Conformational studies of proline-, thiaproline- and dimethylsilaproline-containing diketopiperazines.

As proline plays an important role in biologically active peptides, many analogues of this residue have been developed to modulate the proportion of cis and trans conformers. A correlation between the pyrrolidine ring shape and structural properties of proline has been established. Diketopiperazine (DKP) is the model of choice to study the influence of the proline ring modification. In this contribution, cyclo(Gly-Pro) and two analogues cyclo(Sip-Pro) and cyclo(Thz-Pro) have been studied with proton NMR. We showed that both analogues with heteroatoms in gamma position, silicon and sulfur respectively, display a more rigid five-member ring. The usual flexibility of proline ring is restrained in both cases and only the two C(beta)-exo and C(beta)-endo conformations are observed.     

Conformational studies of N-Tyr-MIF-1 in aqueous solution by 1H nuclear magnetic resonance spectroscopy.

N-Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2) is an endogenous brain peptide with multiple effects on animal behavior. However, there have been no studies on the conformation of this tetrapeptide. In this report, we studied the conformation of N-Tyr-MIF-1 in aqueous solution by conventional one-dimensional and two-dimensional (COSY and NOESY) 1H nuclear magnetic resonance spectroscopy at 300 MHz. A complete set of assignments for the resolved resonances and approximate assignments for the overlapping resonances were made. The results demonstrate that N-Tyr-MIF-1 is in slow exchange between two conformers, most likely determined by the cis and trans states of the proline residue. The minor conformation represents 30 +/- 3% of the population over the temperature range from 3 degrees to 73 degrees. In the major conformation, the tyrosine aromatic ring appears to be close enough to interact directly with the proline pyrrolidine ring, as indicated by a strong temperature dependence of the proline C beta H, C delta H and C delta H' chemical shifts. In contrast, this interaction of the tyrosine and proline rings is not present in the minor conformation.     

Flexible-geometry conformational energy maps for the amino acid residue preceding a proline.

Previously calculated conformational energy maps suggest that the alpha-helical conformation for the residue preceding a proline is disfavored relative to the extended conformation by more than 7 kcal/mol. In known protein structures this conformation is observed, however, to occur for about 9% of all prolines. In addition, introduction or removal of prolines at theoretically unfavorable positions in proteins and peptides can have modest effects on stability and structure. To investigate the discrepancy between calculation and experiment, we have determined how the conformation of the proline affects the calculated energy. We have also explored the effect of bond length and bond angle relaxation on the conformational energy map. The conformational energy of the preceding residue is found to be unaffected by the conformation of the proline, but the effect of allowing covalent bond relaxation is dramatic. If bond lengths and angles, and dihedral angles within the pyrrolidine ring, are allowed to relax, a calculated energy difference between the alpha and beta conformations of 1.1 kcal/mol is obtained, in reasonable agreement with experiment. The detailed shape of the calculated energy surface is also in excellent agreement with the observed conformational distributions in known protein structures.     

Rational design of protein stability: effect of (2S,4R)-4-fluoroproline on the stability and folding pathway of ubiquitin

Background

Many strategies have been employed to increase the conformational stability of proteins. The use of 4-substituted proline analogs capable to induce pre-organization in target proteins is an attractive tool to deliver an additional conformational stability without perturbing the overall protein structure. Both, peptides and proteins containing 4-fluorinated proline derivatives can be stabilized by forcing the pyrrolidine ring in its favored puckering conformation. The fluorinated pyrrolidine rings of proline can preferably stabilize either a C^γ-exo or a C^γ-endo ring pucker in dependence of proline chirality (4R/4S) in a complex protein structure. To examine whether this rational strategy can be generally used for protein stabilization, we have chosen human ubiquitin as a model protein which contains three proline residues displaying C^γ-exo puckering.

Methodology/Principal Findings

While (2S,4R)-4-fluoroproline ((4R)-FPro) containing ubiquitinin can be expressed in related auxotrophic Escherichia coli strain, all attempts to incorporate (2S,4S)-4-fluoroproline ((4S)-FPro) failed. Our results indicate that (4R)-FPro is favoring the C^γ-exo conformation present in the wild type structure and stabilizes the protein structure due to a pre-organization effect. This was confirmed by thermal and guanidinium chloride-induced denaturation profile analyses, where we observed an increase in stability of −4.71 kJ·mol⁻¹ in the case of (4R)-FPro containing ubiquitin ((4R)-FPro-ub) compared to wild type ubiquitin (wt-ub). Expectedly, activity assays revealed that (4R)-FPro-ub retained the full biological activity compared to wt-ub.

