Spatial structure of the cardioactive octapeptide

The spatial structure of the cardioactive octapeptide Pro1-Gln2-Asp3-Pro4-Phe5-Leu6-Arg7-Ile8-NH2 was investigated using the theoretical conformational analysis. The low-energy conformations of the octapeptide molecule were found, the values of dihedral angles of the backbone and side chains of the amino acid residues constituting the peptide were determined, and the energies of intra-and interresidual interactions were estimated. It was shown that the spatial structure of this molecule represent six stable low-energy forms of the main chain.     

Assignment of histidine resonances in the 1H NMR (500 MHz) spectrum of subtilisin BPN' using site-directed mutagenesis

A spin-echo pulse sequence has been used to resolve the six histidine C-2H protons in the 500-MHz NMR spectrum of subtilisin BPN'. Five of these residues have been substituted by site-directed mutagenesis, and this has enabled a complete assignment of these protons to be obtained. Analysis of the pH titration curves of these signals has provided microscopic pKas for the six histidines in this enzyme. The pKas of the histidine residues in subtilisin BPN' have been compared with the values obtained for the histidines in the homologous enzyme from Bacillus licheniformis (subtilisin Carlsberg). Four of the five conserved histidines titrate with essentially identical pKa's in the two enzymes. It therefore appears that the assignments made for these residues in subtilisin BPN' can be transferred to subtilisin Carlsberg. On the basis of these assignments, the one histidine that titrates with a substantially different pKa in the two enzymes can be assigned to histidine-238. This difference in pKa has been attributed to a Trp to Lys substitution at position 241 in subtilisin Carlsberg.     

Spatial structure of Thr-Pro-Ala-Glu-Asp-Phe-Met-Arg-Phe-NH2 molecule

The spatial structure of cardioactive Thr-Pro-Ala-Glu-Asp-Phe-Met-Arg-Phe-NH₂ molecule has been investigated using a theoretical conformational analysis. The low-energy conformations of the molecule were found, the values of the backbone and side T-T chain dihedral angles of amino acid residues constituting the peptide were determined, and the energies of intra- and interresidual interactions were estimated. It is revealed that the spatial structure of this molecule can exist in 11 stable backbone forms.     

On the multiple-minima problem in the conformational analysis of polypeptides. IV. Application of the electrostatically driven Monte Carlo method to the 20-residue membrane-bound portion of melittin

The conformational space of the membrane-bound portion of melittin has been searched using the electrostatically driven Monte Carlo (EDMC) method with the ECEPP/2 (empirical conformational energy program for peptides) algorithm. The former methodology assumes that a polypeptide or protein molecule is driven toward the native structure by the combined action of electrostatic interactions and stochastic conformational changes associated with thermal movements. The algorithm produces a Monte Carlo search in the conformational hyperspace of the polypeptide using electrostatic predictions and a random sampling technique, combined with local minimization of the energy function, to locate low-energy conformations. As a result of 8 test calculations on the 20-residue membrane-bound portion of melittin, starting from six arbitrary and two completely random conformations, the method was able to locate a very low-energy region of the potential with a well-defined structure for the backbone. In all of the cases under study, the method found a cluster of similar low-energy conformations that agree well with the structure deduced from x-ray diffraction experiments and with one computed earlier by the build-up procedure.     

Spatial structure of isoleucine pentapeptides Glu-Phe-Leu-Arg-Ile-NH2 and Pro-Phe-Tyr-Arg-Ile-NH2

The spatial structure of two cardioactive isoleucine pentapeptides Glu-Phe-Leu-Arg-Ile-NH2 (I) and Pro-Phe-Tyr-Arg-Ile-NH2 (II) have been investigated using the theoretical conformational analysis. The low-energy conformations of these molecules were found, the values of dihedral angles of the backbone and side chains of the amino acid residues constituting these peptides were determined, and the energies of intra- and interresidual interactions were estimated. It was revealed that the spatial structure of molecule I can exist as five and that of molecule II as seven stable backbone forms.     

