RNA double helices generated from crystal structures of double helical dinucleoside phosphates.

The dinucleoside phosphates ApU and GpC form right-handed anti-parallel double helical fragments within their crystal lattices. Using a least squares procedure, we have generated the extended double helices which these fragments represent. ApU corresponds to a double helix with 11.9 residues per turn and a pitch of 28. 1Å. The GpC double helix has 10.4 residues per turn and a pitch of 26. 9Å.     

Characterization at atomic resolution of peptide helical structures.

A survey of literature for the various types of helices experimentally observed in high-resolution single crystal x-ray diffraction analyses of peptides has allowed to determine accurate conformational and helical parameters for the various secondary structures such as the alpha-helix, the 3(10)-helix, the fully extended conformation (2(5)-helix) and the beta-bend ribbon spiral. For each of these structures the characteristic phi, psi conformational parameters, n, the number of residues per turn, h, the height per residues and p, the pitch of the helix are described.     

3(10)-helices in proteins are parahelices

The 3(10)-helix is characterized by having at least two consecutive hydrogen bonds between the main-chain carbonyl oxygen of residue i and the main-chain amide hydrogen of residue i + 3. The helical parameters--pitch, residues per turn, radius, and root mean square deviation (rmsd) from the best-fit helix--were determined by using the HELFIT program. All 3(10)-helices were classified as regular or irregular based on rmsd/(N - 1)1/2 where N is the helix length. For both there are systematic, position-specific shifts in the backbone dihedral angles. The average phi, psi shift systematically from approximately -58 degrees, approximately -32 degrees to approximately -90 degrees, approximately -4 degrees for helices 5, 6, and 7 residues long. The same general pattern is seen for helices, N = 8 and 9; however, in N = 9, the trend is repeated with residues 6, 7, and 8 approximately repeating the phi, psi of residues 2, 3, and 4. The residues per turn and radius of regular 3(10)-helices decrease with increasing length of helix, while the helix pitch and rise per residue increase. That is, regular 3(10)-helices become thinner and longer as N increases from 5 to 8. The fraction of regular 3(10)-helices decreases linearly with helix length. All longer helices, N > or = 9 are irregular. Energy minimizations show that regular helices become less stable with increasing helix length. These findings indicate that the definition of 3(10)-helices in terms of average, uniform dihedral angles is not appropriate and that it is inherently unstable for a polypeptide to form an extended, regular 3(10)-helix. The 3(10)-helices observed in proteins are better referred to parahelices.     

Studies on the Structure of Feather Keratin: II. A β-Helix Model for the Structure of Feather Keratin

The assumption that the proline residues in feather keratin, which comprise 12 per cent of the total, are periodically located along the polypeptide chain is shown to lead to an essentially unique structure for this fibrous protein. The structure is based on a β-helix; i.e., an extended chain which coils slowly to form a helix of relatively large pitch. Such helices tend to aggregate by hydrogen bonding to form cylindrical units, which in turn can aggregate further into cable-like structures. This model has been tested with respect to its predictions concerning the x-ray diffraction pattern, infrared spectrum, mechanical properties, and chemical behavior of feather keratin. Preliminary results indicate that it is better capable of accounting for the data than previously proposed structures.     

The effects of changes in ring geometry on computer models of amylose

A review of single-crystal studies shows that α-D-glucopyranose, residues of which constitute the monomeric units of amylose, is flexible within the constraints of the Cl conformation, and that the internal differences among the rings are most clearly indicated by the variety in ring-torsion angles (or conformation angles). An index of the cumulative effect of changes in these angles is provided by the length of the virtual bond, O-1—O-4, and classification of residue geometries by virtual bond-length permits a systematic selection of suitable residues for the construction of models of amylose. By the use of D-glucopyranose residues having different geometries, it is possible to build models of (a) V-amylose helices having 6, 7, and 8 residues per turn, (b) single and double helical B-amyloses, and (c) KBr-amylose, all of which satisfy reasonable stereochemical criteria. Because no single residue can satisfactorily model all of the well known polymorphs of amylose, it is suggested that structural determinations that utilize a rigid residue approximation should make use of the full range of known, residue geometries.     

