Influence of intramolecular interactions on conformational and dynamic properties of analogs of heptapeptide AFP<Subscript>14–20</Subscript>

Conformational and dynamic properties of proteins and peptides play an important role in their functioning. However, mechanisms that underlie this influence have not been fully elucidated. In the present work we computationally constructed analogs of heptapeptide AFP_14–20 (LDSYQCT) — one of the biologically active sites of human α-fetoprotein (AFP) — to study their conformational and dynamic properties using molecular dynamics simulation. Analogs were obtained by point substitutions of amino acid residues taking into account differences in their physicochemical properties and also on the basis of analysis of amino acid substitutions in the AFP_14–20-like motifs revealed in different physiologically active proteins. It is shown that changes in conformational mobility of amino acid residues of analogs are due to disruption or arising of intramolecular interactions that, in turn, determine existence of steric restrictions during rotation around covalent bonds of the peptide backbone. Substitution of an amino acid by another one with significant difference in physicochemical properties may not lead to remarkable changes in conformational and dynamic properties of the peptide if intramolecular interactions remain unchanged.     

Peptide models of electrostatic interactions in proteins: NMR studies on two beta-turn tetrapeptides containing Asp-His and Asp-Lys salt bridges

Two model peptides Boc-Asp-Pro-Aib-X-NHMe [X = His (1) and X = Lys (2)] were synthesized to simulate intramolecular electrostatic interactions between ionizable side chains. Conformational analysis by 270-MHz 1H NMR in (CD3)2SO reveals that the backbone secondary structures of these two peptides are stabilized by two strong intramolecular hydrogen bonds, involving the consecutive carboxy-terminal NH groups. 1H NMR chemical shifts were measured in 1, 2, and a protected derivative, Boc-Asp(OBzl)-Pro-Aib-His-NHMe (3). These shifts were also measured for the model compounds Ac-Lys-NHMe, Boc-Asp-NHMe, and Boc-His-NHMe in their different states of ionization. An analysis of the chemical shifts of the ionization-sensitive reporter resonances suggests the formation of a strong intramolecular salt bridge in the lysyl peptide 2 and a bridge of moderate strength in the histidyl peptide 1. A comparison of the temperature dependence of chemical shifts in peptides 1-3 suggests that intramolecular salt bridge formation results in diminished backbone flexibility. The results establish that proximity effects confer far greater stability to intramolecular ion pair interactions vis-a-vis their intermolecular counterparts. The salt bridge interaction in peptide 1 displays a remarkable sensitivity to the dielectric constant of the solvent medium. The results suggest that these peptides are good simulators of the role of salt bridges in the structural dynamics of proteins.     

Contribution of individual amino acids within MHC molecule or antigenic peptide to TCR ligand potency

The TCR recognition of peptides bound to MHC class II molecules is highly flexible in some T cells. Although progress has been made in understanding the interactions within the trimolecular complex, to what extent the individual components and their amino acid composition contribute to ligand recognition by individual T cells is not completely understood. We investigated how single amino acid residues influence Ag recognition of T cells by combining several experimental approaches. We defined TCR motifs for CD4+ T cells using peptide synthetic combinatorial libraries in the positional scanning format (PS-SCL) and single amino acid-modified peptide analogues. The similarity of the TCR motifs defined by both methods and the identification of stimulatory antigenic peptides by the PS-SCL approach argue for a contribution of each amino acid residue to the overall potency of the antigenic peptide ligand. In some instances, however, motifs are formed by adjacent amino acids, and their combined influence is superimposed on the overall contribution of each amino acid within the peptide epitope. In contrast to the flexibility of the TCR to interact with different peptides, recognition was very sensitive toward modifications of the MHC-restriction element. Exchanges of just one amino acid of the MHC molecule drastically reduced the number of peptides recognized. The results indicate that a specific MHC molecule not only selects certain peptides, but also is crucial for setting an affinity threshold for TCR recognition, which determines the flexibility in peptide recognition for a given TCR.     

