). 1H NMR NH chemical shifts in CDCl3 suggest a predominantly anti orientation of the C=O and pyrrole NHs, which is opposite to the orientation observed in the crystal.

Synthesis of delta(E)Phe-containing tripeptide via photoisomerization and its conformation in solution.

A new synthetic route to (E)-beta-phenyl-alpha,beta-dehydroalanine (delta(E)Phe)-containing peptide was presented via photochemical isomerization of the corresponding (Z)-beta-phenyl-alpha,beta-dehydroalanine (delta(Z)Phe)-containing peptide. By applying this method to Boc-Ala-delta(Z)Phe-Val-OMe (Z-I: Boc, t-butoxycarbonyl; OMe, methoxy), Boc-Ala-delta(E)Phe-Val-OMe (E-I) was obtained. The identification of peptide E-I was evidenced by 1H-nmr, 13C-nmr, and uv absorption spectroscopy, elemental analysis, and hydrogenation. The conformation of peptide E-I in CDCl3 was investigated by 1H-nmr spectroscopy (solvent dependence of NH chemical shift and difference nuclear Overhauser effect). Interestingly, peptide E-I differed from peptide Z-I in the hydrogen-bonding mode. Namely, for peptide Z-I, only Val NH participates in intramolecular hydrogen bonding, which leads to a type II beta-turn conformation supported by hydrogen bonding between CO(Boc) and NH(Val). On the other hand, for peptide E-I, two NHs, delta(E)Phe NH and Val NH, participate in intramolecular hydrogen bonding. In both peptides, a remarkable NOE (approximately 11-13%) was observed for Ala C(alpha) H-deltaPhe NH pair. Based on the nmr data and conformational energy calculation, it should be concluded that peptide E-I takes two consecutive gamma-turn conformations supported by hydrogen bonding between CO(Boc) and NH(delta(E)Phe), and between CO(Ala) and NH(Val) as its plausible conformation.

Synthesis, microbicidal activity, and solution structure of the dodecapeptide from bovine neutrophils

The dodecapepetide sequence R–L–C–R–I–V–V–I–R–V–C–R with a disulfide bridge between the cysteine residues found in bovine neutrophils was synthesized by solid-phase procedures. Its antimicrobial activity against oral microorganisms such as Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Streptococcus mutans, and Streptococcus gordonii was examined, and its structural features were examined by CD and determined by two-dimensional (2D) nmr. The strains P. gingivalis (W50 and 381), A. actinomycetemcomitans (Y4 and 67), S. gordonii (DL1), and S. mutans (GS5) are found to be highly sensitive to this peptide at 2–2.5 μM concentrations, suggesting that the dodecapeptide is a potent antibiotic for oral pathogens. The weak negative n–σ* band observed at ∼265–270 nm in the CD spectra of this peptide provides evidence for the presence of a disulfide bridge. The negative n–π* band at ∼200 nm and the positive π–π* band at 185 nm suggest a folded structure for this peptide. The negative n–π* shifts from 200 to 206 nm with an increase in intensity in dipalmitoylphosphotidylcholine vesicles, suggesting that the peptide might associate to form higher order aggregates in lipid medium. The assignment of backbone and side-chain proton resonances has been accomplished by the combined analysis of 2D total correlated and nuclear Overhauser effect spectroscopy. The temperature dependence of amide NH chemical shifts and 1H–2H exchange effect on amide NH resonances indicate the involvement of amide NH groups of Cys3, Ile5, Ile8, Val10, and Arg12 in intramolecular hydrogen bonding. The coupling constant (J) values, the set of medium-, short-, and long-range nuclear Overhauser effects, and the results of restrained structure calculation using the distance geometry algorithm for nmr applications provide evidence for a folded, loop-like structure with a type I (III) β-turn involving Ile5, Val6, Val7, and Ile8, and two antiparallel β-strands involving the N-terminal Arg1, Leu2, Cys3, and Val4 and the C-terminal Arg9, Val10, Cys11, and Arg12 residues. The structure of the dodecapeptide mimics the amphiphilic structure of large 30–35 residue defensins and the peptide appears to exhibit similar antimicrobial potency. © 2000 John Wiley & Sons, Inc. Biopoly 53: 281–292, 2000

Structures of ovine corticotropin-releasing factor and its Ala32 mutant as studied by CD and NMR techniques.

