Synthesis and conformational analysis of aib-containing peptide modelling for N-glycosylation site in N-glycoprotein

A tetrapetide containing an Aib residue, Boc-Asn-Aib-Thr-Aib-OMe, was synthesized as a peptide model for the N-glycosylation site in N-glycoproteins. Backbone conformation of the peptide and possible intramolecular interaction between the Asn and Thr side chains were elucidated by means of n.m.r. spectroscopy. Temperature dependence of NH proton chemical shift and NOE experiments showed that Boc-Asn-Aib-Thr-Aib-OMe has a tendency to form a β-turn structure with a hydrogen bond involving Thr and Aib⁴ NH groups. Incorporation of Aib residues in the peptide model promotes folding of the peptide backbone. With folded backbone conformation, carboxyamide protons of the Asn residue are not involved in hydrogen bond network, while the OH group of the Thr residue is a candidate for a hydrogen bond in DMSO-d₆ solution.     

Structural engineering of the HIV-1 protease molecule with a beta-turn mimic of fixed geometry.

An important goal in the de novo design of enzymes is the control of molecular geometry. To this end, an analog of the protease from human immunodeficiency virus 1 (HIV-1 protease) was prepared by total chemical synthesis, containing a constrained, nonpeptidic type II' beta-turn mimic of predetermined three-dimensional structure. The mimic beta-turn replaced residues Gly16,17 in each subunit of the homodimeric molecule. These residues constitute the central amino acids of two symmetry-related type I' beta-turns in the native, unliganded enzyme. The beta-turn mimic-containing enzyme analog was fully active, possessed the same substrate specificity as the Gly16,17-containing enzyme, and showed enhanced resistance to thermal inactivation. These results indicate that the precise geometry of the beta-turn at residues 15-18 in each subunit is not critical for activity, and that replacement of the native sequence with a rigid beta-turn mimic can lead to enhanced protein stability. Finally, the successful incorporation of a fixed element of secondary structure illustrates the potential of a "molecular kit set" approach to protein design and synthesis.     

Stereochemical studies on cyclic peptides: Detailed energy minimization studies on hydrogen bonded all-trans cyclic pentapeptide backbones

Conformational studies have been carried out on hydrogenbonded all-trans cyclic pentapeptide backbone. Application of a combination of grid search and energy minimization on this system has resulted in obtaining 23 minimum energy conformations, which are characterized by unique patterns of hydrogen bonding comprising of β- and γ-turns. A study of the minimum energy conformationsvis-a-vis non-planar deviation of the peptide units reveals that non-planarity is an inherent feature in many cases. A study on conformational clustering of minimum energy conformations shows that the minimum energy conformations fall into 6 distinct conformational families. Preliminary comparison with available X-ray structures of cyclic pentapeptide indicates that only some of the minimum energy conformations have formed crystal structures. The set of minimum energy conformations worked out in the present study can form a consolidated database of prototypes for hydrogen bonded backbone and be useful for modelling cyclic pentapeptides both synthetic and bioactive in nature. This is part XV of the series. Part XIV in this series is Ramakrishnanet al 1987.     

Molecular Dynamics Simulations of β-turn Forming Tetra- and Hexapeptides

Abstract

It was previously shown that the structural ensemble of model peptides DDKG and GKDG (H. Ishii et al. Biopolymers 24, 2045–2056, 1985), DEKS (A. Otter et al. J. Biomol. Struct. Dyn. 7, 455–476, 1989) NPGQ (F. R. Carbone et al. Int. J. Pept. Protein. Res. 26, 498–508, 1985), SALN (H. Santa et al. J. Biomol. Struct. Dyn. 16, 1033–1041, 1999), SYPFDV and SYPYDV (J. Yao et al. J. Mol. Biol. 243, 736–753, 1994), VP^DAH and VP^DSH (B. Imperiali et al. J. Am. Chem. Soc. 114, 3182–3188, 1992) in solution contains a significant—or in some cases dominant—proportion of β-turn conformation. In this study, a protein database was searched for the above, unprotected sequences which incorporate only L-amino acid residues. Simulated annealing and 25 ns MD simulations of structures were also performed. The DSSP and STRIDE secondary structure-assigning algorithms and clustering were used to analyze trajectories and i, i+3 hydrogen bonds were also sought. The DSSP analysis showed a fluctuation between β-turn and random meander structure, although bend structures were not detected because of the insufficient length of peptide chains. This alternating trend was confirmed when the STRIDE algorithm was used to analyze trajectories, but STRIDE assigned more turn structures. The population of the strongest clusters was above 40% and the middle structures adopted β-turn structure for most sequences. These results are in good agreement with previous experimental results and support the idea of the ultra-marginal stability of turns in the absence of stabilizing long-range interactions of the neighboring segments of a polypeptide chain. However, interactions between the side-chains in tetrapeptides could also contribute to turn stability and result in unusual stability in some cases. Our observations suggest that such interactions are the consequence rather than the driving force of turn formation.     