Conclusions/Significance

The results fully confirm the general applicability of incorporating fluoroproline derivatives for improving protein stability. In general, a rational design strategy that enforces the natural occurring proline puckering conformation can be used to stabilize the desired target protein.     

The planar conformation of a strained proline ring: a QM/MM study

QM and QM/MM energy calculations have been carried out on an atomic resolution structure of liganded triosephosphate isomerase (TIM) that has an active site proline (Pro168) in a planar conformation. The origin of the planarity of this proline has been identified. Steric interactions between the atoms of the proline ring and a tyrosine ring (Tyr166) on one side of the proline prevent the ring from adopting the up pucker (chi1 is approximately -30 degrees), while the side chain of a nearby alanine (Ala171) forbids the down pucker (chi1 is approximately +30 degrees). To obtain a proline conformation that is in agreement with the experimentally observed planar state, a quantum system of sufficient size is required and should at least include the nearby side chains of Tyr166, Ala171, and Glu129 to provide enough stabilization. It is argued that the current force fields for structure optimization do not describe strained protein fragments correctly. The proline is part of a catalytic loop that closes upon ligand binding. Comparison of the proline conformation in different TIM X-ray structures, indicates that in the closed conformation of TIM the proline is planar or nearly planar, while in the open conformation it is down puckered. This suggests that the planarity possibly plays a role in the overall catalytic cycle of TIM, presumable acting as a reservoir of energy that becomes available upon loop opening.     

Effects of ring contraction on the conformational preferences of α-substituted proline analogs

The structural consequences derived from the incorporation of either a methyl or a phenyl group at the α carbon of proline were recently investigated by quantum mechanical calculations (J Org Chem 2008, 73, 3418). In this work, the effect produced by contraction of the pyrrolidine ring on such α-substituted proline analogs has been explored using the same computational methods. Specifically, the intrinsic conformational preferences of the N-acetyl-N'-methylamide derivatives of the lower proline homolog L-azetidine-2-carboxylic acid (Aze), characterized by a four- instead of a five-membered ring, and its α-methyl (αMeAze) and α-phenyl (αPhAze) derivatives have been determined using quantum mechanical calculations and compared to those observed before for the proline counterparts. Replacement of the pyrrolidine ring by an azetidine cycle leads to a reduction of the conformational flexibility, especially for the Aze and αMeAze derivatives, which should be attributed to the quasi-planar geometry of the four-membered ring. Furthermore, the azetidine nitrogen shows pyramidalization, which depending on the peptide backbone conformation favors the formation of an attractive N-H···N interaction or alleviates a severe steric hindrance. Calculations on different environments predict that the tendency of αMeAze to adopt γ-turns is higher than that of unsubstituted Aze and α-methylproline, this feature being in full agreement with the experimental observations available.     

Stabilization of the biologically active conformation of the principal neutralizing determinant of HIV-1(IIIB) containing a cis-proline surrogate: 1H NMR and molecular modeling study

The V3 loop is part of the gp120 glycoprotein, an extracellular protein located on the membrane of the human immunodeficiency virus (HIV-1). This loop is significantly important in many biological processes of the virus and contains the principal neutralizing determinant (PND). The PND is one of the most variable regions of the envelope, and this is probably related to the ability of the HIV virus to escape the immunologic defenses of the target host. Particular attention has been paid to the central part of the V3 loop which contains a highly conserved GPGR/GPGQ sequence and represents the binding site for antibodies. Many attempts have been made to design synthetic peptides as mimics of the V3 loop capable of eliciting immune response. However, this strategy suffers from the great conformational flexibility small peptides have in solution, which together with bioavailability represents the most important limitation to the usefulness of synthetic peptides as drugs and as synthetic immunogens. The use of conformationally constrained peptides can alleviate this problem. Early works using NMR studies have shown that a V3(IIIB) loop-derived peptide is conformationally heterogeneous when free in water. Upon complexation with 0.5beta, a monoclonal neutralizing antibody specific for the HIV-1(IIIB) strain, it adopts a beta-hairpin conformation with the central proline forming a type VIb beta-turn. In this study, we report the design and characterization of a conformationally restricted peptide with a sequence identical to that previously described, but with thiazolidine derivatives replacing the proline. The affinity of the 2,2-dimethylthiazolidine derivative for 0.5beta demonstrates that this moiety can successfully be used to mimic the proline in a cis conformation. This peptide not only displays a high propensity to adopt a beta-hairpin conformation but also retains the type VIb RGPG beta-turn similar to that found in the native complex. These compounds could help in elaborating more efficient immunogens for HIV-1 synthetic vaccine development.     

