Base-stacking interactions in double-helical DNA structures: experiment versus theory

Atom-atom potential energy calculations have been undertaken for deriving stacking energies in double-helical structures. A comparison between the energy patterns of A- and B-type double-helical fragments determined by single-crystal X-ray diffraction methods versus idealized uniform models based on fibre diffraction data shows that the van der Waals stacking energy is largely sensitive to local changes in the relative orientation of adjacent base pairs. The sequence-dependent conformational variability observed in the high-resolution structures appears to be a consequence of the equipartitioning of the stacking energy along the double helix. The large energy variations expected for a uniform structure are dampened considerably in the observed structures by means of local changes in conformational features such as helix rotation and roll angles between base pairs.     

Molecular structures for microbial polysaccharides: conformation of the Klebsiella serotype K25 capsular polysaccharide

Fibre type X-ray diffraction patterns have been obtained from oriented, semi-crystalline films prepared from the sodium salt of the capsular polysaccharide of Klebsiella serotype K25. This molecule has a tetrasaccharide repeating structure consisting of a disaccharide backbone and a disaccharide side chain. The backbone contains a di-equatorially 1,4 linked β-d-glucose residue followed by a di-equatorially 1,3 linked β-d-galactose residue. The side chain is attached to the axial O(4) position of the galactose residue and consists of a di-equaltorially 1,2 linked β-d-glucoronic acid with a β-d-glucose residue attached terminally. An interesting feature of the backbone linkage geometry of this polysaccharide is its similarity with those of the animal connective tissue polydisaccharides. Analysis of diffraction patterns gives rise to an extended three fold helical conformation with an axially projected advance per chemical repeat of 0.97 nm. Molecular models have been computer generated using least squares techniques to optimize interatomic contacts and simultaneously meet the observed helical parameters. A left handed helix with inter-residue stabilizing hydrogen bonds was found to be most favourable and comparison of this model with other relevant polysaccharide structures is male.     

Interpretation of the low-angle meridional neutron diffraction patterns from collagen fibres in terms of the amino acid sequence

Measurement of fully corrected, low angle meridional neutron diffraction intensities from native collagen fibres, in a full range of H₂O/D₂O contrasts, is described. The observed contrast dependence of the intensities of the first 12 orders of 670 A (D) axial periodicity is fitted to a general quadratic theory of contrast variation. The observed first order contrast dependence is compared to predictions based on the amino acid sequence, assuming different extents of chemical H/D exchange, and found to be consistent with complete non-carbon linked H/D exchange except for 1 to 1.6 hydrogen atoms per gly-X-Ytriplet involved in H-bonding. Both X-ray and neutron diffraction data in a variety of contrasts are consistent with a unique model for the axially projected structure of native collagen fibrils based on the amino acid sequence. This model if characterized by average axial residua translations in the NH₂ and COOH terminal, non-triple helical telopeptides, expressed as multiples of the triple helical residue translation, of 0.85 ± 0.05 and 0.7 ± 01 respectively, with D = 235 ± 1 residues.     

Base-Stacking Interactions in Double-Helical DNA Structures: Experiment Versus Theory

Abstract

Atom-atom potential energy calculations have been undertaken for deriving stacking energies in double-helical structures. A comparison between the energy patterns of A- and B-type double-helical fragments determined by single-crystal X-ray diffraction methods versus idealized uniform models based on fiber diffraction data shows that the van der Waals stacking energy is largely sensitive to local changes in the relative orientation of adjacent base pairs. The sequence-dependent conformational variability observed in the high-resolution structures appears to be a consequence of the equipartitioning of the stacking energy along the double helix. The large energy variations expected for a uniform structure are dampened considerably in the observed structures by means of local changes in conformational features such as helix rotation and roll angles between base pairs.     

An energetic evaluation of a “Smith” collagen microfibril model

An energy minimized three-dimensional structure of a collagen microfibril template was constructed based on the five-stranded model of Smith (1968), using molecular modeling methods and Kollman force fields (Weiner and Kollman, 1981). For this model, individual molecules were constructed with three identical polypeptide chains ((Gly-Pro-Pro) _n , (Gly-Prop-Hyp) _n , or (Gly-Ala-Ala) _n , wheren=4, 12, and 16) coiled into a right-handed triple-helical structure. The axial distance between adjacent amino acid residues is about 0.29 nm per polypeptide chain, and the pitch of each chain is approximately 3.3 residues. The microfibril model consists of five parallel triple helices packed so that a left-handed superhelical twist exists. The structural characteristics of the computed microfibril are consistent with those obtained for collagen by X-ray diffraction and electron microscopy. The energy minimized Smith microfibril model for (Gly-Pro-Pro)₁₂ has an axial length of about 10.2 nm (for a 36 amino acid residue chain), which gives an estimated D-spacing (234 amino acids per chain) of approximately 66.2 nm. Studies of the microfibril models (Gly-Pro-Pro)₁₂, (Gly-Pro-Hyp)₁₂, and (Gly-Ala-Ala)₁₂ show that nonbonded van der Waals interactions are important for microfibril formation, while electrostatic interactions contribute to the stability of the microfibril structure and determine the specificity by which collagen molecules pack within the microfibril.     

