PROTEIN DISULFIDE ANALYSIS AND DESIGN

The significant contribution of naturally occurring disulfide bonds to protein stability has encouraged development of methods to engineer non-native disulfides in proteins. These have yielded mixed results. We summarize applications of the program MODIP for disulfide engineering. The program predicts sites in proteins where disulfides can be stably introduced. The program has also been used as an aid in conformational analysis of naturally occurring disulfides in a-helices, antiparallel and parallel beta-strands. Disulfides in a-helices occur only at N-termini, where the first cysteine residue is the N-cap residue of the helix. The disulfide occurs as a CXXC motif and can possess redox activity. In antiparallel beta-strands, disulfides occur exclusively at non-hydrogen bonded (NHB) registered pairs of antiparallel beta-sheets with only 1 known natural example occurring at a hydrogen bonded (HB) registered pair. Conformational analysis suggests that disulfides between HB residue pairs are under torsional strain. A similar analysis to characterize disulfides in parallel beta-strands was carried out. We observed that only 9 instances of cross-strand disulfides exist in a non-redundant dataset. Stereochemical analysis shows that while tbe chi(square) angles are similar to those of other disulfides, the chi(1) and chi(2) angles show more variation and that one of tbe strands is generally an edge strand.

Analysis of the conformations of septanoside sugars

Conformational analysis of unnatural seven-membered sugars, namely, septanoses and septanosides are discussed herein. The conformational properties of these sugars in the solid state, solution phase and computational methods are presented. The analyses reveal that conformations of septanosides are diverse and largely unpredictable, as compared furanosides and pyranosides.

Stereochemistry of Peptides Containing 1-AminocyclopentanecarboxylicA cid ( Am5): Solution and Solid-state Conformations of Boc-Acc '-Acc'-NHMe

The conformational analysis of a protected homodipeptide of 1-aminocyclopentanecarboxylic acid (Acc5) has been carried out. 1H-nmr studies establish a -turn conformation for Boc-Acc5-Acc5-NHMe in chloroform and dimethylsulfoxide solutions involving the methylamide NH in an intramolecular hydrogen bond. Supportive evidence for the formation of an intramolecular hydrogen bond is obtained from ir studies. X-ray diffraction studies reveal a type III -turn conformation in the solid state stabilized by a 4 1 hydrogen bond between the Boc CO and methylamide NH groups. The , values for both Acc5 residues are close to those expected for an ideal 310-helical conformation ( ± 60°, ±30°).

Type II beta-turn conformation of pivaloyl-L-prolyl-alpha-aminoisobutyryl-N-methylamide: Theoretical, spectroscopic, and X-ray studies

Pivaloyl-L-Pro-Aib-N-methylamide has been shown to possess one intramolecular hydrogen bond in (CD3)2SO solution, by 1H-nmr methods, suggesting the existence of beta -turns, with Pro-Aib as the corner residues. Theoretical conformational analysis suggests that Type II beta-turn conformations are about 2 kcal mol-1 more stable than Type III structures. A crystallographic study has established the Type II beta-turn in the solid state. The molecule crystallizes in the space group P21 with a = 5.865 Å, b = 11.421 Å, c = 12.966 Å, beta = 97.55°, and Z = 2. The structure has been refined to a final R value of 0.061. The Type II -turn conformation is stabilized by an intramolecular 4 1 hydrogen bond between the methylamide NH and the pivaloyl CO group. The conformational angles are Pro = -57.8°, Pro = 139.3°, Aib = 61.4°, and Aib = 25.1°. The Type II beta-turn conformation for Pro-Aib in this peptide is compared with the Type III structures observed for the same segment in larger peptides.