Conformation-Determining role for the N-acetyl group in the O-glycosidic linkage, α-GalNAc-Thr

An ¹H-nmr study of 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-D-galactopyranose (AcGalNAc) glycosylated Thr-containing tripeptides in Me₂SO-d₆ solution reveals two mutually exclusive intramolecular hydrogen bonds. In Z-Thr(AcGalNAc)-Ala-Ala-OMe, there is an intramolecular hydrogen bond between the Thr amide proton and the sugar N-acetyl carbonyl oxygen. The strength of this hydrogen bond will be dependent on the amino acid residues on the Thr C terminal side to some undetermined distance. In Ac-Thr(AcGalNAc)-Ala-Ala-OMe, a different intramolecular hydrogen bond between the sugar N-acetyl amide proton and the Thr carbonyl oxygen exists. The choice of hydrogen bonds seems dependent on the bulkiness of the residues on the Thr N terminal side. The consequence of such strong hydrogen bonds is a clearly defined orientation of the sugar moiety with respect to the peptide backbone. In the former, the plane of the sugar pyranose ring is roughly oriented perpendicularly to the peptide backbone. The latter orientation is where the plane of the sugar ring is roughly in line with the peptide backbone. In both orientations, the sugar moiety can increase the shielding of the neighboring amino acid residues from the solvent. The idea that the amino acid residues near the glycosylated Thr influence orientation of the sugar moiety with respect to the peptide backbone and in turn possibly hinder peptide backbone flexibility has interesting implications in the conformational as well as the biological role of O-glycoproteins.     

An approach to the problem of the structuro-functional organization of natural peptides

Theory and computational scheme of three-dimensional structure and dynamic conformational properties of naturally occurring peptides are proposed basing on a known amino acid sequence. The diverse biological activity of a low-molecular peptide is shown to arise from a restricted number of preferable spatial structures which may occur under physiological conditions. Each particular function of an oligopeptide is connected to a definite spatial structure, belonging to the set of low-energy conformations from one biological activity of a peptide shift of the conformational equilibrium caused by a change of environmental conditions. This shift is provided for by specific intramolecular interactions, alternative in their nature, which stabilize a particular structure. An approach is suggested which enables to construct a synthetic analog with the predetermined physiologically active conformation, prior to all chemical and biological tests.     

Integration of QSAR modelling and QM/MM analysis to investigate functional food peptides with antihypertensive activity

Antihypertensive peptides derived from dietary proteins have long been recognised as an important source of developing functional foods with blood pressure-lowering effect. However, most of such peptides exhibit diverse tastes, such as sweet, bitter, sour and salty, which is a non-negligible aspect considered in the food development process. In the present study, several predictive quantitative structure–activity relationship (QSAR) models that correlate peptide's structural features with their multi-bioactivities and bitter taste are established at both sequence and structure levels, and the models are then used to conduct extrapolation on thousands of randomly generated, structurally diverse peptides with chain lengths ranging from two to six amino acid residues. Based on the statistical results gained from QSAR modelling, the relationship between the antihypertensive activity and bitter taste of peptides at different sequence lengths is investigated in detail. Moreover, the structural basis, energetic property and biological implication underlying peptide interactions with angiotensin-converting enzyme (ACE), a key target of antihypertensive therapy, are analysed at a complex three-dimensional structure level by using a high-level hybrid quantum mechanics/molecular mechanics scheme. It is found that (a) bitter taste is highly dependent on peptide length, whereas ACE inhibitory potency has only a modest correlation with the length, (b) dipeptides and tripeptides perform a moderate relationship between their ACE inhibition and bitterness, but the relationship could not be observed for those peptides of more than three amino acid residues and (c) the increase in sequence length does not cause peptides to exhibit substantial enhancement of antihypertensive activity; this is particularly significant for longer peptides such as pentapeptides and hexapeptides.     

The spin label amino acid TOAC and its uses in studies of peptides: chemical, physicochemical, spectroscopic, and conformational aspects

We review work on the paramagnetic amino acid 2,2,6,6-tetramethyl-N-oxyl-4-amino-4-carboxylic acid, TOAC, and its applications in studies of peptides and peptide synthesis. TOAC was the first spin label probe incorporated in peptides by means of a peptide bond. In view of the rigid character of this cyclic molecule and its attachment to the peptide backbone via a peptide bond, TOAC incorporation has been very useful to analyze backbone dynamics and peptide secondary structure. Many of these studies were performed making use of EPR spectroscopy, but other physical techniques, such as X-ray crystallography, CD, fluorescence, NMR, and FT-IR, have been employed. The use of double-labeled synthetic peptides has allowed the investigation of their secondary structure. A large number of studies have focused on the interaction of peptides, both synthetic and biologically active, with membranes. In the latter case, work has been reported on ligands and fragments of GPCR, host defense peptides, phospholamban, and β-amyloid. EPR studies of macroscopically aligned samples have provided information on the orientation of peptides in membranes. More recent studies have focused on peptide-protein and peptide-nucleic acid interactions. Moreover, TOAC has been shown to be a valuable probe for paramagnetic relaxation enhancement NMR studies of the interaction of labeled peptides with proteins. The growth of the number of TOAC-related publications suggests that this unnatural amino acid will find increasing applications in the future.     