High‐resolution crystal structures reveal a mixture of conformers of the Gly61‐Asp62 peptide bond in an oxidized flavodoxin from Bacillus cereus

Flavodoxins (Flds) are small proteins that shuttle electrons in a range of reactions in microorganisms. Flds contain a redox‐active cofactor, a flavin mononucleotide (FMN), and it is well established that when Flds are reduced by one electron, a peptide bond close to the FMN isoalloxazine ring flips to form a new hydrogen bond with the FMN N5H, stabilizing the one‐electron reduced state. Here, we present high‐resolution crystal structures of Flavodoxin 1 from Bacillus cereus in both the oxidized (ox) and one‐electron reduced (semiquinone, sq) state. We observe a mixture of conformers in the oxidized state; a 50:50 distribution between the established oxidized conformation where the peptide bond is pointing away from the flavin, and a conformation where the peptide bond is pointing toward the flavin, approximating the conformation in the semiquinone state. We use single‐crystal spectroscopy to demonstrate that the mixture of conformers is not caused by radiation damage to the crystal. This is the first time that such a mixture of conformers is reported in a wild‐type Fld. We therefore carried out a survey of published Fld structures, which show that several proteins have a pronounced conformational flexibility of this peptide bond. The degree of flexibility seems to be modulated by the presence, or absence, of stabilizing interactions between the peptide bond carbonyl and its surrounding amino acids. We hypothesize that the degree of conformational flexibility will affect the Fld ox/sq redox potential.     

Conformational plasticity and dynamics in the generic protein folding catalyst SlyD unraveled by single-molecule FRET

The relation between conformational dynamics and chemistry in enzyme catalysis recently has received increasing attention. While, in the past, the mechanochemical coupling was mainly attributed to molecular motors, nowadays, it seems that this linkage is far more general. Single-molecule fluorescence methods are perfectly suited to directly evidence conformational flexibility and dynamics. By labeling the enzyme SlyD, a member of peptidyl-prolyl cis-trans isomerases of the FK506 binding protein type with an inserted chaperone domain, with donor and acceptor fluorophores for single-molecule fluorescence resonance energy transfer, we directly monitor conformational flexibility and conformational dynamics between the chaperone domain and the FK506 binding protein domain. We find a broad distribution of distances between the labels with two main maxima, which we attribute to an open conformation and to a closed conformation of the enzyme. Correlation analysis demonstrates that the conformations exchange on a rate in the 100 Hz range. With the aid from Monte Carlo simulations, we show that there must be conformational flexibility beyond the two main conformational states. Interestingly, neither the conformational distribution nor the dynamics is significantly altered upon binding of substrates or other known binding partners. Based on these experimental findings, we propose a model where the conformational dynamics is used to search the conformation enabling the chemical step, which also explains the remarkable substrate promiscuity connected with a high efficiency of this class of peptidyl-prolyl cis-trans isomerases.     

Conformational preferences of substituted prolines in the collagen triple helix

Researchers have recently questioned the role hydroxylated prolines play in stabilizing the collagen triple helix. To address these issues, we have developed new molecular mechanics parameters for the simulation of peptides containing 4(R)-fluoroproline (Flp), 4(R)-hydroxyproline (Hyp), and 4(R)-aminoproline (Amp). Simulations of peptides based on these parameters can be used to determine the components that stabilize hydroxyproline over proline in the triple helix. The dihedrals F-C-C-N, O-C-C-N, and N-C-C-N were built using a N-beta-ethyl amide model. One nanosecond simulations were performed on the trimers [(Pro-Pro-Gly)(10)](3), [(Pro-Hyp-Gly)(10)](3), [(Pro-Amp-Gly)(10)](3), [(Pro-Amp(1+)-Gly)(10)](3), and [(Pro-Flp-Gly)(10)](3) in explicit solvent. The results of our simulations suggest that pyrrolidine ring conformation is mediated by the strength of the gauche effect and classical electrostatic interactions.     