Tertiary structure of myelopeptides. II. Conformational analysis of Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro, Val-Val-Tyr-Pro-Asp, and Val-Asp-Pro-Pro

Theoretical conformational analysis was used to study the spatial structure and conformational properties of myelopeptides, bone marrow peptide mediators. The low-energy conformations of myelopeptides MP-4 (Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro), MP-5 (Val-Val-Tyr-Pro-Asp), and MP-6 (Val-Asp-Pro-Pro) were found; the values of dihedral angles of backbone and side chains of the amino acid residues were determined; and the energies of intra- and interresidual interactions were estimated.     

The interaction of a tyrosyl residue and carboxyl groups in the specific interaction between Streptomyces subtilisin inhibitor and subtilisin BPN'. A chemical modification study.

An ultraviolet absorption difference spectrum that is typical of a change in ionization state (pKa 9.7 leads to greater than 11.5) of a tyrosyl residue has been observed on the binding between Streptomyces subtilisin inhibitor (SSI) and subtilisin BPN' [EC 3.4.21.14] at alkaline pH, ionic strength 0.1 M, at 25 degrees C (Inouye, K., Tonomura, B., and Hiromi, K., submitted). When the complex of SSI and subtilisin BPN' is formed at an ionic strength of 0.6 M and pH 9.70, the characteristic features of the protonation of a tyrosyl residue in the difference spectrum are diminished. These results suggest that the pKa-shift of a tyrosyl residue observed at alkaline pH and lower ionic strength results from an electrostatic interaction. Nitration of tyrosyl residues of SSI and of subtilisin BPN' was performed with tetranitromethane (TNM). By measurements of the difference spectra observed on the binding of the tyrosyl-residue-nitrated SSI and the native subtilisin BPN', and on the binding of the native SSI and the tyrosyl-residue-nitrated subtilisin BPN' and alkaline pH, the tyrosyl residue in question was shown to be one out of the five tyrosyl residues of pKa 9.7 of the enzyme. This tyrosyl residue was probably either Tyr 217 or Tyr 104 on the basis of the reactivities of tyrosyl residues of the enzyme with TNM and their locations on the enzyme molecule. Carboxyl groups of SSI were modified by covalently binding glycine methyl ester with the aid of water-soluble carbodiimide, in order to neutralize the negative charges on SSI. In the difference spectrum which was observed on the binding of subtilisin BPN' and the 5.3-carboxyl-group-modified SSI at alkaline pH, the characteristic features of the protonation of a tyrosyl residue were essentially lost, and the difference spectrum is rather similar to that observed on the binding of the native SSI and the enzyme at neutral pH. This phenomenon indicates that the pKa of a tyrosyl residue of the enzyme is shifted upwards by interaction with carboxyl group(s) of SSI on the formation of the enzyme-inhibitor complex.     

Spatial structure of myelopeptides: I. Conformational analysis of MP-1, MP-2, and MP-3

Theoretical conformational analysis was used to study the spatial structure and conformational properties of myelopeptides, bone-marrow peptide mediators. The low-energy conformations of three hexapeptides MP-1 (Phe-Leu-Gly-Phe-Pro-Thr), MP-2 (Leu-Val-Val-Tyr-Pro-Trp), and MP-3 (Leu-Val-Cys-Tyr-Pro-Gln) were found, the values of dihedral angles of the backbone and side chains of the amino acid residues constituting these peptides were determined, and the energies of intra- and interresidual interactions were estimated.     

Photoreactivity of histidyl residues in subtilisins Novo and DY. Photooxidation of subtilisins

Subtilisins Novo and DY were photoinactivated in the presence of methylene blue according to first order kinetics. The competitive inhibitor N alpha-benzoyl-L-arginine protected significantly against inactivation. Under the conditions employed in this study a selective photooxidation of the active site histidine 64 was achieved. Rate constants of 0.32 X 10(-2), s-1 and 0.35 X 10(-2), s-1, were calculated for the Novo enzyme and subtilisin DY, respectively. Apparent pKa values of the catalytically important imidazole group of 7.0 +/- 0.1 (s. Novo) and 7.1 +/- 0.1 (s. DY) were directly determined. The histidyl residues in the two proteases, except the active site histidine, which is the first target of photooxidation, are "buried" in the interior of the protein globule. Conformational studies suggested that the photoreactive histidine is not involved in the stabilization of the protein conformation.     