Theoretical-Studies On The Conformations Of Acyclic Alditols

The conformational analysis by energy calculation is described for some acyclic sugars such as D-glucitol, D-mannitol and galactitol. Planar Zig-zag conformation is the most favoured conformation for the three alditols. However, the energy difference between the ‘bent-chain’ and ‘straight-chain’ conformation is less in the case of D-glucitol (0.9 Kcal Mole-1)compared to those of D-mannitol (~2.4 Kcal mole-1)and galactitol (~2.5 Kcal Mole-1).The solvent accessibility studies favour bent –chain conformation for D-glucitol and straight-chain conformation for D-mannitol and glactitol. These conformations, arrived at by theorticle analysis are compared with those abseverd in the solid state determined by X=ray differaction techinique and their acetylated derivatives in solution by NMR technique. These studies suggest that, when the energy difference between straight and bent conformations is small, latticc energy (in the case of solids) and solvent (in the case of solutions) do play a dominant role on the favoured conformations.

Is 3'-nucleotide rigid?

Conformational analysis of nucleic acids and polynucleotides is far more complex than that of proteins and polypeptides, due to five single bond rotations in addition to the sugar puckerings in the monomer. Sundaralingam1 proposed the concept of the 'rigid' nucleotides from analysis of crystal structure data, with the flexibility allowed only about the phosphodiester bonds. However, the crystal structure of deoxyguanosine-5'−phosphate2,3 indicates at gt conformation about the C-4'−C-5' bond against gg in a conformationally rigid nudeotide1. Jack et al. 4 considered the flexibility of nucleotides in tRNA about the C-4'−C-5' bond, thereby introducing the concept of 'non-rigid' ribonucleotides. Conformational flexibility of the f uranose ring in DNA and RNA and their energetics using classical and quantum chemical methods have been reported5−8. We have examined the flexibility of 3'-nucleotides. alpha, the most important of the conformational parameters defining the 3'-end of a nucleotide unit9, has a value in the range 195°−270° in all the 3'-nucleotides, dinucleoside monophosphates and higher oligomers which have been surveyed. A survey of the proposed structures of polyribonudeotides10,11 also shows the values of a to be greater than 200°. However, the structures proposed for B-DNA by Arnott and Hukins12,13 and D-DNA by Arnott et al. 14 have values of alpha of 155° and 141° respectively, much lower than the lowest observed value. The structure for B-DNA has two strong, short contacts (C-2'...OP-1 = 2.64 Å and HC-2"...OP-1 = 1.79 Å) which lead to an energetically unfavourable conformation. Hence, it is of interest to investigate whether, by allowing flexibility to the sugar moiety in the nucleotide unit, it is possible to make the structure energetically favourable. Here, conformational energy calculations were carried out to determine the range of alpha which would give rise to energetically favoured conformations with different sugar puckerings. Our analysis has shown that the theoretically obtained range is nearly the same as the preferred range in crystals, indicating the flexibility of the 3'-nucleotides.

A Case Study of the Conformation of Poly(alpha-aminoisobutyric acid): alpha- or 310-Helix

The relative stabilities of a- and Blo-helical structures for polymers of a-aminoisobutyric acid (Aib) have been worked out, using the classical potential energy functions. To make a comparative study, we have used Buckingham "6-exp" and Kitaigorodsky's potential functions. Conformational analysis of the dipeptide segment with Aib residue indicates the necessity for nonplanar distortion of the peptide unit, which is a common feature in the observed crystal structures with Aib residues. In the range of Aw -10 to +loo studied, a-helical conformations are preferred in the region -3" < Aw < +loo, and Blo-helical conformations are preferred in the region -3" > Aw > -10'. Minimum energy conformations for right-handed structures are found in the +ue region of Aw and correspondingly for left-handed structures in the -ue region of Aw. For Aw - 6", a-helical structures have four- or near fourfold symmetry with h - 1.5 A. Such a helix with n = 4 and h = 1.5 A is termed an a'-helix. This structure is found to be consistent with the electron diffraction data of Malcolm3 and energetically more favorable than the standard 310-helix.