Relationship between proton–proton nmr coupling constants and substituent electronegativities. III. Conformational analysis of proline rings in solution using a generalized Karplus equation

The relationship between published vicinal proton–proton coupling constants and the pseudorotation properties of the pyrrolidine ring in L -proline, 4-hydroxy-L -proline, 4-fluoro-L -proline, and several linear and cyclic model proline peptides is investigated. Compared to earlier studies, several important improvements are incorporated: (1) a new empirical generalization of the classical Karplus equation is utilized, which allows a valid correction for the effects of electronegativity and orientation of substitutents on ³J_HH; (2) an empirical correlation between proton–proton torsion angles and the pseudorotational parameters P and τ_m is derived; and (3) the best fit of the conformational parameters to the experimental coupling constants is obtained by means of a computerized iterative least-squares procedure. Two pseudorotation ranges were considered, classified as type N (χ₂ positive sign) and type S (χ₂ negative sign). The conformational equilibrium is fully described in terms of four geometrical parameters (P_N, τ_N, P_S, τ_S) and the equilibrium constant K. The present results indicate that, in general, the geometrical properties found in x-ray studies of proline and hydroxyproline residues are well preserved in solution. Several novel features are encountered, however. It is demonstrated that the proline ring occurs in a practically 1:1 conformational equilibrium between well-defined N- and S-type forms. Introduction of an amide group at the C-terminal end has no observable effect on this equilibrium, but the formation of a peptide bond at the imino nitrogen site results in a pronounced, but not exclusive, preference for an S-type form which is roughly 1.1 kcal/mol more stable than its N-type counterpart. The hydroxyproline ring system in neutral or acidic medium displays a pure N-type state, but N-acetylation results in the appearance of a minor (S-type) conformation. Cyclic proline dipeptides similarly exist in a biased conformational equilibrium. The major form (77–88%) corresponds to the N-type conformer observed in the solid state; the minor S-form has not been observed before. In contrast, cyclic hydroxyproline dipeptides display complete conformational purity. Ranges of endocyclic torsion angles deduced for the various classes of pyrrolidine derivatives in solution are presented. Each torsion appears confined to a surprisingly narrow range, comprising about 4°–8° in most cases. In all, the proline ring is far less “floppy” than hitherto assumed.     

Solution NMR study of the monomeric form of p13suc1 protein sheds light on the hinge region determining the affinity for a phosphorylated substrate.

Cyclin-dependent kinase subunit (CKS) proteins bind to cyclin-dependent kinases and target various proteins to phosphorylation and proteolysis during cell division. Crystal structures showed that CKS can exist both in a closed monomeric conformation when bound to the kinase and in an inactive C-terminal beta-strand-exchanged conformation. With the exception of the hinge loop, however, both crystal structures are identical, and no new protein interface is formed in the dimer. Protein engineering studies have pinpointed the crucial role of the proline 90 residue of the p13(suc1) CKS protein from Schizosaccharomyces pombe in the monomer-dimer equilibrium and have led to the concept of a loaded molecular spring of the beta-hinge motif. Mutation of this hinge proline into an alanine stabilizes the protein and prevents the occurrence of swapping. However, other mutations further away from the hinge as well as ligand binding can equally shift the equilibrium between monomer and dimer. To address the question of differential affinity through relief of the strain, here we compare the ligand binding of the monomeric form of wild-type S. pombe p13(suc1) and its hinge mutant P90A in solution by NMR spectroscopy. We indeed observed a 5-fold difference in affinity with the wild-type protein being the most strongly binding. Our structural study further indicates that both wild-type and the P90A mutant proteins adopt in solution the closed conformation but display different dynamic properties in the C-terminal beta-sheet involved in domain swapping and protein interactions.     

The alphaM1 segment of the nicotinic acetylcholine receptor exhibits conformational flexibility in a membrane environment

The transmembrane domain of the nicotinic acetylcholine receptor (nAChR) is predominantly alpha-helical, and of the four distinctly different transmembrane M-segments, only the helicity of M1 is ambiguous. In this study, we have investigated the conformation of a membrane-embedded synthetic M1 segment by solid-state nuclear magnetic resonance (NMR) methods. A 35-residue peptide representing the extended alphaM1 domain 206-240 of the Torpedo californica nAChR was synthesized with specific 13C - and 15N-labelled amino acids, and was incorporated in different phosphatidylcholine model membranes. The chemical shift of the isotopic labels was resolved by magic angle spinning (MAS) NMR and could be related to the secondary structure of the alphaM1 analog at the labelled sites. Our results show that the membrane-embedded alphaM1 segment forms an unstable alpha-helix, particularly near residue Leu18 (alphaLeu223 in the entire nAChR). This non-helical tendency was most pronounced when the peptide was incorporated in fully hydrated phospholipid bilayers, with an estimated 40-50% of the peptides having an extended conformation at position Leu18. We propose that the conserved proline residue at position 16 in the alphaM1 analog imparts a conformational flexibility on the M1 segments that could enable membrane-mediated modulation of nAChR activity.     