Hydrogen-deuterium (H/D) exchange mapping of Abeta 1-40 amyloid fibril secondary structure using nuclear magnetic resonance spectroscopy

We describe here details of the hydrogen-deuterium (H/D) exchange behavior of the Alzheimer's peptide Abeta(1)(-)(40), while it is a resident in the amyloid fibril, as determined by high-resolution solution NMR. Kinetics of H/D exchange in Abeta(1)(-)(40) fibrils show that about half the backbone amide protons exchange during the first 25 h, while the other half remain unexchanged because of solvent inaccessibility and/or hydrogen-bonded structure. After such a treatment for 25 h with D(2)O, fibrils of (15)N-enriched Abeta were dissolved in a mixture of 95% dimethyl sulfoxide (DMSO) and 5% dichloroacetic acid (DCA) and successive heteronuclear (1)H-(15)N HSQC spectra were collected to identify the backbone amides that did not exchange in the fibril. These studies showed that the N and C termini of the peptide are accessible to the solvent in the fibril state and the backbone amides of these residues are readily exchanged with bulk deuterium. In contrast, the residues in the middle of the peptide (residues 16-36) are mostly protected, suggesting that that many of the residues in this segment of the peptide are involved in a beta structure in the fibril. Two residues, G25 and S26, exhibit readily exchangeable backbone amide protons and therefore may be located on a turn or a flexible part of the peptide. Overall, the data substantially supports current models for how the Abeta peptide folds when it engages in the amyloid fibril structure, while also addressing some discrepancies between models.     

Prediction of the native conformation of a polypeptide by a statistical-mechanical procedure. II. Average backbone structure of enkephalin

The average conformation of Met-enkephalin was determined by using an adaptive, importance-sampling Monte Carlo algorithm (SMAPPS—Statistical Mechanical Algorithm for Predicting Protein Structure). In the calculation, only the backbone dihedral angles (? and ψ) were allowed to vary; i.e., all side-chain (χ) and peptide-bond (ω) dihedral angles were kept fixed at the values corresponding to a low-energy structure of the pentapeptide. The total conformational energy for each randomly generated structure of the polypeptide was obtained by summing over the interaction energies of all pairs of nonbonded atoms of the whole molecule. The interaction energies were computed by the program ECEPP/2 (Empirical Conformational Energy Program for Peptides). Solvent effects were not included in the computation. The calculation was repeated until a total of 10 independent average conformations were established. The regions of conformational space occupied by the average structures were compared with the regions of low conditional free energy obtained by SMAPPS in the first paper of this series. Such a comparison provides an analysis of the capacity of SMAPPS to adjust the Monte Carlo search to regions of highest probability. The results demonstrate that the ability of SMAPPS to focus the Monte Carlo search is excellent. Finally, the 10 independent average conformations and the mean of the 10 average structures were utilized as the initial conformations for a direct energy minimization of the pentapeptide. Of the 11 final energy-minimized structures, three of the conformations were found to be equivalent to the conformation of lowest energy determined previously. In addition, all but two of the remaining energy-minimized structures were found to correspond to one of the two other conformations of high probability obtained in the first paper of this series. These results indicate that a set of independent average conformations can provide a rational, unbiased choice for the initial conformation, to be used in a direct energy minimization of a polypeptide. The final energy-minimized structures consequently constitute a set of low-energy conformations, which include the global energy minimum.     

Calculation of protein conformation by the build-up procedure. Application to bovine pancreatic trypsin inhibitor using limited simulated nuclear magnetic resonance data

Low-energy conformations of a set of tetrapeptides derived from the small protein bovine pancreatic trypsin inhibitor (BPTI) were generated by a build-up procedure from the low-energy conformations of single amino acid residues. At each stage, various-size fragments were built up from all combinations of smaller ones, the total energies were then minimized, and the low-energy conformations were retained for the next stage. The energies of the tetrapeptides were re-ordered by including the effects of hydration. No information other than the amino acid sequence was used to obtain the low-energy conformations of the hydrated tetrapeptides. The latter were then supplemented with a limited set of simulated NMR distance information, derived from the X-ray structure of BPTI, to provide a basis for building the rest of the whole protein molecule by the same procedure. A total of 189 upper bounds, plus 12 pairs of upper and lower bounds pertaining to the location of the three disulfide bonds in this molecule, were used. Four sets of conformations of the entire molecule were generated by utilizing different combinations of smaller fragments. It was possible to obtain low-energy conformations with small rms deviations, 1.1 to 1.4 A for the alpha-carbons, from the structure derived by X-ray diffraction. The average deviations of the backbone dihedral angles were also low, viz. 23 degrees to 26 degrees.     

