X-ray fibre diffraction study of conformational changes in hyaluronate induced in the presence of sodium,potassium and calcium cations

Hyaluronate purified from all cations by ion exchange chromatography was introduced to the cations sodium, potassium and calcium in a controlled way. The conformations formed in the presence of these ions were studied as a function of ionic strength, hydrogen ion activity, humidity and temperature using X-ray fibre diffraction. In sodium hyaluronate above pH 4.0 a contracted helix is found which approximates to a four-fold helix with an axial rise per disaccharide of 0.84 nm. There is no requirement for water molecules in the unit cell as the Na⁺ can be coordinate by the hyaluronate chains alone. On crystallizing hyaluronate below pH 4.0 an extended 2-fold helix with an axial rise per disaccharide of 0.98 nm is formed. In the presence of potassium above pH 4.0 a conformation similar, but not identical, to that of sodium was found where the helix backbone is again four-fold with an axial rise per disaccharide h=0.90 nm. To maintain the coordination of the potassium ion, four water molecule/disaccharide are required and on removal of these the conformation is destabilized going to a new helix where n = 4 and h = 0.97 nm. Below pH 4.0 the conformation is a contracted 4-fold helix with h = 0.82 nm. In this structure two antiparallel chains intertwine to form a double helix. The packing of the double helical units is stabilized by water molecules, the unit cell requiring 8 water molecules/disaccharide. Formation of the calcium hyaluronate complex above pH 3.5 yields a three-fold helix with h = 0.95 nm. The requirement for water in the unit cell to maintain full crystallinity is high, at 9 water molecules/disaccharide; however, on removal of this water, though the crystallinity is disrupted, the conformation remains constant. The acid form of calcium-hyaluronate yields an equivalent conformation to that of sodium under the same condition, i.e. a helix with n = 2, h = 0.98 nm. The presence of small quantities of calcium in what are otherwise potassium or sodium solutions of hyaluronate yield the 3-fold conformation for hyaluronate. Thus calcium has an important role to play in deciding the dominating conformation present in hyaluronate. The variety of conformations yielded by the different cations indicates a subtle interaction between hyaluronate and its environment, in which the balance between the cations will control to some degree the interactions between hyaluronate chains and thus affect the mechanical properties of the matrix which they form. The conformations of individual chains are all stabilized in varying degrees by intra-chain hydrogen bonds.     

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.     

Glycosaminoglycan conformation: do aqueous molecular dynamics simulations agree with x-ray fiber diffraction?

Glycosaminoglycan-protein interactions are biologically important and require an appreciation of glycan molecular shape in solution, which is presently unavailable. In previous studies we found strong similarity between aqueous molecular dynamics (MD) simulations and published x-ray diffraction refinements of hyaluronan. We have applied a similar approach here to chondroitin and dermatan, attempting to clarify some of the issues raised by the x-ray diffraction literature relating to chondroitin and dermatan sulfate. We predict that chondroitin has the same beta(1-->4) linkage conformation as hyaluronan, and that their average beta(1-->3) conformations differ. This is explained by changes in hydrogen-bonding across this linkage, resulting from its axial hydroxyl, causing a different sampling of left-handed helices in chondroitin (2.5- to 3.5-fold) as compared with hyaluronan (3.0- to 4.0-fold). Few right-handed helices, which lack intramolecular hydrogen-bonds, were sampled during our MD simulations. Thus, we propose that the 8-fold helix observed in chondroitin-6-sulfate, represented in the literature as an 8(3) helix (right-handed), though it has never been refined, is more likely to be 8(5) (left-handed) helix. Molecular dynamics simulations implied that (4)C(1) and (2)S(O), but not (1)C(4), forms of iduronate could be used in refinements of dermatan x-ray fiber diffraction patterns. Current models of 8-fold dermatan sulfate chains containing (4)C(1) iduronate refine to right-handed helices, which possess no intramolecular hydrogen-bonds. However, MD simulations predict that models containing (2)S(O) iduronate could provide better (8(5) helix) starting structures for refinement. Thus, the 8-fold dermatan sulfate refinement (8(3) helix) could be in error.     

Hyaluronic acid: molecular conformations and interactions in two sodium salts.

A detailed structure for the tetragonal form (a = b = 0.989 nm, c, fibre axis, = 3.394 nm) of sodium hyaluronate has been obtained by analysing X-ray fibre diffraction data using new molecular modelling techniques. Two polysaccharide chains pass through each unit cell, one at the corner and one at the centre. The chains are anti-parallel to one another. Each chain is a left-handed, 4-fold helix of disaccharide units. There are intramolecular hydrogen bonds stabilising each glycosidic linkage. Octahedrally co-ordinated sodium ions link, by O … Na⁺ … O bridges, neighbouring polysaccharide chains that are further linked by hydrogen bonds. No double-helix model (as originally proposed for this structure) has been found to be free of unacceptable non-bonded contacts or to fit the diffraction intensities as closely.The tetragonal form, which is stable at zero relative humidity, contains no detectable water molecules. At higher relative humidities a related orthorhombic form is observed in which only the a dimension of the lattice is different (a = 1.153 nm, b = 0.989 nm, c = 3.386 nm). In this form the hyaluronate helix is 2-fold with tetrasaccharide units conformationally similar to the 4-fold helix of the tetragonal form. The Na⁺ … O binding and hydrogen bonds lost on expansion of the tetragonal lattice are all replaced in the orthorhombic structure by bridges through water molecules, four of which associated with each tetrasaccharide.     