Synthesis of novel unnatural amino acid as a building block and its incorporation into an antimicrobial peptide

Considering the biological mechanism and in vivo stability of antimicrobial peptides, we designed and synthesized novel unnatural amino acids with more positively charged and bulky side chain group than lysine residue. The unusual amino acids, which were synthesized by either solution phase or solid phase, were incorporated into an antimicrobial peptide. Its effect on the stability, activity, and the structure of the peptide was studied to evaluate the potential of these novel unnatural amino acids as a building block for antimicrobial peptides. The incorporation of this unusual amino acid increased the resistance of the peptide against serum protease more than three times without a decrease in the activity. Circular dichroism spectra of the peptides indicated that all novel unnatural amino acids must have lower helical forming propensities than lysine. Our results indicated that the unnatural amino acids synthesized in this study could be used not only as a novel building block for combinatorial libraries of antimicrobial peptides, but also for structure–activity relationship studies about antimicrobial peptides.     

Large scale ab initio modeling of structurally uncharacterized antimicrobial peptides reveals known and novel folds

Antimicrobial resistance within a wide range of infectious agents is a severe and growing public health threat. Antimicrobial peptides (AMPs) are among the leading alternatives to current antibiotics, exhibiting broad spectrum activity. Their activity is determined by numerous properties such as cationic charge, amphipathicity, size, and amino acid composition. Currently, only around 10% of known AMP sequences have experimentally solved structures. To improve our understanding of the AMP structural universe we have carried out large scale ab initio 3D modeling of structurally uncharacterized AMPs that revealed similarities between predicted folds of the modeled sequences and structures of characterized AMPs. Two of the peptides whose models matched known folds are Lebocin Peptide 1A (LP1A) and Odorranain M, predicted to form β-hairpins but, interestingly, to lack the intramolecular disulfide bonds, cation-π or aromatic interactions that generally stabilize such AMP structures. Other examples include Ponericin Q42, Latarcin 4a, Kassinatuerin 1, Ceratotoxin D, and CPF-B1 peptide, which have α-helical folds, as well as mixed αβ folds of human Histatin 2 peptide and Garvicin A which are, to the best of our knowledge, the first linear αββ fold AMPs lacking intramolecular disulfide bonds. In addition to fold matches to experimentally derived structures, unique folds were also obtained, namely for Microcin M and Ipomicin. These results help in understanding the range of protein scaffolds that naturally bear antimicrobial activity and may facilitate protein design efforts towards better AMPs.     

Effects of amino acid substitutions in and around the arginine-glycine-aspartic acid (RGD) sequence on fertilization and parthenogenetic development in mature bovine oocytes

Integrins have been shown to be involved in the process of fertilization and many integrin-ligand interactions are mediated through the recognition of an arginine-glycine-aspartic acid (RGD) sequence. Despite the fact the RGD domain is a principal player in determining the functional characteristics of an adhesive protein, increasing evidence has accumulated implicating the amino acids flanking the RGD sequence in determining the functional properties of the RGD-containing protein. A set of linear peptides in which the amino acid sequence in and around the RGD tri-peptide was modified was synthesized to better understand the specificity of the RGD-receptor interaction. Mature oocytes were fertilized in vitro in the presence of RGD-containing and RGD-modified peptides. Both the RGD-containing and RGD-modified peptides impaired the ability of sperm to fertilize bovine oocytes, illustrated by a reduction in cleavage. The linear modified RGD containing peptides were also examined for their ability to induce parthenogenetic development with the objective of providing a linear RGD peptide with greater biological activity than the one (GRGDSPK) used previously (Campbell et al., 2000). The data demonstrate the specificity of the receptor for the RGD sequence, further implicate the involvement of integrins in the process of bovine fertilization, and illustrate the importance of the amino acids surrounding the RGD sequence in determining the binding and functional properties of RGD-containing peptides. The data support the findings that a linear RGD peptide can block fertilization and that amino acids around the RGD sequence have an impact on the biological activity of the receptor.     