Type II beta-turn conformation of pivaloyl-L-prolyl-a-aminoiso-butyryl-N-methylamide: Theoretical, spectroscopic and X-ray studies

Pivaloyl-L-Pro-Aib-N-methylamihdaes been shown to possess one intramolecular hydrogen bond in (CD&SO solution, by 'H-nmr methods, suggesting the existence of p-turns, with Pro-Aib as the corner residues. Theoretical conformational analysis suggests that Type II P-turn conformations are about 2 kcal mol-' more stable than Type 111 structures. A crystallographic study has established the Type I1 /%turn in the solid state. The molecule crystallizes in the space group P21 with a = 5.865 8, b = 11.421 A, c = 12.966 A, /3 = 97.55", and 2 = 2. The structure has been refined to a final R value of 0.061. The Type I1 p-turn conformation is stabilized by an intramolecular 4 - 1 hydrogen bond between the methylamide NH and the pivaloyl CO group. The conformational angles are @pro= -57.8", $pro = 139.3', @Aib = 61.4', and $Ajb = 25.1'. The Type 11 /%turn conformation for Pro-Aib in this peptide is compared with the Type I11 structures observed for the same segment in larger peptides.

Conformationally restricted formyl methionyl tripeptide chemoattractants: a three-dimensional structure-activity study of analogs incorporating a C alpha,alpha-dialkylated glycine at position 2

The conformationally restricted CHO-L-Met-Xxx-L-Phe-OY (where Xxx = Aib, Ac3c, Ac5c, Ac6c, and Ac7c; Y = H, Me) tripeptides, analogs of the chemoattractant CHO-L-Met-L-Leu-L-Phe-OH, have been synthesized in solution by classical methods and fully characterized. Compounds were compared to determine the combined effect of backbone conformational preferences and side-chain bulkiness on the relation of three-dimensional structure to biological activity. Each peptide was tested for its ability to induce granule enzyme secretion from rabbit peritoneal polymorphonuclear leukocytes. In parallel, a conformational analysis on the CHO-blocked peptide and their tertbutyloxycarbonylated synthetic precursors was performed in the crystal state and in solution using X-ray diffraction, infrared absorption, and 1H nuclear magnetic resonance. The biological and conformational data are discussed in relation to the proposed model of the chemotactic peptide receptor of rabbit neutrophils.

Stereochemistry of peptides containing 1-aminocyclopentane carboxylic acid (Acc5). Solution and solid state conformations of Boc-Acc5-Acc5-NHMe.

The conformational analysis of a protected homodipeptide of 1-aminocyclopentanecarboxylic acid (Acc5) has been carried out. 1H-nmr studies establish a ?-turn conformation for Boc-Acc5-Acc5-NHMe in chloroform and dimethylsulfoxide solutions involving the methylamide NH in an intramolecular hydrogen bond. Supportive evidence for the formation of an intramolecular hydrogen bond is obtained from ir studies. X-ray diffraction studies reveal a type III ?-turn conformation in the solid state stabilized by a 4 ? 1 hydrogen bond between the Boc CO and methylamide NH groups. The ?,? values for both Acc5 residues are close to those expected for an ideal 310-helical conformation (?? ± 60°, ?? ±30°).

Stereochemical studies on cyclic peptides. Part XI. Conformation of cyclic pentapeptides having intramolecular 3 leads to 1 hydrogen bonds.

Conformational analysis of cyclic pentapeptides having two intra-ring 3 leads to 1 hydrogen bonds has been carried out. It is found that the structure can easily be formed with trans planar peptide units without causing significant angular strain at the alpha-carbon atoms. Four different types of conformations designated Types I--IV are possible for the backbone structure. Details of these four types of conformations and also the accommodating possibility of these types for allglycyl and all-alanyl residues are presented. Three of the four types have relatively low energies for glycyl residues whereas the other one has a slightly higher energy. When alanyl residues are introduced at the five alpha-carbon atoms, the types that are energetically favourable depend upon the sequence of isomers. Energy calculations have also been carried out for the combinations of glycyl, L- and D-alanyl residues. The theoretical results are compared with available experimental observations both from solution and solid state studies.