An energetic evaluation of a "Smith" collagen microfibril model.

An energy minimized three-dimensional structure of a collagen microfibril template was constructed based on the five-stranded model of Smith (1968), using molecular modeling methods and Kollman force fields (Weiner and Kollman, 1981). For this model, individual molecules were constructed with three identical polypeptide chains ((Gly-Pro-Pro) _n , (Gly-Prop-Hyp) _n , or (Gly-Ala-Ala) _n , wheren=4, 12, and 16) coiled into a right-handed triple-helical structure. The axial distance between adjacent amino acid residues is about 0.29 nm per polypeptide chain, and the pitch of each chain is approximately 3.3 residues. The microfibril model consists of five parallel triple helices packed so that a left-handed superhelical twist exists. The structural characteristics of the computed microfibril are consistent with those obtained for collagen by X-ray diffraction and electron microscopy. The energy minimized Smith microfibril model for (Gly-Pro-Pro)₁₂ has an axial length of about 10.2 nm (for a 36 amino acid residue chain), which gives an estimated D-spacing (234 amino acids per chain) of approximately 66.2 nm. Studies of the microfibril models (Gly-Pro-Pro)₁₂, (Gly-Pro-Hyp)₁₂, and (Gly-Ala-Ala)₁₂ show that nonbonded van der Waals interactions are important for microfibril formation, while electrostatic interactions contribute to the stability of the microfibril structure and determine the specificity by which collagen molecules pack within the microfibril.     

Structure of collagen with a new net of hydrogen bonds]

A conformational variability of the collagen triple helix was studied with the methods of molecular mechanics. The Rich-Crick model with one hydrogen bond per tripeptide fragment or the model with two hydrogen bonds per tripeptide fragment were used for tripeptides forming the primary structure of the protein. Imino acid and amino acid residues were located in the second position of the tripeptide fragments in the first and second cases, respectively. Conformations on domain boundaries, which had alternating structures with one and two hydrogen bonds per tripeptide, were particularly studied. Essentially all types of collagen backbone composed of amino acid residues most frequently occurring in this protein were considered. A new model was suggested that combined elements of the Rich-Crick model and our new approach. This was shown to be stereochemically valid, energetically advantageous, and consistent with the experimental data. It was conclusively demonstrated that the primary structure of collagen determines its tertiary structure.     

Structure of type I and type III heterotypic collagen fibrils: an X-ray diffraction study

The molecular packing arrangement within collagen fibrils has a significant effect on the tensile properties of tissues. To date, most studies have focused on homotypic fibrils composed of type I collagen. This study investigates the packing of type I/III collagen molecules in heterotypic fibrils of colonic submucosa using a combination of X-ray diffraction data, molecular model building, and simulated X-ray diffraction fibre diagrams. A model comprising a 70-nm-diameter D- (approximately 65 nm) axial periodic structure containing type I and type III collagen chains was constructed from amino acid scattering factors organised in a liquid-like lateral packing arrangement simulated using a classical Lennard-Jones potential. The models that gave the most accurate correspondence with diffraction data revealed that the structure of the fibril involves liquid-like lateral packing combined with a constant helical inclination angle for molecules throughout the fibril. Combinations of type I:type III scattering factors in a ratio of 4:1 gave a reasonable correspondence with the meridional diffraction series. The attenuation of the meridional intensities may be explained by a blurring of the electron density profile of the D period caused by nonspecific or random interactions between collagen types I and III in the heterotypic fibril.     

Computation of 3D structure and distribution of helical parameters for D-period segments of human fibrillar collagen III

The structures of D-period segments of collagen (234 amino residues or ～1/4 of whole length) are established by methods of molecular mechanics and geometry analysis. Each D-period segment proves to have a unique spatial structure. The distributions of local helical parameters along the molecule are calculated. It is found that a second hydrogen bond is formed in every case when the second residue in the tripeptide G-X-Y is an amino acid. With such a combined H-bond network, all the peptide CO groups of glycines and of the third residues in tripeptides have quasi-equivalent positions on the surface of the collagen molecule. The local deformations of the polyproline II helix in the triple complex give rise to the observed modulation of structure at the macromolecular level, which may be important for the mutual orientation of collagen molecules during fibrillogenesis.     