Role of peptide backbone conformation on biological activity of chemotactic peptides

To investigate the role of peptide backbone conformation on the biological activity of chemotactic peptides, we synthesized a unique analog of N-formyl-Met-Leu-Phe-OH incorporating the C alpha,alpha disubstituted residue, dipropylglycine (Dpg) in place of Leu. The conformation of the stereochemically constrained Dpg analog was examined in the crystalline state by x-ray diffraction and in solution using NMR, IR, and CD methods. The secretagogue activity of the peptide on human neutrophils was determined and compared with that of a stereochemically constrained, folded type II beta-turn analog incorporating 1-aminocyclohexanecarboxylic acid (Ac6c) at position 2 (f-Met-Ac6c-Phe-OMe), the parent peptide (f-Met-Leu-Phe-OH) and its methyl ester derivative (f-Met-Leu-Phe-OMe). In the solid state, the Dpg analog adopts an extended beta-sheet-like structure with an intramolecular hydrogen bond between the NH and CO groups of the Dpg residue, thereby forming a fully extended (C5) conformation at position 2. The phi and psi values for Met and Phe residues are significantly lower than the values expected for an ideal antiparallel beta conformation causing a twist in the extended backbone both at the N and C termini. Nuclear magnetic resonance studies suggest the presence of a significant population of the peptide molecules in an extended antiparallel beta conformation and the involvement of Dpg NH in a C5 intramolecular hydrogen bond in solutions of deuterated chloroform and deuterated dimethyl sulfoxide. IR studies provide evidence for the presence of an intramolecular hydrogen bond in the molecule and the antiparallel extended conformation in chloroform solution. CD spectra in methanol, trifluoroethanol, and trimethyl phosphate indicate that the Dpg peptide shows slight conformational flexibility, whereas the folded Ac6c analog is quite rigid. The extended Dpg peptide consistently shows the highest activity in human peripheral blood neutrophils, being approximately 8 and 16 times more active than the parent peptide and the folded Ac6c analog, respectively. However, the finding that all four peptides have ED50 (the molar concentration of peptide to induce half-maximal enzyme release) values in the 10(-8)-10(-9) M range suggests that an induced fit mechanism may indeed be important in this ligand-receptor interaction. Moreover, it is also possible that alterations in the backbone conformation at the tripeptide level may not significantly alter the side chain topography and/or the accessibility of key functional groups important for interaction with the receptor.     

Systematic peptide engineering and structural characterization to search for the shortest antimicrobial peptide analogue of gaegurin 5

As part of an effort to develop new, low molecular mass peptide antibiotics, we searched for the shortest bioactive analogue of gaegurin 5 (GGN5), a 24-residue antimicrobial peptide. Thirty-one kinds of GGN5 analogues were synthesized, and their biological activities were analyzed against diverse microorganisms and human erythrocytes. The structural properties of the peptides in various solutions were characterized by spectroscopic methods. The N-terminal 13 residues of GGN5 were identified as the minimal requirement for biological activity. The helical stability, the amphipathic property, and the hydrophobic N terminus were characterized as the important structural factors driving the activity. To develop shorter antibiotic peptides, amino acid substitutions in an inactive 11-residue analogue were examined. Single tryptophanyl substitutions at certain positions yielded some active 11-residue analogues. The most effective site for the substitution was the hydrophobic-hydrophilic interface in the amphipathic helical structure. At this position, tryptophan was the most useful amino acid conferring favorable activity to the peptide. The introduced tryptophan played an important anchoring role for the membrane interaction of the peptides. Finally, two 11-residue analogues of GGN5, which exhibited strong bactericidal activity with little hemolytic activity, were obtained as property-optimized candidates for new peptide antibiotic development. Altogether, the present approach not only characterized some important factors for the antimicrobial activity but also provided useful information about peptide engineering to search for potent lead molecules for new peptide antibiotic development.     

Synthesis of differentially protected N-acylated reduced pseudodipeptides as building units for backbone cyclic peptides.