ZNC(C)PR的构象及分子动力学模拟

本文用分子动力学方法和质子间距约束结合建立了ZNC(C)PR的构象。质子间距是由二维核磁共振的NOE效应得到的。结果表明ZNC(C)PR分子的主链是伸展的,但Asn2的侧链与Arg5的侧链相互靠近,使整个分子成紧密结构。在所建模型的基础上,对ZNC(C)PR进行了分子动力学摸拟。发现第一位的焦谷氨酸环很灵活,这可能意味着焦谷氨酸环对ZNC(C)PR的活性并不重要,这与实验结果是一致的。     

Changes in conformation and slow refolding kinetics in mutant iso-2-cytochrome c with replacement of a conserved proline residue

Using oligonucleotide-directed mutagenesis, we have produced a mutant form of iso-2-cytochrome c of yeast in which threonine (Thr-71) replaces a conserved proline residue (Pro-71) located between two short alpha-helical segments in the native protein. Optical spectroscopy indicates that, at pH 7.2, Thr-71 iso-2-cytochrome c folds to a nonnative conformation possibly related to the alkaline form of the native protein. On titration to pH 5.2, Thr-71 iso-2-cytochrome c regains many of the optical properties of the normal protein. We have shown that the proline residue at position 71 has no effect on the kinetics of fluorescence-detected slow refolding. However, between pH 5 and pH 7.2 the amplitude for absorbance-detected slow folding is strongly pH dependent in the mutant protein but is largely independent of pH in the normal protein. We believe this to be due to the folding of Thr-71 iso-2-cytochrome c to a nonnative conformation at pH 7.2 that does not require the slow, absorbance-detected conformational changes observed in folding to the more native-like state at pH 5-6.     

Synthesis and peptide bond orientation in tetrapeptides containing L-azetidine-2-carboxylic acid and L-proline

The role of the amino acid proline in influencing the secondary and tertiary structure of proteins and polypeptides has been an area of active study for many years. We have investigated this problem by incorporating the four-membered ring amino acid, azetidine-2-carboxylic acid, into some proline polypeptides. An adjunct to the synthesis of the peptides was the synthesis of azetidine-2-carboxylic acid and its resolution. We developed an improved synthesis of N-benzhydryl-2-carbobenzyloxy azetidine, an essential intermediate required for the synthesis of L-azetidine-2-carboxylic acid. This amino acid was subsequently obtained via the partial hydrogenation of the N-benzhydryl compound, under mild conditions. Our ability to isolate the intermediate N-benzhydryl-2-carboxylic acid demonstrated that the rate of cleavage of the O-benzyl ester group in this molecule is faster than the cleavage of the N-benzhydryl group. The tetrapeptides, Boc-(L-Pro)3-L-Aze-Opcp, and Boc-(L-Aze-L-Pro)2-Opcp (Boc: t-butoxycarbonyl; Pro: proline; Aze: azetidine-2-carboxyl acid; Opcp: pentachlorophenyl), were prepared using traditional solution peptide synthesis. They were characterized by direct chemical ionization-mass spectrometry, CD spectra, and 13C- and 1H-nmr spectroscopy. The assessment of the secondary structure of the two peptides using the methods noted above has led us to conclude that the compound Boc-(L-Aze-L-Pro)2-Opcp, in trifluoroethanol, has an all-cis peptide bond conformation with phi and psi torsion angles compatible with a left-handed helix. The secondary structure assessment of the peptide Boc-(L-Pro)3-L-Aze-Opcp, in chloroform or trifluoroethanol, leads to an assignment of both cis and trans peptide bonds as being present in the peptide. We have interpreted this latter finding as indicating that the introduction of the azetidine group into a peptide containing three consecutive proline residues in a linear sequence perturbs the normal proline peptide secondary structure in this tetrapeptide.     