On the importance of backbone loop and peptide flip in the Walker sequence in F-1-ATPase action

The Walker sequence, GXXXXGKT, present in all the six subunits of F-1-ATPase exists in a folded form, known as phosphate-binding loop (P-loop). Analysis of the Ramachandran angles showed only small RMS deviation between the nucleotide-bound and nucleotide-free forms. This indicated a good overlap of the backbone loops. The catalytic beta-subunits (chains D, E and F) showed significant changes in the Ramachandran angles and the side chain torsion angles, but not the structural alpha-subunits (chains A, B and C). Most striking among these are the changes associated with Val160 and Gly161 corresponding to a flip in the peptide unit between them when a nucleotide is bound (chains D or F compared to nucleotide-free chain E). The conformational analysis further revealed a hitherto unnoticed hydrogen bond between amide-N of the flipped Gly161 and terminal phosphate-O of the nucleotide. This assigns a role for this conserved amino acid, otherwise ignored, of making an unusual direct interaction between the peptide backbone of the enzyme protein and the incoming nucleotide substrate. Significance of this interaction is enhanced, as it is limited only to the catalytic subunits, and also likely to involve a mechanical rotation of bonds of the peptide unit. Hopefully this is part of the overall events that link the chemical hydrolysis of ATP with the mechanical rotation of this molecule, now famous as tiny molecular motor.

beta gamma-fused turn structures in sugar amino acid (SAA) containing cyclic tetrapeptides with alpha 3 delta architecture

The current manuscript describes conformational analysis of 15-membered cyclic tetrapeptides (CTPs), with alpha 3 delta architecture, containing sugar amino acids (SAA) having variation in the stereocenter at C5 carbon. Conformational analyses of both the series, in protected and deprotected forms, were carried out in DMSO-d(6) using various NMR techniques, supported by restrained MD calculations. It was intriguing to notice that the alpha 3 delta macrocycles got stabilized by both 10-membered beta-turn as well as a seven-membered gamma-turn, fused within the same macrocycle. The presence of fused sub-structures within a 15-membered macrocycle is rare to see. Also, the stereocenter variation at C5 did not affect the fused turn structures and exhibited similar conformations in both the series. The design becomes highly advantageous as fused reverse turn structures are occurring in the cyclic structure with minimalistic size macrocycle and this can be applied to develop suitable pharmacophores in the drug development process. (C) 2014 Elsevier Ltd. All rights reserved.

Tetrahydrofuran amino acid-containing gramicidin S analogues with improved biological profiles

Gramicidin S (GS) is a cyclic cationic antimicrobial peptide (CAP) with a wide spectrum of antibiotic activities whose usage has been limited to topical applications owing to its cytotoxic side effects. We have synthesized tetrahydrofuran amino acid (Taa)-containing GS analogues, and we have carried out conformational analysis and explored their structure activity relationships by evaluating their antitubercular, antibacterial and cytotoxic properties. Two of these analogues showed impressive as well as selective activity against Mycobacterium tuberculosis (MTB) without toxicity towards mammalian Vero cells or human RBCs, and are promising as potential leads.

Conformational restrictions of peptides via backbone modification: Solution and crystal-state analysis of Boc-L-Pro-DZPhe-Gly-NH2

An N-alpha-protected model tripeptide amide containing, in the central position, an alpha,beta-dehydrophenylalanine (Z-configurational isomer), Boc-L-Pro-DELTA-Z-Phe-Gly-NH2 (Boc, tert-butyloxycarbonyl), has been synthesized by solution methods and fully characterized. IR absorption and H-1 NMR studies provided evidence for the occurrence of a significant population of a conformer containing two consecutive, intramolecularly H-bonded (type II-III') beta-bends in solution. However, an X-ray diffraction analysis clearly indicates that only the type-II beta-bend structure survives in the crystal state.