The corticotropin-releasing factor (CRF) is a 41-amino acid peptide-amide hormone, which mediates a general stress-response. It has been reported that the substitution of His-32 in the ovine CRF (oCRF) with Ala brings about a 4.5-fold increase in activity [Kornreich et al. (1992) J. Med. Chem. 35, 1870-76]. Here, we have determined the secondary structure of this Ala-substituted ovine CRF ([Ala32]oCRF) and compare it with that of oCRF using circular dichroism (CD) and NMR techniques in trifluoroethanol (TFE) solution, which is known to stabilize the alpha-helix formation. In contrast to an earlier report, it was observed the alpha-helical structure extends to the C-terminus of oCRF. By analyzing the CalphaH and NH chemical shifts, the properties of local structures of oCRF were elucidated. The oCRF and [Ala32]oCRF have stable alpha-helical structures in the middle region, regardless of pH and temperature, and the alpha-helix initiation regions of these peptides are stabilized as the pH is decreased. However, the [Ala32]oCRF has a more stable alpha-helical structure than oCRF in the vicinity of the substitution region, and it is thought that this is the cause of the increased activity of [Ala32]oCRF.

Synthesis, microbicidal activity, and solution structure of the dodecapeptide from bovine neutrophils.

The dodecapepetide sequence R-L-C-R-I-V-V-I-R-V-C-R with a disulfide bridge between the cysteine residues found in bovine neutrophils was synthesized by solid-phase procedures. Its antimicrobial activity against oral microorganisms such as Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Streptococcus mutans, and Streptococcus gordonii was examined, and its structural features were examined by CD and determined by two-dimensional (2D) nmr. The strains P. gingivalis (W50 and 381), A. actinomycetemcomitans (Y4 and 67), S. gordonii (DL1), and S. mutans (GS5) are found to be highly sensitive to this peptide at 2-2.5 microM concentrations, suggesting that the dodecapeptide is a potent antibiotic for oral pathogens. The weak negative n-sigma* band observed at approximately 265-270 nm in the CD spectra of this peptide provides evidence for the presence of a disulfide bridge. The negative n-pi* band at approximately 200 nm and the positive pi-pi* band at 185 nm suggest a folded structure for this peptide. The negative n-pi* shifts from 200 to 206 nm with an increase in intensity in dipalmitoylphosphotidylcholine vesicles, suggesting that the peptide might associate to form higher order aggregates in lipid medium. The assignment of backbone and side-chain proton resonances has been accomplished by the combined analysis of 2D total correlated and nuclear Overhauser effect spectroscopy. The temperature dependence of amide NH chemical shifts and (1)H-(2)H exchange effect on amide NH resonances indicate the involvement of amide NH groups of Cys3, Ile5, Ile8, Val10, and Arg12 in intramolecular hydrogen bonding. The coupling constant (J(NH-C(alpha)H)) values, the set of medium-, short-, and long-range nuclear Overhauser effects, and the results of restrained structure calculation using the distance geometry algorithm for nmr applications provide evidence for a folded, loop-like structure with a type I (III) beta-turn involving Ile5, Val6, Val7, and Ile8, and two antiparallel beta-strands involving the N-terminal Arg1, Leu2, Cys3, and Val4 and the C-terminal Arg9, Val10, Cys11, and Arg12 residues. The structure of the dodecapeptide mimics the amphiphilic structure of large 30-35 residue defensins and the peptide appears to exhibit similar antimicrobial potency.

Conformational analysis of endothelin-1 analogs with indolizidinone amino acids incorporated at the C-terminus.