The agarose double helix and its function in agarose gel structure

Agarose and eight different derivatives carrying O-methyl, O-sulphate, O-hydroxyethyl or O-carboxyethylidene substituents in various positions were studied by optical rotation, X-ray diffraction and computerised molecular model building methods. All samples showed essentially the same order-disorder transition during gel-sol interconversion. In addition, all the samples that could be made into oriented films or fibres gave X-ray diffraction diagrams corresponding to a common molecular structure. A double helix model for this structure is proposed that has the 0.95 nm axial periodicity observed and a calculated cylindrically averaged Fourier transform in good agreement with the observed (continuous) layer line intensities. Each chain in the double helix forms a lefthanded 3-fold helix of pitch 1.90 nm and is translated axially relative to its partner by exactly half this distance. This model accounts for the sign and magnitude of the optical rotation shift that accompanies the sol-gel transitions and is sterically accessible to each of the various substituted forms. The relationship between agarose gel properties and the double helix is discussed and the structure compared with i-carrageenan.     

Calmodulin activation of target enzymes. Consequences of deletions in the central helix

The central helical region of calmodulin (CaM) includes amino acids 65-92 and serves to separate the two pairs of Ca2(+)-binding sites. This region may impart conformational flexibility and also interact with target proteins. The functional effects of deleting two, three, five, or eight amino acids from the central helix were monitored by examining the activation of phosphodiesterase, smooth muscle myosin light chain (MLC) kinase, and Ca2+/CaM-dependent protein kinase II (CaM kinase II). CaMDM(-8), a calmodulin-deletion mutant with 8 amino acids deleted from the middle of the central helix, failed to activate MLC kinase, phosphodiesterase, or CaM kinase II at physiologically significant concentrations of activator but also had altered electrophoretic mobility and tyrosine fluorescence properties suggesting major changes in the structure of this mutant. Deletion of five amino acids (77-81) resulted in an increase in apparent Kact for phosphodiesterase (150-fold), CaM kinase II (25-fold), and MLC kinase (5-fold) relative to CaM. The maximal autophosphorylation activity of CaM kinase II was also diminished 70% with CaMDM(-5). For phosphodiesterase activation, CaMDM(-2) has a 15-fold increase in apparent Kact while CaMDM(-3) had an apparent Kact value only 3-fold higher than native CaM. In contrast, the activation of MLC kinase by the two (79-80)- and three (79-81)-amino acid deletion mutants were indistinguishable from each other or native CaM. CaMDM(-2) and CaMDM(-3) stimulated CaM kinase II autophosphorylation to 85 and 70%, respectively, of native CaM with less than a 2-fold increase in Kact. Therefore, all deletions in the central helix of CaM reduce the efficiency of phosphodiesterase activation as reflected by substantial alterations in Kact. MLC kinase activation, however, is relatively insensitive to small two or three amino acid deletions. CaM kinase II interacts with the central helix deletion mutants in a complex manner with alterations in both the Kact and the maximum activity. The data suggest the central helix of CaM may serve as a flexible tether for MLC kinase (and to a lesser extent CaM kinase II) but that an extended conformation of CaM, as predicted from the crystal structure, may be required for phosphodiesterase activation.     

A new model for DNA containing A.T and I.C base pairs.

DNA polymers containing exclusively A.T or I.C base pairs frequently exhibit D- or E-type X-ray diffraction patterns when dried. The distribution of intensities in fiber patterns appears to demand helical structures with 7 and 7.5 bp/turn, respectively, but it is not stereochemically possible to wind a right-handed antiparallel B-family helix this tightly. It is a simple matter, however, to build a left-handed helix with 7-7.5 bp/turn by incorporating Hoogsteen pairing into a Z helix framework. X-ray intensities calculated from this novel left-handed Hoogsteen model provide as reasonable a fit to the D-DNA diffraction pattern as do intensities calculated from previously proposed right-handed 8-fold models.     