Backbone cyclization has become an important method for generating or stabilizing the bioactive conformation of peptides without affecting the amino acid side-chains. Up to now, backbone cyclic peptides were mostly synthesized with bridges between N-amino- and N-carboxy-functionalized peptide bonds. To study the influence of a more flexible backbone on the biological activity, we have developed a new type of backbone cyclization which is achieved via the N-functionalized moieties of acylated reduced peptide bonds. As described in our previous publications, the formation of N-functionalized dipeptide units facilitates the peptide assembly compared with the incorporation of N-alkyl amino acids. Besides the racemization-free synthesis of Fmoc-protected pseudodipeptide esters with reduced peptide bonds, the new type of backbone modification allows the use of a great variety of omega-amino- and alpha,omega-dicarboxylic acids differing in chain length and chemical properties. Best results for the coupling of the omega-amino- and alpha,omega-dicarboxylic acids to the reduced peptide bond were obtained by the formation of mixed anhydrides with alkyl chloroformates. Whereas the protecting group combination of Z/OBzl in the dipeptide unit and Boc/OtBu for the N-functionalized moiety leads to the formation of 2-ketopiperazine during hydrogenation, the combination of Fmoc/OtBu and Alloc/OAll is very suitable for the synthesis of backbone cyclic peptides on solid support.     

Flexibility is a mechanical determinant of antimicrobial activity for amphipathic cationic α-helical antimicrobial peptides

Antimicrobial peptides (AMPs) are recognized as the potential substitutions for common antibiotics. Flexibility has been demonstrated to be a dominant on antimicrobial activity of an AMP, similar to the structural parameters such as hydrophobicity and hydrophobic moment as well as positive charge. To better understand the effect of flexibility on antimicrobial activity, we herein examined seventy-eight peptides derived from nine different species. Defined as a weighted average of amino acid flexibility indices over whole residue chain of AMP, flexibility index was used to scale the peptide flexibility and indicated to be a reflection of mechanical properties such as tensile and flexural rigidities. The results demonstrated that flexibility index is relevant to but different from other structural properties, may enhance activity against Escherichia coli for stiff clustered peptides or reduce activity against E. coli for flexible clustered peptides, and its optimum occurs at about − 0.5. This effect of flexibility on antimicrobial activity may be involved to the antimicrobial actions, such as stable peptide-bound leaflet formation and sequent stress concentration in target cell membrane, mechanically. The present results provide a new insight in understanding antimicrobial actions and may be useful in seeking for a new structure–activity relationship for cationic and amphipathic α-helical peptides.     

Non-sequential vasopressin peptides. Stereochemistry and biological activity.

Two cyclic disulfides of structure Cys-Tyr-Arg-Arg-Tyr-Cys-NH2 (1) and Cys-Tyr(Me)-Arg-Arg-Tyr(Me)-Cys-NH2 (2), two nonapeptide derivatives of 1 extended at the C-terminal with Pro-Arg-Gly-NH2 (3) or Pro-D-Arg-Gly-NH2 (4) and derivatives of 3 and 4 having Mpr in position 1, i.e. analogs (5) and (6), respectively, were synthesized, and their stereochemistry and biological activity were studied. All the peptides displayed low dose-dependent uterotonic activity in vitro and antidiuretic activity in vivo. None of the peptides increased the blood pressure of the experimental animals. Compounds 2, 4 and 6 showed a low inhibitory effect on AVP pressor activity; compound 6, in addition, displays a significant and long-lasting vasodepressor effect. NMR measurements indicated the existence of hydrogen bond between the amino acid residues in positions 2,5 and 3,4 of peptides 1 and 2, and side-chain interactions between amino acid residues in positions 2,3 and 4,5 of peptide 1. No such side-chain interactions were detected in peptide 2.     

Alanine Scanning Studies of the Antimicrobial Peptide Aurein 1.2

Antimicrobial peptides (AMPs) are compounds widely distributed in nature that display activity against a broad spectrum of pathogens. Amphibian skin, as an organ rich in pharmacologically active peptides, appears to be an interesting source of novel AMPs. Aurein 1.2 (GLFDIIKKIAESF-NH₂) is a short 13-residue antimicrobial peptide primarily isolated from the skin secretions of Australian bell frogs. In this study, the alanine scan of aurein 1.2 was performed to investigate the effect of each amino acid residue on its biological and physico-chemical properties. The biological studies included determination of minimum inhibitory concentration, activity against biofilm, and inhibitory effect on its formation. Moreover, the hemolytic activity as well as serum stability was determined. The hydrophobicity of peptides and their self-assembly were investigated using reversed-phase chromatography. In addition, their helicity was calculated from circular dichroism spectra. The results not only provided information on structure-activity relationship of aurein 1.2 but also gave insights into design of novel analogs of AMPs in the future.