In light of the fact that a beta-turn conformation at the C-terminus of endothelin-1 (ET-1) could be responsible for activity at the ET(B) receptor, the incorporation of (3S, 6S, 9S)-2-oxo-3-amino-1-azabicyclo[4.3.0]nonane-9-carboxylic acid (IAA) should provide an elegant method to establish whether a formyl group on Trp21 plays a role in stabilizing a beta-turn. Eight linear ET-1 analogs, four formylated and four nonformylated, ET-1-(Leu17-Asp18-IAA-Trp21); ET-1-(Leu17-IAA-Ile20-Trp21); ET-1-(Leu17-Asp18-Pro19-Ile20-Trp21) and ET-1-(Leu17-Asp 8-Ile19-Pro20-Trp21) have been analyzed by high-resolution nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. Two-dimensional double quantum filtered correlation spectroscopy (DQFCOSY), total correlation spectroscopy (TOCSY) and nuclear Overhausen enhancement spectroscopy (NOESY) were resolved and analyzed for each molecule. Interspatial distance constraints were derived from the intensity of the NOESY connectivities, The formation of hydrogen bonding was monitored from the temperature-dependence of the NH chemical shifts. Molecular models calculated by means of distance geometry, simulated annealing and energy minimization, suggested a global elongated structure for the formylated analogs and a folded arrangement for the nonformylated derivatives, but no hydrogen bonding was detected at the C-terminus of ET-1 analogs.

An advantage for use of isotope labeling and NMR chemical shifts to analyze the structure of four homologous IgG-binding domains of staphylococcal protein A

Because of the complexity arising from the large molecular size and the amino acid sequence homologies of IgG-binding domains of Staphylococcal Protein A (SpA), we have introduced a combination of stable isotope labeling and both qualitative and quantitative investigations of the structural dependence of the NMR chemical shifts for its structure analysis. In order to enable selective isotope labeling with high efficiency, a mutated low molecular weight Protein A (LPA; MWt=27 kDa) which consists of E, D, A, B and 13 residues of the C-domain was used in this study. Amide proton chemical shifts, measured using uniformly 15N-labeled LPA and LPA labeled selectively with 15N-alanine, show that the turn between helices 1 and 2, and its tertiary interactions with helix 3, are very similar in all domains. This contradicts previous results obtained using independent structure calculations on isolated domains. The close similarity in NH and 15N chemical shifts of alanine residues in the interdomain linker suggests that the linker maintains a similar structure both in isolated domains and in the intact protein. We show that the high-field shifted methyl signal of Ala 48 is affected by the ring-current effect arising from Phe 30, and has a very similar helical environment in all four domains. Thus, helix 3 is present in all domains, as we previously reported [Kikuchi et al., J Biochem Biophys Method, 1999:38:203–208], even though it is not observed in the crystal structure [Deisenhofer J. Biochemistry 1981;20:2361–2370].

NMR Structures of a Nonapeptide from DNA Binding Domain of Human Polymerase-α Determined by Iterative Complete-Relaxation-Matrix Approach

Nuclear magnetic resonance structures of a nonapeptide, ERFKCPCPT, selected from the DNA binding domain of human polymerase-a, were determined by complete relaxation matrix analysis of transverse NOE data. The structures exhibit a type III turn with residues KCPC, and the remaining residues exhibit non-ordered structures. The turn was confirmed by α, N (i,i+3) connectivity, a low temperature coefficient of NH chemical shift (−3.1 × 1O−3) of the fourth residue, 3JNHα coupling constants, and characteristic CD peaks at 228 and 200 nm. Furthermore, ø and ψ dihedral angles for the i + 1, and i + 2 residues of the tum are found to be−80 and−41 and−60 and−40 degrees. The first proline residue is trans- while the second exists in both eis- and trans- configurations, with trans- being more than 80% populated. The trans-configuration was established from C5α-P6α correlation and ø and ψ angles of the proline. The five-membered proline ring is in DOWN puckered (C-β-exo/C-γ-endo) conformation. The structure of the peptide reveals that the two cysteine thiols are−5 A° apart and appropriately positioned to covalently bind cis-diamminedichloroplatinum(II), a widely used anti-cancer drug.