Structural organization of [met]enkephalin and endorphin molecules. III. Theoretical conformation analysis of beta-endorphin

Conformational energy calculations were carried out for beta-endorphin. Its spatial structure can be described by nine low-energy conformations. The calculations yielded the values of all dihedral angles of the backbone and side chains of these forms as well as intra- and inter-residue interaction energies.     

Protein three-dimensional structure generation with an empirical hydrophobic penalty function

Given current computational environments, it is worthwhile to establish amino acid residue-level functions which approximate protein folds quite well. Such functions must be the interim steps toward protein three-dimensional structure prediction, I have shown that an empirical hydrophobic penalty function of protein, derived from the number of residues in a sphere around each residue, could be utilized to distinguish the correctly folded structure from the incorrect ones. In order to assess the predictive power of the penalty function, I had generated conformations by randomly changing main chain dihedral angles, and applied the penalty function to them. If only a local region was allowed to change its conformation, native like structures could be generated within a reasonable computational time. In global simulations, however, a considerable number of nonnative conformations, which gave as small a penalty value as that of the native protein, were found. Although some of the conformations were compact and globular, they were quite different from the native structure in that they lacked most of the secondary structures. This result shows that the penalty function alone cannot define the native structure, and that substructure information may help the penalty function to reach the correctly folded structure.     

An experimental analysis of species sorting and mass effects in freshwater bacterioplankton

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Spatial structure of myelopeptides: II. Conformational analysis of MP-4, MP-5, and MP-6

Theoretical conformational analysis was used to study the spatial structure and conformational properties of myelopeptides, bone-marrow peptide mediators. The low-energy conformations of myelopeptides MP-4 (Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro), MP-5 (Val-Val-Tyr-Pro-Asp), and MP-6 (Val-Asp-Pro-Pro) were found; the values of dihedral angles of backbone and side chains of the amino acid residues were determined; and the energies of intra- and interresidual interactions were estimated.__________Translated from Bioorganicheskaya Khimiya, Vol. 31, No. 2, 2005, pp. 140–146.Original Russian Text Copyright © 2005 by Ismailova, Akhmedov, Abbasli, Godjaev.     

A dynamic N-capping motif in cytochrome b5: evidence for a pH-controlled conformational switch

Apocytochrome b5 is a marginally stable protein exhibiting under native conditions a slow conformational exchange in its C-terminal region. The affected elements of secondary structure include a 3(10)-helix containing at its N-terminus a histidine Ncap and a subsequent proline. Participation of the neutral histidine side-chain in backbone amide capping lowers the imidazole pKa. To explore the nature of the conformational exchange in the protein and determine whether it is related to cis-trans isomerization of the His-Pro bond, three octapeptides encompassing the helix were synthesized and studied by NMR spectroscopy. One corresponded to the wild-type sequence, the second was the D-histidine epimer, and the third contained an alanine in place of the proline. It was found that the rates of cis-trans interconversion in the proline-containing peptides were slower than the rates of the conformational exchange in the protein. In addition, the wild-type peptide hinted at a predisposition for Ncap formation when in the trans configuration. Analysis of the pH response of the peptides and protein suggested that at pH near neutral, the conformational exchange detected in the protein involved only species with a trans His-Pro bond and could be approximated with a three-state model by which the terminal helix sampled a locally unfolded state. This state, which contained an uncapped histidine with a normal pKa, partitioned into neutral and protonated populations according to pH. The intrinsic conformational bias of the wild-type peptide and the pH-driven equilibria illustrated how a 3(10)-element could serve as a nucleation site for structural rearrangement.     

Studies on the heterotropic interaction of hemoglobin. I. Mass spectrometric method for determination of the pKa of the beta-146 histidine residue in human hemoglobin.