Utilization of 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide (EDCI)/1-hydroxybenzotriazole (HOBt) as a polymerizing agent

The commonly used coupling reagents in peptide synthesessuch as dicyclohexylcabodiimide, diisopropylcarbodiimide and3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide with or without1-hydroxybenzotriazole or N-hydroxysuccinimide have been used as polymerizing agents in the synthesis of elastic/plastic protein-based polymers. It was found that 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide with 1-hydroxybenzotriazole gave equally good polymers comparable toconventional p-nitrophenol approach. Further, we present here the polymerization and characterization of structural andfunctional properties of poly(Val-Pro-Gly-Val-Gly), which is themost striking repeating sequence in the bovine and porcine elastins. The polymers obtained by both p-nitrophenol and 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide approach werecharacterized by carbon-13 and proton nuclear magnetic resonancespectroscopy, differential scanning calorimetry, circular dichroism and fourier transform infrared spectroscopy. These results conclude that poly(Val-Pro-Gly-Val-Gly) obtained by bothmethods were identical in all respects of physical and chemicalproperties indicates that 3-ethyl-1(N,N-dimethyl)aminopropylcarbodiimide with 1-hydroxybenzotriazole method can be conveniently employed to synthesize these polymers.     

Thermodynamic and structural characterization of Asn and Ala residues in the disallowed II' region of the Ramachandran plot

Residue Asn47 at position L1 of a type II' beta-turn of the alpha-spectrin SH3 domain is located in a disallowed region of the Ramachandran plot (phi = 56 +/- 12, psi = -118 +/- 17). Therefore, it is expected that replacement of Asn47 by Gly should result in a considerable stabilization of the protein. Thermodynamic analysis of the N47G and N47A mutants shows that the change in free energy is small (approximately 0.7 kcal/mol; approximately 3 kJ/mol) and comparable to that found when mutating a Gly to Ala in a alpha-helix or beta-sheet. X-ray structural analysis of these mutants shows that the conformation of the beta-turn does not change upon mutation and, therefore, that there is no relaxation of the structure, nor is there any gain or loss of interactions that could explain the small energy change. Our results indicate that the energetic definition of II' region of the Ramachandran plot (phi = 60 +/- 30, psi = -115 +/- 15) should be revised for at least Ala and Asn in structure validation and protein design.     

The identification of conserved interactions within the SH3 domain by alignment of sequences and structures

The SH3 domain, comprised of approximately 60 residues, is found within a wide variety of proteins, and is a mediator of protein-protein interactions. Due to the large number of SH3 domain sequences and structures in the databases, this domain provides one of the best available systems for the examination of sequence and structural conservation within a protein family. In this study, a large and diverse alignment of SH3 domain sequences was constructed, and the pattern of conservation within this alignment was compared to conserved structural features, as deduced from analysis of eighteen different SH3 domain structures. Seventeen SH3 domain structures solved in the presence of bound peptide were also examined to identify positions that are consistently most important in mediating the peptide-binding function of this domain. Although residues at the two most conserved positions in the alignment are directly involved in peptide binding, residues at most other conserved positions play structural roles, such as stabilizing turns or comprising the hydrophobic core. Surprisingly, several highly conserved side-chain to main-chain hydrogen bonds were observed in the functionally crucial RT-Src loop between residues with little direct involvement in peptide binding. These hydrogen bonds may be important for maintaining this region in the precise conformation necessary for specific peptide recognition. In addition, a previously unrecognized yet highly conserved beta-bulge was identified in the second beta-strand of the domain, which appears to provide a necessary kink in this strand, allowing it to hydrogen bond to both sheets comprising the fold.     

The use of norbornene derivatives in the synthesis of conformationally constrained peptides and pseudo-peptides

Summary The desymmetrisation ofendo-norborn-5-ene-2,3-dicarboxylic anhydride by proline esters has been used to prepare conformationally constrained pseudo-peptides with two peptide chains parallel to one another. A Curtius rearrangement on the desymmetrication adduct produced the corresponding isocyanate which was used to prepare both a peptide incorporating anendo-2-amino-3-carboxy-norborn-5-ene unit, and a pseudo-peptide with two peptide chains parallel to one another but offset by the presence of a urea unit. The conformational analysis of the resulting peptides was carried out, and the norbornene unit was found to induce the formation of β-turns and parallel β-sheets.     

Energy minimization studies on α-turns

Using a grid search technique, the entire conformational space of a system of four linked peptide units (tetrapeptide) was scanned to pick out geometrically possible 5→1 type hydrogen-bonded conformations defined as an α-turn. The energy minimization of these conformations led to 23 distinct minimum energy conformations (MECs) falling in 13 different classes. The presence of β and γ turn type hydrogen bonds along with 5→1 type hydrogen bond gave conformational variability in a given class. The occurrence of bifurcated hydrogen bonding network was a characteristic feature of most of the MECs. In many prototype MECs non-glycyl residues such as Ala and Pro could be accommodated. Comparison of MECs with the α-turn examples that are observed in proteins showed that the conformationally worked out MECs occurred in isolation in proteins, with the α-helical α-turn being distinctly the most predominant. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

A conformational study in solution of pro-somatostatin fragments by NMR and computational methods

The results of a conformational study by nuclear magnetic spectroscopy and computational methods on a series of point-mutated synthetic peptides, containing 14 amino acid residues and mimicking the region containing the Arg-Lys dibasic cleavage site of pro-somatostatin, have confirmed the possible role of a well defined secondary structure in the recognition phenomenon by processing enzymes. The importance of the residues located near the Arg-Lys dibasic site in the C-terminal region of the pro-hormone for the cleavage of the precursor into somatostatin-14 has been confirmed. The present structural analysis indicates the occurrence of two β-turns in the 4–7 and 11–14 regions, flanking the cleavage site, for all the peptides recognized as substrates by the processing enzyme. Interestingly, in the point-mutated analogue not processed by the enzyme and containing the replacement of proline by alanine in position 5 the first β-turn is displaced by one residue and involves the Ala⁵-Arg⁸ segment. This observation may explain the lack of recognition by the maturation enzyme. © 1998 European Peptide Society and John Wiley & Sons, Ltd.