Nativelike structure and stability in a truncation mutant of a protein minidomain: the peripheral subunit-binding domain.

Despite its small size, the peripheral subunit-binding domain from the dihydrolipoamide acetyltransferase component of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex adopts a unique, compact structure. To determine whether the full 43 residue sequence is required for the domain to adopt a stable, nativelike structure, 3 proteins of different lengths were prepared. Psbd41 corresponds to residues 3-43 of the domain, psbd36 spans residues 6-41, and psbd33 comprises residues 7-39. Psbd41 folds in a cooperative, two-state fashion with a Tm of 53 degrees C and a stability at 25 degrees C of 2.2 kcal mol-1. Psbd36 is nearly as stable with a Tm of 48 degrees C and a stability of 1.8 kcal mol-1. Similar m-values and heat capacities suggest that psbd36 and psbd41 bury approximately the same surface area. Minimal differences in CalphaH and NH chemical shifts between psbd41 and psbd36 show that the two sequences adopt the same tertiary fold. On a per residue basis, DeltaH degrees and DeltaC degrees p fall within the range typical for single-domain globular proteins. Psbd33 is significantly less stable. It is not fully folded at 25 degrees C, and at all temperatures it shows broadened NMR lines. ANS titrations provide evidence that this is due to an equilibrium between nativelike and unfolded molecules rather than formation of a molten globule. The fraction of psbd33 molecules which are folded appear to adopt the same structure as the full-length domain. Thus, although more than the 33 residue core is required to form a fully stable native structure, the entire sequence is not required for folding.

17O chemical shifts and deuterium isotope effects on13C chemical shifts of intramolecularly hydrogen-bonded compounds

17O chemical shifts were measured in 40 enamines activated in the β-position by C=O, COO, NO2, SO and SO2 groups. Data for the oxygen-containing series of o-hydroxyacyl aromatics are also included for comparison. Intramolecular hydrogen bonding in the enamines is discussed in terms of the acceptor and donor groups and the separating link. 17O chemical shifts, the two-bond deuterium isotope shifts on C-α 13C shifts and 1H NH or OH chemical shifts are correlated to show the interrelations of these parameters in elucidating intramolecular hydrogen bonds and their strength in a wide variety of compounds. 17O chemical shifts in open-chain compounds are shown to reflect intramolecular hydrogen bonding by a change to lower frequency whereas for five-membered rings steric effects cause higher frequency chemical shifts. © 1998 John Wiley & Sons, Ltd.

Stabilization of a C7 equatorial gamma turn in DMSO-d6 by a ditryptophan crosslink

Covalent crosslinks can control local peptide conformation. In tripeptide sequences of the general formula Cys-Xxx-Cys, cysteine disulfides have been previously shown to enforce a C7 equatorial γ-turn conformation (also referred to as an inverse γ-turn). Much less is known about the effects of dityrosine and ditryptophan crosslinks on local peptide structure. In a series of tripeptides, ditryptophan crosslinks were formed using the two-step process of acid-promoted Mannich dimerization followed by oxidative aromatization. In these peptides, with the general formula Trp-Xxx-Trp (Xxx≠Gly), ditryptophan crosslinks were found to stabilize a C7 equatorial γ-turn conformation in DMSO-d6. Rigorous support for a C7 equatorial conformation in the crosslinked sequence Trp-Pro-Trp came from a variety of 1H NMR experiments and molecular modelling. Interproton distances were derived from NOE buildups that were determined through a series of double pulsed field gradient spin echo (DPFGSE) experiments. In addition, the small temperature dependence of the i+2 NH chemical shifts (Δδ/ΔT<2ppm/°C) provided further support for the intramolecular hydrogen bond which defines a γ-turn.