A new D-DNA form of poly(dA-dT).poly(dA-dT): an A-DNA type structure with reversed Hoogsteen pairing

The D-DNA double helix model of poly(dA-dT).poly(dA-dT) proposed in the literature is not in accordance with some notable experimental facts and physicochemical conditions to which it is related. Thus, the fibre X-ray diffraction pattern of D-DNA obtained at a relative humidity lower than that giving the A-DNA form is singularly not taken into account when one assumes that there is only one D structure of B-DNA type. We rather suggest that there are actually two different forms of D-DNA, namely D(A) which partakes in the D-A-B transitions and D(B) associated with the D-B change of conformation. Although these two DNA structures have the same helical parameters (pitch and number of residues per turn), in agreement with X-ray data, their detailed conformations are considerably different. Whereas D(B) is indeed the structure generally defined as D-DNA, a critical analysis based on a comparison between different possible DNA double helices leads us to propose dihedral angles, a set of atomic coordinates and a stereo view of another new form of D-DNA, the D(A) structural model. It is a right-handed double helix with a dinucleotide as the repeat unit. The furanose rings are of the A-DNA type (C3' endo) and the bases are hydrogen bonded according to the reversed Hoogsteen pairing. Such a disposition renders the D(A) model unsuitable for poly(dI-dC).poly(dI-dC), the other alternating polynucleotide observed in the D(B) structure. The consistency of these two different D-DNA structures of poly(dA-dT).poly(dA-dT) with the general aspects of hydration and helix-helix transitions of DNA, as well as with the conformational variability of AT base sequences, is discussed.     

X-ray diffraction and electron microscope studies on the structure of bacterial F pili.

F pili are hollow cylinders with 80 Å outer diameter and 20 Å inner diameter. Both X-ray fibre diffraction and optical diffraction of electron micrographs show a strong layer-line corresponding to a spacing of 32 Å, to which a J₄ Bessel function is assigned on the basis of the optical diffraction. X-ray diffraction patterns show near-meridional intensity on a layer-line corresponding to a spacing of 12.8 Å, to which a J₁ Bessel function is assigned. Mass per length measurements on unstained specimens in the scanning transmission electron microscope give 3000 daltons/Å, indicating that the 11,200 dalton pilin subunits are 3.7 Å apart along the axial direction of the pili. These observations show that the pilus structure can be represented as four coaxial helices of pitch 128 Å with the pilin subunits elongated and overlapping along the line of these helices. Each of these helices of subunits is translated axially with respect to its neighbour, to give a basic helix of 3.6 units per turn of 12.8 Å pitch. Radial electron density calculations indicate a 50 Å diameter girdle of hydrophobic amino acids between the inner and outer diameters of the protein shell. A molecular model of the structure at low resolution is presented.     

Formation of cytoplasmic turns between two closely spaced transmembrane helices during membrane protein integration into the ER membrane

The helical hairpin, two closely spaced transmembrane helices separated by a short turn, is a recurring structural element in integral membrane proteins, and may serve as a compact unit that inserts into the membrane en bloc. Previously, we have determined the propensities of the 20 natural amino acids, when present in the middle of a long hydrophobic stretch, to induce the formation of a helical hairpin with a lumenally exposed turn during membrane protein assembly into the endoplasmic reticulum membrane. Here, we present results from a similar set of measurements, but with the turn placed on the cytoplasmic side of the membrane. We find that a significantly higher number of turn-promoting residues need to be present to induce a cytoplasmic turn compared to a lumenal turn, and that, in contrast to the lumenal turn, the positively charged residues Arg and Lys are the strongest turn-promoters in cytoplasmic turns. These results suggest that the process of turn formation between transmembrane helices is different for lumenal and cytoplasmic turns.     

Structure studies of amylose-V complexes and retrograded amylose by action of alpha amylases, and a new method for preparing amylodextrins