Backbone dynamics in dihydrofolate reductase complexes: role of loop flexibility in the catalytic mechanism

To elucidate the influence of local motion of the polypeptide chain on the catalytic mechanism of an enzyme, we have measured (15)N relaxation data for Escherichia coli dihydrofolate reductase in three different complexes, representing different stages in the catalytic cycle of the enzyme. NMR relaxation data were analyzed by the model-free approach, corrected for rotational anisotropy, to provide insights into the backbone dynamics. There are significant differences in the backbone dynamics in the different complexes. Complexes in which the cofactor binding site is occluded by the Met20 loop display large amplitude motions on the picosecond/nanosecond time scale for residues in the Met20 loop, the adjacent betaF-betaG loop and for residues 67-69 in the adenosine binding loop. Formation of the closed Met20 loop conformation in the ternary complex with folate and NADP(+), results in attenuation of the motions in the Met20 loop and the betaF-betaG loop but leads to increased flexibility in the adenosine binding loop. New fluctuations on a microsecond/millisecond time scale are observed in the closed E:folate:NADP(+) complex in regions that form hydrogen bonds between the Met20 and the betaF-betaG loops. The data provide insights into the changes in backbone dynamics during the catalytic cycle and point to an important role of the Met20 and betaF-betaG loops in controlling access to the active site. The high flexibility of these loops in the occluded conformation is expected to promote tetrahydrofolate-assisted product release and facilitate binding of the nicotinamide ring to form the Michaelis complex. The backbone fluctuations in the Met20 loop become attenuated once it closes over the active site, thereby stabilizing the nicotinamide ring in a geometry conducive to hydride transfer. Finally, the relaxation data provide evidence for long-range motional coupling between the adenosine binding loop and distant regions of the protein.     

Conformational analysis of methyl 5-O-methyl septanosides: effect of glycosylation on conformer populations

Methyl 5-O-methyl-alpha-d-glycero-d-idoseptanoside (3) and methyl 5-O-methyl-beta-d-glycero-d-guloseptanoside (4) were investigated as (1-->5)-linked di-/oligoseptanoside mimetics. Here we report the synthesis of 3 and 4 and describe their preferred solution conformations through a combination of ab initio/DFT calculations and (1)H (3)J(H,H) NMR coupling constant analysis. The conformations of 3 and 4 observed in this study are discussed in comparison to those of the parent (C5 hydroxy) compounds 1 and 2. The results indicate that methyl 5-O-methyl-alpha-septanoside 3 is relatively rigid and adopts the same (3,4)TC(5,6) conformation as 1. Methyl 5-O-methyl-beta-septanoside 4 is somewhat less rigid than its parent septanoside (2). In addition to the (6,O)TC(4,5) conformation adopted by 2, beta-septanoside 4 also populates the adjacent (3,4)TC(5,6) conformation. Glycosylation at C5 on beta-septanoside 4 therefore increases its overall flexibility and allows access to alternative ring conformations.     

Inter-chain Proline: Proline Contacts Contribute to the Stability of the Triple Helical Conformation

Abstract

The triple helical conformation observed in the collagen group of proteins is related to the presence of large numbers of imino residues and is derived from the stereochemical properties of these residues. The triple helix is stabilized by increasing numbers of these residues. Hydrogen bonds are usually considered to be a major factor in the formation and stability of protein conformation, however, imino residues are not hydrogen bond donors. We have evaluated the role of these residues in stabilizing the triple helix by re-examining two X-ray based structures of the triple helical polypeptide (Pro-Pro- Gly)₁₀ using molecular mechanics calculations. The two minimized structures are comparable in energy and have helical parameters close to the starting values for each starting structure. Our studies suggest that clusters of close van der Waals contacts between proline residues in adjacent chains contribute significantly to the stability of the triple helix. Preliminary NMR studies support this concept. We propose that non-bonded interactions between proline residues may be a significant stabilizing force in the triple helix generated by (Pro-Pro-Gly)₁₀.     

The multiple conformations of a cyclic disaccharide: DI-β-d-glucopyranose 1,6′:1′,6-dianhydride hexaacetate

The conformational behaviour of a cyclic disaccharide, di-β-d-glucopyranose 1,6′:1′,6-dianhydride hexaacetate, has been investigated. Because this molecule can exist only with the glucose rings in the unusual flexible forms, such conformational parameters as pseudorotation phase-angles have been used. Within a given number of approximations, the conformational space available for the whole system can be explored by considering only one two-dimensional map. Detailed investigations have shown that three stable conformations may be proposed. Among these, two correspond to minima found in the solid state. In one form, the six-membered rings adopt a boat conformation, whereas a skew conformation is found for the other form. However, these two conformations cannot be considered to be unique models of the conformation in solution; they both produce sets of proton-proton coupling-constants inconsistent with observed n.m.r.-spectroscopic results. At least the third form, having the six-membered rings in skew conformations, has to be taken into account. Deviations from coupling constants-molecular conformation relationships are thought to originate from ring strain.