Synergistic interactions between the genetically modified bacterial polysaccharide P2 and carob or konjac mannan

Rheological studies have confirmed that the bacterial polysaccharide P2, a genetically modified variant of the Acetobacter xylinum polysaccharide acetan, undergoes synergistic gelation with either of the plant polysaccharides carob or konjac mannan. X-ray fibre diffraction data shows that P2 can form a 5-fold helical structure of pitch 4.7nm and an axial rise per disaccharide repeat of 0.92nm. Optical rotation data demonstrate that P2 undergoes a coil-helix transition in solution and that deacylation enhances the stability of the helical structure in solution. Studies made on mixtures prepared at different temperatures and ionic strengths suggest that denaturation of the P2 helix favours interaction and gelation. Deacetylation of P2 enhances gelation. X-ray diffraction data for oriented fibres prepared from deacetylated P2-konjac mannan mixed films reveal a 6-fold helical structure of pitch 5.54nm with an axial rise per disaccharide repeat also of 0.92nm. This mixed helix provides direct evidence for binding between the two polysaccharides. P2 contains two sites of acetylation: one on the backbone and one on the sidechain. The former site of acetylation inhibits helix formation for P2. It is suggested that this site of acetylation also inhibits formation of the mixed helix, explaining the enhanced gelation of mixtures on deacetylation.     

Two types of tripeptide conformation in collagen. Calculation of the structure of (Gly-Pro-Ser)n and (Gly-Val-Hyp)n polytripeptides

Conformational analysis of polypeptides (Gly-Pro-Ser)n and (Gly-Val-Hyp)n was carried out for collagen-like triple helical complexes (coiled coils with screw symmetry). The lowest energy structure of the first polymer (helical parameters t 52,8, h 0,282 nm) is very close to that of (Gly-Pro-Hyp)n. The hydroxyl group of a serine residue does not form any intramolecular hydrogen bonds in this structure. (Gly-Val-Hyp)n triple complex is shown to unwind to t 7,7, h 0,297 nm as a result of optimization procedure. These findings confirm the assumption, made earlier on the basis of conformational analysis of (Gly-Pro-Hyp)n, (Gly-Pro-Ala)n, (Gly-Ala-Hyp)n, (Gly-Ala-Ala)n, that the collagen triple helix contains stable wound triplets with proline in the second position, while the absence of imino acid in the 2nd position facilitates the unwinding of the triple helix. Thus, a collagen helix appears to have different parameters for the sites differing in the amino acid sequence. The values measured in the X-ray experiments (h 0,29 nm, t' 36) should be considered as a result of averaging. The model allows to reconcile the X-ray data for collagen and crystalline (Gly-Pro-Pro)10 oligomer.     

Construction and design of single stranded collagen-like structure

Polytheonamide B, a 48 residue long highly cytotoxic polypeptide extracted from marine sponges contains amino acids of alternate chirality and the N-terminal region is rich in t-Leu residues. The aim of this study is to analyze the effect of these alternate chiralities and conformational behavior of various model peptides containing t-Leu, in order to explore their role in designing bioactive peptides that shall offer advantages comparable to polytheonamide B, while circumventing its limitations. The conformational behavior of various peptides constructed from t-Leu of the form Ac-(L/D-X-L/D-Y)n-NHMe, where X = Gly/Ala/Leu and Y = t-Leu has been studied and compared with the corresponding peptides containing Leu residue. The results show that the helix driving capacity of L and D forms of t-Leu is less than that of Leu residue. In poly t-Leu peptides, the population of collagen/inverse collagen-type structures or right/left handed-helical structures for L and D forms respectively is found to be chain length-dependent. The stability of the helical structures is increased by -2 kcal per residue over the collagen-type structure in poly t-Leu peptides with chain length greater than five residues. Molecular view of peptides in collagen-type structure shows that the bulky side chains of t-Leu residues mask the NH moieties of the peptide bond, while the carbonyl groups lying along the helical groove are accessible to the small solvent molecules. Molecular model building suggests that one ethylene glycol molecule interacts by forming hydrogen bonds with carbonyl groups of two adjacent t-Leu residues. To the best of our knowledge, this is the first study of its own kind on the construction of a single-strand collagen/inverse collagen-type structure using unusual amino acid residues. Such synthetic collagen mimetic peptides shall exhibit specific affinity to natural collagen under controlled thermal conditions (heat or laser treatment) and hence can be explored as a new targeting method to attach therapeutic drugs to collagens in the living tissues and to biomaterials that incorporate natural collagens.     