     

Modulation of fibrillation of hIAPP core fragments by chemical modification of the peptide backbone

The well-ordered cross β-strand structure found in amyloid aggregates is stabilized by many different side chain interactions, including hydrophobic interactions, electrostatic charge and the intrinsic propensity to form β-sheet structures. In addition to the side chains, backbone interactions are important because of the regular hydrogen-bonding pattern. β-Sheet breaking peptide analogs, such as those formed by N-methylation, interfere with the repetitive hydrogen bonding pattern of peptide strands. Here we test backbone contributions to fibril stability using analogs of the 6-10 residue fibril core of human islet amyloid polypeptide, a 37 amino acid peptide involved in the pathogenesis of type II diabetes. The Phe-Gly peptide bond has been replaced by a hydroxyethylene or a ketomethylene group and the nitrogen-atom has been methylated. In addition, we have prepared peptoids where the side chain is transferred to the nitrogen atom. The backbone turns out to be extremely sensitive to substitution, since only the minimally perturbed ketomethylene analog (where only one of the -NH- groups has been replaced by -CH(2)-) can elongate wildtype fibrils but cannot fibrillate on its own. The resulting fibrils displayed differences in both secondary structure and overall morphology. No analog could inhibit the fibrillation of the parent peptide, suggesting an inability to bind to existing fibril surfaces. In contrast, side chain mutations that left the backbone intact but increased backbone flexibility or removed stabilizing side-chain interactions had very small effect on fibrillation kinetics. We conclude that fibrillation is very sensitive to even small modifications of the peptide backbone.     

Peptide inhibitors of fibronectin, laminin, and other adhesion molecules: unique and shared features

Synthetic peptides can specifically inhibit the function of certain adhesive glycoproteins in vitro and in vivo. We have compared the relative activities of a set of six variant synthetic peptides based on the sequence of fibronectin in terms of their ability to inhibit the interactions of fibroblasts with fibronectin, spreading factor/vitronectin, laminin, and native collagen gels. BHK (baby hamster kidney) and chick embryo fibroblasts spreading on these adhesive molecules displayed distinctive patterns of sensitivity to inhibition by this panel of peptides, which depended on the adhesive molecule rather than the cell type. For fibronectin, Gly-Arg-Gly-Asp-Ser was considerably more active than Arg-Gly-Asp-Ser, whereas these two peptides displayed little difference in activity in inhibiting cell adhesion to spreading factor. For both proteins, the inverted peptide sequence Ser-Asp-Gly-Arg was also moderately active, whereas closely related peptides containing a transposition, a deletion, or a single, conserved amino acid substitution were much less active. For inhibiting interactions with laminin or native type I collagen gels, Gly-Arg-Gly-Asp-Ser was only weakly active, but the inverted peptide Ser-Asp-Gly-Arg unexpectedly continued to display inhibitory activity for both attachment proteins in both cell types. Our results indicate that different adhesive processes depend on distinct peptide recognition events by a cell. However, there may be a possible common denominator among attachment proteins in a moderate sensitivity to Ser-Asp-Gly-Arg. Our study also underscores the importance of examining a full set of peptide analogs when these novel inhibitors are used to characterize biological processes.     

Cyclic peptides — Small and big and their conformational aspects

Cyclic peptides form an interesting class of compounds for study by conformational analysis, by virtue of their unique conformational features and biological properties. The small cyclic peptides having 3-6 peptide units in their ring, show a variety of conformational characteristics such as occurrence ofcis peptide units, flexibility of peptide dimension and variety in hydrogen bonding. The different possible conformations of cyclic tri- and hexa-peptides are given and certain specific conformational features are discussed for cyclic tetra and pentapeptides. For higher cyclic peptides, the hydrogen bonding requirement for stability of the backbone of the ring, is seen to be kept to a minimum. These various features and their significance are examined and discussed in the light of energy minimization studies and analysis of available experimental data.