A mass spectrometric method was developed to determine pH-dependent hydrogen-deuterium exchange at the C-2 position of the imidazole ring of histidine, after converting the amino acid to the methylthiohydantoin derivative. The amount of deuterium exchange in N-acetyl-histidine estimated by the present method was confirmed to be in good agreement with that determined by NMR spectrometry. N-Acetylhistidine was deuterated at various pH's. From the amount of deuterium exchange, a pseudo-first order rate constant (kpsi) was calculated. A pKa value of 7.2 for the amino acid was obtained from the relation between kpsi and pH. This method was applied to estimate the pKa value of beta-146 histidine in human hemoglobin. Human hemoglobin deuterated at various pH's was digested with carboxypeptidase A [EC 3.4.12.2] to release the beta-146 histidine. The amount of deuterium exchange in the isolated histidine was determined to obtain kpsi. From these measurements pKa values of 7.0 for the histidine in oxyhemoglobin and of 8.2 for that in deoxyhemoglobin were found at 36.5 degrees, respectively.     

Characterizing a partially folded intermediate of the villin headpiece domain under non-denaturing conditions: contribution of His41 to the pH-dependent stability of the N-terminal subdomain

The contribution of interactions involving the imidazole ring of His41 to the pH-dependent stability of the villin headpiece (HP67) N-terminal subdomain has been investigated by nuclear magnetic resonance (NMR) spin relaxation. NMR-derived backbone N-H order parameters (S2) for wild-type (WT) HP67 and H41Y HP67 indicate that reduced conformational flexibility of the N-terminal subdomain in WT HP67 is due to intramolecular interactions with the His41 imidazole ring. These interactions, together with desolvation effects, contribute to significantly depress the pKa of the buried imidazole ring in the native state. 15N R1rho relaxation dispersion data indicate that WT HP67 populates a partially folded intermediate state that is 10.9 kJ mol(-1) higher in free energy than the native state under non-denaturing conditions at neutral pH. The partially folded intermediate is characterized as having an unfolded N-terminal subdomain while the C-terminal subdomain retains a native-like fold. Although the majority of the residues in the N-terminal subdomain sample a random-coil distribution of conformations, deviations of backbone amide 1H and 15N chemical shifts from canonical random-coil values for residues within 5A of the His41 imidazole ring indicate that a significant degree of residual structure is maintained in the partially folded ensemble. The pH-dependence of exchange broadening is consistent with a linear three-state exchange model whereby unfolding of the N-terminal subdomain is coupled to titration of His41 in the partially folded intermediate with a pKa,I=5.69+/-0.07. Although maintenance of residual interactions with the imidazole ring in the unfolded N-terminal subdomain appears to reduce pKa,I compared to model histidine compounds, protonation of His41 disrupts these interactions and reduces the difference in free energy between the native state and partially folded intermediate under acidic conditions. In addition, chemical shift changes for residues Lys70-Phe76 in the C-terminal subdomain suggest that the HP67 actin binding site is disrupted upon unfolding of the N-terminal subdomain, providing a potential mechanism for regulating the villin-dependent bundling of actin filaments.     

Local propensities and statistical potentials of backbone dihedral angles in proteins

The following three issues concerning the backbone dihedral angles of protein structures are presented. (1) How do the dihedral angles of the 20 amino acids depend on the identity and conformation of their nearest residues? (2) To what extent are the native dihedral angles determined by local (dihedral) potentials? (3) How to build a knowledge-based potential for a residue's dihedral angles, considering the identity and conformation of its nearest residues? We find that the dihedral angle distribution for a residue can significantly depend on the identity and conformation of its adjacent residues. These correlations are in sharp contrast to the Flory isolated-pair hypothesis. Statistical potentials are built for all combinations of residue triplets and depend on the dihedral angles between consecutive residues. First, a low-resolution potential is obtained, which only differentiates between the main populated basins in the dihedral angle density plots. Minimization of the dihedral potential for 125 test proteins reveals that most native alpha-helical residues (89%) and a large fraction of native beta-sheet residues (47%) adopt conformations close to their native one. For native loop residues, the percentage is 48%. It is also found that this fraction is higher for residues away from the ends of alpha or beta secondary structure elements. In addition, a higher resolution potential is built as a function of dihedral angles by a smoothing procedure and continuous functions interpolations. Monte Carlo energy minimization with this potential results in a lower fraction for native beta-sheet residues. Nevertheless, because of the higher flexibility and entropy of beta structures, they could be preferred under the influence of non-local interactions. In general, most alpha-helices and many beta-sheets are strongly determined by the local potential, while the conformations in loops and near the end of beta-sheets are more influenced by non-local interactions.