Heterotrimeric type I collagen C-telopeptide conformation as docked to its helix receptor

The amino (N-telo) and carboxyl (C-telo) telopeptides of type I collagen play crucial roles, in vivo and in vitro, in the assembly of collagen fibrils, regulating the axial alignment of the molecules within a fibril, azimuthal orientations of neighboring molecules, and cross-link formation. High-resolution structures of the telopeptides are not available from X-ray diffraction studies, but computational methods permitted prediction of the N-telo structure within a fibril. Here, using a suite of molecular modeling software, the more complex heterotrimeric C-telo of human type I collagen has been built from the correct sequences and energy minimized and the energy minimum confirmed by molecular dynamics. The receptor triple helix was modeled on the basis of the Protein Data Bank coordinates of a collagen-like sequence. Docking of the heterotrimeric C-telopeptide to its receptor showed that hydrophobic interactions involving the short alpha2 C-telopeptide are crucial determinants of its azimuthal orientation within the docked structure. A docked C-telo can interact with only one neighboring helix. The two alpha1(I) C-telo chains in the alpha1(Alpha)-alpha2-alpha1(B) chain stagger do not have identical docked conformations, and one of the alpha1(I) C-telo chains appears to be favored for formation of a cross-link between its K(16C) and a helix K87. Prior studies showed that a docked N-telopeptide can interact with two adjacent collagen monomers, forming a tightly packed region. A recent X-ray analysis showed the N- and C-telo regions pack differently, with the C-telo region being less densely packed than N-telo regions. This difference between N- and C-telopeptide docked structures demonstrates how unique and specific packing can occur in the fibril at each boundary of the type I collagen gap region.     

Conformation of keratan sulphate

X-ray diffraction patterns from stretched films of keratan sulphate, isolated from bovine cornea, indicate that the molecules are twofold helices with an axial rise per disaccharide residue of 0.945 nm. These helices are oriented with their twofold screw axes parallel and about equally spaced, but are not further organized into regular crystalline arrays. Computer methods were used to construct a molecular model with the observed symmetry and axial rise per disaccharide residue, with standard bond lengths, bond angles and pyranose ring conformations and with a hydrogen bond of length 0.270 nm between O(3) of N-acetylglucosamine and O(5) of galactose. This model has no unacceptably short non-bonded interatomic distances. The intensity distribution of the diffraction pattern calculated from the co-ordinates of the model is in reasonable agreement with the observed intensity distribution. This keratan sulphate model is an extended polysaccharide chain fringed with charged sulphate side groups, and is similar to those that have already been reported for chondroitin sulphates and dermatan sulphate, paralleling the similarities in covalent structure and biological occurrence among these substances.     

Folding of a small helical protein using hydrogen bonds and hydrophobicity forces

A reduced protein model with five to six atoms per amino acid and five amino acid types is developed and tested on a three-helix-bundle protein, a 46-amino acid fragment from staphylococcal protein A. The model does not rely on the widely used Go approximation, which ignores non-native interactions. We find that the collapse transition is considerably more abrupt for the protein A sequence than for random sequences with the same composition. The chain collapse is found to be at least as fast as helix formation. Energy minimization restricted to the thermodynamically favored topology gives a structure that has a root-mean-square deviation of 1.8 A from the native structure. The sequence-dependent part of our potential is pairwise additive. Our calculations suggest that fine-tuning this potential by parameter optimization is of limited use.     

X-ray-diffraction patterns from chondroitin 4-sulphate, dermatan sulphate and heparan sulphate

Ordered conformations from the sodium salts of chondroitin 4-sulphate, dermatan sulphate and heparan sulphate were observed by X-ray diffraction. Chondroitin 4-sulphate shows similar threefold helical character to that previously reported for chondroitin 6-sulphate and hyaluronates. Dermatan sulphate forms an eightfold helix with an axial rise per disaccharide of 0.93nm, which favours the l-iduronic acid moiety in the normal C1 chair form. The layer-line spacing and axial projection in heparan sulphate of 1.86nm favours a tetrasaccharide repeat with glycosidic linkages alternating beta-d-(1-->4) and alpha-d-(1-->4).     