Human-salivary, porcine-pancreatic, and Bacillus subtilis alpha amylases were used to study the structure of amylose-V complexes with butyl alcohol, tert-butyl alcohol, 1,1,2,2-tetrachloroethane, and 1-naphthol, and of retrograded amylose. Alpha amylase hydrolyzes the amorphous, folding areas on the surfaces of the lamella of packed helices, with the formation of resistant, amylodextrin fragments. Their degree of polymerization (d.p.) corresponds to the diameter of the helices and the folding length of the chain. The resistant fragments were fractionated on a column of Bio-Gel A-0.5m. Gel filtration of human-salivary and porcine-pancreatic alpha amylase hydrolyzates gave resistant fragments whose peak fractions, i.e., the three pooled fractions from the gel-filtration column with the highest amount of carbohydrate, had a d.p. of 75 +/- 4 for the amylose complex with butyl alcohol, 90 +/- 3 for those with tert-butyl alcohol and tetrachloroethane, and 123 +/- 2 for that with 1-naphthol. These d.p. values correspond to helices of six residues per turn with a folding length of 10 nm, seven residues per turn with a folding length of 10 nm, and eight residues per turn with a folding length of 12 nm (or nine residues per turn with a folding length of 10 nm), respectively. Acid hydrolysis of retrograded amylose gave a resistant fragment having an average d.p. of 32, human-salivary and porcine-pancreatic alpha amylases gave a resistant fragment of d.p. 43, and Bacillus subtilis alpha amylase gave a resistant fragment of d.p. 50. A structure for retrograded amylose is proposed in which there are crystalline, double-helical regions that are 10 nm long, interspersed with amorphous regions. The amorphous regions are hydrolyzed by acid and by alpha amylases, leaving the crystalline regions intact. The differences in the sizes of the resistant amylodextrins depend on the differences in the specificities of the hydrolyzing agents: acid hydrolyzes right up to the edge of the crystalline region, whereas the alpha amylases hydrolyze up to some point several D-glucosyl residues away from the crystalline region, leaving "stubs" on the ends of the amylodextrins whose sizes are dependent on the sizes of the binding sites of the individual alpha amylases.(ABSTRACT TRUNCATED AT 400 WORDS)     

Spatial structure of gramicidin A in organic solvents. 1H-NMR analysis of conformation heterogeneity in ethanol

Structural features of double helices formed by polypeptides with alternating L- and D-amino acid residues were analysed. It was found that the map of short distances (less than 4 A) between protons of the two backbones is unique for each double helix type and even its fragment implies unambiguously parameters of the helix (i.e. parallel or antiparallel, handedness, pitch of helix, relative shift of polypeptide chains). By analysis of two-dimensional 1H-NMR spectra (COSY, RELSY, HOHAHA, NOESY), proton resonances of [Val1]gramicidin A (GA) in the ethanol solution were assigned. The results obtained show that the solution contains five stable conformations of GA in comparable concentrations. Monomer of GA is in a random coil conformation. Specific maps of short interproton distances for the other four species (1-4) were obtained by means of two dimensional nuclear Overhauser effect spectroscopy. The maps as well as spin-spin couplings of the H-NC alpha-H protons and solvent accessibilities of the individual amide groups correspond to four types of double helices pi pi LD 5,6 with 5.6 residues per turn. The double helices are related to the Veatch species 1-4 of GA. Species 1 and 2 are left-handed parallel double helices increase increase pi pi LD 5,6 with different relative shift of polypeptide chains. Species 3 is a left-handed antiparallel double helix increase decrease pi pi LD 5,6 and species 4 is a right-handed parallel double helix increase increase LD 5,6. In the dimers helices are fixed by the maximum number (28) of interbackbone hydrogen bonds NH...O = C possible for these structures. Species 1, 3 and 4 have C2 symmetry axes. Relationship between gramicidin A spatial structures induced by various media is discussed.     

ω-Helices in Proteins

A modification of the α-helix, termed the ω-helix, has four residues in one turn of a helix. We searched the ω-helix in proteins by the HELFIT program which determines the helical parameters—pitch, residues per turn, radius, and handedness—and p = rmsd/(N ? 1)^1/2 estimating helical regularity, where “rmsd” is the root mean square deviation from the best fit helix and “N” is helix length. A total of 1,496 regular α-helices 6–9 residues long with p ≤ 0.10 Å were identified from 866 protein chains. The statistical analysis provides a strong evidence that the frequency distribution of helices versus n indicates the bimodality of typical α-helix and ω-helix. Sixty-two right handed ω-helices identified (7.2% of proteins) show non-planarity of the peptide groups. There is amino acid preference of Asp and Cys. These observations and analyses insist that the ω-helices occur really in proteins.     

Polynucleotides. 13. Stoichiometric and thermodynamic studies of polynucleotide helices with non-complementary residues

Stoichiometry and thermodynamic properties of polyadenylate–polyuridylate helices containing varying proportions of near-randomly distributed non-complementary uridine residues were charactrized from an analysis of their mixing curves and melting profiles measured at 259 nm and at appropriate longer wavelength isochromic points. The noncomplementary residues in this homopolymer–copolymer system (in which the homopolymer has the capacity to readjust with respect to the residues with which it is in opposition) show absolute preference for an extrahelical conformation even when situated in … AAUAA … sequences and must occur therefore as single loops. As the frequency of extrahelical residues in creases, the electrostatic energy of these complexes becomes greater, and is particularly severe for the three-stranded helices. Thus, an adenyl-ate-uridylate copolymer containing 35.2 mole percent uridine residues does not form a three-stranded complex with polyuridylate even in 0.7M Na⁺at O°C. The imperfections introduced into the helix lattice by extrahelical residues decrease the cooperativity of thermal denaturation as well as T_m. However, for the helices with extrahelical residues in low frequency (～1 per helix turn) only small increases in concentration of charge-neutralizing ions are required to bring T_m to the level of their perfect analogs. Two-stranded helices with a higher density of extra helical residues (～5 per helix turn) show [Na⁺] dependence of T_m characteristic of perfect three-stranded helices. These findings together with the absence of an effect of these imperfections on the hypochromicity per base-pair suggest only minimal disruption of helix continuity or distortion of stacking interactions that normally in volve the base pairs or triplets.     

1H-NMR study of gramicidin A transmembrane ion channel. Head-to-head right-handed, single-stranded helices

The structure of [Val1]gramicidin A incorporated into sodium dodecyl-d25 sulphate micelles has been studied by two-dimensional proton NMR spectroscopy. Analysis of nuclear Overhauser effects, spin-spin couplings and solvent accessibility of NH groups show that the conformation of the Na+ complex of gramicidin A in detergent micelles, which in many ways mimic the phospholipid bilayer of biomembranes, is an N-terminal to N-terminal (head-to-head) dimer (Formula: see text) formed by two right-handed, single-stranded beta 6.3 helices with 6.3 residues per turn, differing from Urry's structure by handedness of the helices.     

Possible nucleosome arrangements in the higher-order structure of chromatin

Consideration has been given to possible sequences of nucleosomes which can produce a ‘thick fibre’-like structure. Only a few basic requirements were imposed: (i) the thick fibre is a regular single helix with about 7 nucleosomes per turn; (ii) the nucleosomes are equidistant along the polynuclesome chain; (iii) the helix is flexible having variable pitch. It was found that in addition to the straightforward sequential arrangement there is only one other nonsequential arrangement which satisfies these requirements. This is a helix with around 8 nucleosomes per turn in which all nucleosomes are identically placed. It is possible in the region of 200 to 218 ± 10 base pairs (b.p.) DNA repeats lengths. The linker DNA is straight or almost straight and crosses the internal ‘hollow’ cylinder which is not occupied by nucleosomes. This structure satisfies the experimental data for the distance distribution function, and the observed mass per unit length and changes noted in the mass per unit length. Further, if it is assumed that the core particle axis of symmetry is in the plane of the two linkers and bisects them then this makes the core particles oblique to the thick fibre radii with alternate angles of ± 20 to 30°. This orientation of the nucleosomes can explain the DNA digestion patterns obtained with DNase II and with DNase I.     

Interaction of the Escherichia coli HU protein with DNA. Evidence for formation of nucleosome-like structures with altered DNA helical pitch

Nuclease digestion studies of DNA bound to the histone-like protein HU show that cuts in each strand of the DNA double helix are made with a periodicity of 8.5 base-pairs. By contrast, similar digestions of DNA in eukaryotic nucleosomes show a repeat of 10.4 base-pairs. This and other results (including circular dichroism studies) are consistent with the proposal that the pitch of the DNA double helix in the HU complex is reduced from a repeat length of 10.5 to 8.5 base-pairs per helical turn. Simultaneously, the DNA in the HU-DNA complex containing two dimers of HU per 60 base-pairs has its linking number decreased by 1.0 turn per 290 base-pairs. From these changes it is calculated that HU imposes a DNA writhe of 1.0 per three to four monomers of HU. The results suggest a model in which DNA is coiled in left-handed toroidal supercoils on the HU complex, having a stoichiometry resembling that of the half-nucleosome of eukaryotic chromatin. An important distinction is that HU complexes can restrain the same number of DNA superhelical turns as eukaryotic nucleosomes, yet the DNA retains more negative torsional tension, just as is observed in prokaryotic chromosomes in vivo. Another distinction is that HU-DNA complexes are less stable, having a dissociation half-life of 0.6 min in 50 mM-NaCl. This last property may explain prior difficulties in detecting prokaryotic nucleosome-like structures.     