Solution structures of the core light-harvesting alpha and beta polypeptides from Rhodospirillum rubrum: implications for the pigment-protein and protein-protein interactions

We have determined the solution structures of the core light-harvesting (LH1) alpha and beta-polypeptides from wild-type purple photosynthetic bacterium Rhodospirillum rubrum using multidimensional NMR spectroscopy. The two polypeptides form stable alpha helices in organic solution. The structure of alpha-polypeptide consists of a long helix of 32 amino acid residues over the central transmembrane domain and a short helical segment at the N terminus that is followed by a three-residue loop. Pigment-coordinating histidine residue (His29) in the alpha-polypeptide is located near the middle of the central helix. The structure of beta-polypeptide shows a single helix of 32 amino acid residues in the membrane-spanning region with the pigment-coordinating histidine residue (His38) at a position close to the C-terminal end of the helix. Strong hydrogen bonds have been identified for the backbone amide protons over the central helical regions, indicating a rigid property of the two polypeptides. The overall structures of the R.rubrum LH1 alpha and beta-polypeptides are different from those previously reported for the LH1 beta-polypeptide of Rhodobacter sphaeroides, but are very similar to the structures of the corresponding LH2 alpha and beta-polypeptides determined by X-ray crystallography. A model constructed for the structural subunit (B820) of LH1 complex using the solution structures reveals several important features on the interactions between the LH1 alpha and beta-polypeptides. The significance of the N-terminal regions of the two polypeptides for stabilizing both B820 and LH1 complexes, as clarified by many experiments, may be attributed to the interactions between the short N-terminal helix (Trp2-Gln6) of alpha-polypeptide and a GxxxG motif in the beta-polypeptide.     

X-ray diffraction study into the effects of liming on the structure of collagen

The manufacture of parchment from animal skin involves processes that remove hair, fats, and other macromolecules. Although it is well understood that the collagen fibers "open up" during processing, this study uses small and wide-angle X-ray diffraction to measure quantitatively the changes induced at the nanoscopic and microscopic levels. The axial rise per residue distance within the collagen molecules is unaffected by salt and lime treatments. Salting of the hides appears to remove noncollagenous materials. The intermolecular lateral packing distance between the hydrated collagen molecules (1.4 nm) increases after salting ( approximately 1.5 nm) and liming ( approximately 1.55 nm); drying is responsible for a reduction to approximately 1.2 nm in all samples. The axial staggered array (d spacing) is reduced by 1 nm after liming and is unaffected by drying. The average fibril diameter increases from 103.2 to 114.5 nm following liming, and the fibril-to-fibril distance increases from 122.6 to 136.1 nm.     

Interpretation of the small-angle X-ray diffraction of collagen in view of the primary structure of the alpha1 chain.

The small-angle X-ray diffraction pattern of collagen has been calculated using the sequence of the alpha 1 chain and a Hodge-Petruska scheme for the packing of the collagen molecules. The molecular stagger giving the best fit of calculated-to observed structure factors has been found to be 236 or 237 amino acid residues for three tendon collagens. But this result depends on the appoximation that the molecular conformation is uniform throughout the molecule. A comparison of the observed and calculated electron density profiles in axial projection leads to a corrected model, in which the COOH-terminal telopeptide is contracted in a way suggesting a saddle-shaped electron density distribution near the collagenase site.     

Glutaraldehyde-induced changes in the axially projected fine structure of collagen fibrils

The fine structure of the collagen fibril, as seen in axial projection, is changed by treatment with glutaraldehyde. The changes are detectable in electron-optical staining patterns and in the intensities of the low-angle meridional X-ray diffraction maxima. Current knowledge of the amino acid sequence of collagen and of the axial arrangement of molecules in fibrils permits interpretation in terms of specific alterations to the axial distribution of electron density along the fibril. Analysis of fibril staining patterns from glutaraldehyde-treated calf skin collagen shows that uptake of staining ions in positive staining patterns is inhibited at residues known to interact with glutaraldehyde (lysyl, hydroxylysyl and probably histidyl side-chains) and on other charged residues in the immediate neighbourhood of the glutaraldehyde-reactive residues. This can be seen as a "stain-exclusion effect" due to the presence of bulky polymeric complexes of glutaraldehyde molecules at cross-linking sites. Such stain exclusion accounts for the drastic changes in the negative staining pattern following treatment with glutaraldehyde. The intensity changes observed in the low-angle meridional X-ray reflections from rat tail tendon, similarly treated, also can be explained by the presence of these bulky complexes. Existing data have been used to predict a model of the altered electron density profile indicating the axial distribution of glutaraldehyde along a D-period of moist tendon collagen.