Visualisation of a 2'-5' parallel stranded double helix at atomic resolution: crystal structure of cytidylyl-2',5'-adenosine

X-ray crystallographic studies on 3'-5' oligomers have provided a great deal of information on the stereochemistry and conformational flexibility of nucleic acids and polynucleotides. In contrast, there is very little information available on 2'-5' polynucleotides. We have now obtained the crystal structure of Cytidylyl-2',5'-Adenosine (C2'p5'A) at atomic resolution to establish the conformational differences between these two classes of polymers. The dinucleoside phosphate crystallises in the monoclinic space group C2, with a = 33.912(4)A, b = 16.824(4)A, c = 12.898(2)A and beta = 112.35(1) with two molecules in the asymmetric unit. Spectacularly, the two independent C2'p5'A molecules in the asymmetric unit form right handed miniature parallel stranded double helices with their respective crystallographic two fold (b axis) symmetry mates. Remarkably, the two mini duplexes are almost indistinguishable. The cytosines and adenines form self-pairs with three and two hydrogen bonds respectively. The conformation of the C and A residues about the glycosyl bond is anti same as in the 3'-5' analog but contrasts the anti and syn geometry of C and A residues in A2'p5'C. The furanose ring conformation is C3' endo, C2' endo mixed puckering as in the C3'p5'A-proflavine complex. A comparison of the backbone torsion angles with other 2'-5' dinucleoside structures reveals that the major deviations occur in the torsion angles about the C3'-C2' and C4'-C3' bonds. A right-handed 2'-5' parallel stranded double helix having eight base pairs per turn and 45 degrees turn angle between them has been constructed using this dinucleoside phosphate as repeat unit. A discussion on 2'-5' parallel stranded double helix and its relevance to biological systems is presented.     

Modeling the ion channel structure of cecropin.

Atomic-scale computer models were developed for how cecropin peptides may assemble in membranes to form two types of ion channels. The models are based on experimental data and physiochemical principles. Initially, cecropin peptides, in a helix-bend-helix motif, were arranged as antiparallel dimers to position conserved residues of adjacent monomers in contact. The dimers were postulated to bind to the membrane with the NH2-terminal helices sunken into the head-group layer and the COOH-terminal helices spanning the hydrophobic core. This causes a thinning of the top lipid layer of the membrane. A collection of the membrane bound dimers were then used to form the type I channel structure, with the pore formed by the transmembrane COOH-terminal helices. Type I channels were then assembled into a hexagonal lattice to explain the large number of peptides that bind to the bacterium. A concerted conformational change of a type I channel leads to the larger type II channel, in which the pore is formed by the NH2-terminal helices. By having the dimers move together, the NH2-terminal helices are inserted into the hydrophobic core without having to desolvate the charged residues. It is also shown how this could bring lipid head-groups into the pore lining.     

A hydrogen bonded chain in bacteriorhodopsin by computer modelling approach

The seven alpha-helical segments of Bacteriorhodopsin (BR) passing through the membrane are investigated for a continuous Hydrogen Bonded Chain (HBC). The study is carried out by computer modelling approach. It is assumed that the seven helices are placed as (AGFEDCB), which has been accepted as the best model by several groups. Helices A, D, E and G are considered to be present in right handed alpha-helical conformation. The inter-orientation of these helices are represented by Eulerian angles alpha, beta and gamma. For the helices B, C and F which contain Proline in the middle, several conformational possibilities were considered. In these cases apart from the Eulerian angles alpha, beta and gamma, the dihedral angles phi p-1 and psi p-1 of the residues that are succeeded by Proline residue in the helical regions were also used in fixing the position of the helices with respect to each other. All these parameters were varied to fit with the top, middle and bottom distances reported by electron diffraction studies. Good fit was obtained for all right handed alpha-helical conformations and also for helices B, C and F with a left handed turn at the residue preceeding proline. Hence two structures were analysed for continuous HBC. Structure I which contained all the seven helices in right handed alpha-helical conformation and Structure II, which had the helices A, D, E and G in right handed conformation and the helices B, C and F in right handed alpha-helical conformation with a left handed turn at the residue preceeding proline. All possible staggered conformations were considered for the side chains and the inter atomic distances were analysed for Hydrogen bonds. It was possible to obtain a continuous chain in both the structures which includes most of the residues found to be important by the experiments. However Lys-216 has to be considered in two different conformations to connect the cytoplasmic side with the extra cellular side. The overall height spanned by HBC is about 25A. The chains obtained by both the structures I and II are analysed in terms of the conformational parameters. It has also been possible to place the retinal in the region as predicted by the experiments. The Tryptophan residues which affect the spectral characteristics can be aligned on either side of the retinal.