The cell envelope of Thermoproteus tenax: three-dimensional structure of the surface layer and its role in shape maintenance

The sulphur-dependent archaebacterium Thermoproteus tenax has a cylindrical cell shape variable in length, but constant in diameter. Its whole surface is covered by a regular protein layer (S-layer). The lattice has p6 symmetry and a lattice constant of 32.8 nm. The three-dimensional reconstruction from a tilt series of isolated and negatively stained S-layer shows a complex mass distribution of the protein: a prominent, pillar-shaped protrusion is located at the 6-fold crystallographic axis with radiating arms connecting neighbouring hexamers in the vicinity of the 3-fold axis. The base vectors of the S-layer lattice have a preferred orientation with respect to the longitudinal axis of the cell. The layer can be seen as a helical structure consisting of a right-handed, two-stranded helix, with the individual chains running parallel. Supposing that new S-layer protein is inserted at lattice faults (wedge disclinations) near the poles, growing of the layer would then proceed by moving a disclination at the end of the helix. The constant shape of the cell, as well as the particular structure of the layer, strongly suggest that this S-layer has a shape-maintaining function.     

Calcium regulation of gelsolin and adseverin: a natural test of the helix latch hypothesis

The gelsolin family of actin filament binding proteins have highly homologous structures. Gelsolin and adseverin, also known as scinderin, are the most similar members of this family, with adseverin lacking a C-terminal helix found in gelsolin. This helix has been postulated to serve as a calcium-sensitive latch, keeping gelsolin inactive. To test this hypothesis, we have analyzed the kinetics of severing by gelsolin, adseverin, and a gelsolin truncate which lacks the C-terminal latch. We find that the relationship between severing rate and calcium ion concentration differs between gelsolin and adseverin, and suggest that calcium controls one rate-limiting step in the activation of adseverin and two in the activation of gelsolin. In contrast, both proteins are activated equally by protons, and have identical severing kinetics at pHs below 6.3. The temperature sensitivity of severing by adseverin and gelsolin is remarkably different, with gelsolin increasing its severing rate 8-fold per 10 degrees C increase in temperature and adseverin increasing its rate only 2-fold per 10 degrees C increase in temperature. Analysis of the gelsolin construct lacking the C-terminal helix demonstrates that this helix is responsible for the regulatory differences between gelsolin and adseverin. These results support the C-terminal latch hypothesis for the calcium ion activation of gelsolin.     

A calorimetric study of the triple helix formation: interaction of poly(C) - guanosine - protonated poly(C).]

The triple helix formation of poly(C) - guanosine - poly(C+) was investigated by the help of an LKB scanning micro-calorimeter. The existence of the triple helix could also be shown by recording the melting curves. The ultraviolet absorption at different wave lengths namely 275 nm, 260 nm, and 245 nm was plotted as a function of the temperature. Furthermore formation of the triple helix was shown by plotting the ultraviolet absorption at 245 nm during the increasing addition of guanosine solution to a fixed amount of poly(C) in the solution. Finally the formation of the triple helix was demonstrated by plotting the ultraviolet absorption at 245 nm of a certain mixture of the components while the pH value of the solution was continuously lowered. All these methods show that the monomer interacts with the polymer double helix to form a triple helix. The calorimetric measurements show that the reaction enthalpy is concentration dependent. Above a threshold concentration a rapid increase of the reaction enthalpy is observed. This increase occurs in a very narrow concentration interval. Above this interval a final value of the reaction enthalpy is reached. The amount of the reaction enthalpy for the interaction of guanosine with poly(C) - poly(C+) double helix is 5.5 Kcal (mol base triplet)-1.     

Polarization selectivity of interaction of DNA molecules with X-ray radiation

The optimum form of a long helical molecule, which DNA is, has been calculated in terms of the classical electromagnetic theory. Three different methods of classical electrodynamics are used: the theory of dipole radiation of electromagnetic waves, the energetic power approach, and a helical model of molecules of chiral medium. In all three cases, an identical result for the optimum geometrical form of a long spiral molecule has been obtained. The lead angle between the tangent to the helix and the plane normal to the axis of the helix should be equal to 24.5 degrees. This condition imposes restrictions on the radius and the pitch of the helical molecule. The experimentally measured geometrical characteristics of the DNA molecule satisfy the theoretically calculated condition precisely enough. Having the optimum geometrical form, the DNA molecule is not influenced by a circularly right-polarized electromagnetic wave in the soft X-ray range λ = 7–8 nm. This wave, for which the right-handed DNA molecule is “transparent,” should propagate orthogonally to the helix axis and form a right-handed screw in space. The wave radiated by the right-handed DNA molecule orthogonally to helix axis in the range of λ ≈ 7–8 nm has, accordingly, the left-handed circular polarization. The polarization selectivity of the DNA molecule by the action of X-ray radiation is exhibited strongly enough in the wavelength range of λ ≈ 1–35 nm. The results obtained are valid for any distribution of electric currents in DNA, i.e., for any sequence of nitrogenous bases in DNA.     

Structure of Limulus telson muscle thick filaments

Computer analysis of electron micrographs of negatively stained thick filaments isolated from the telson levator muscle of the horseshoe crab (Limulus polyphemus) has shown that they have a four-stranded helical structure. The repeating units along each helix have a bent extended shape (measuring approximately 20 nm × 8 nm × 8 nm) and are inclined at an angle of about 30 ° to the helical path. At the resolution of this study, it was difficult to establish the exact size of the surface subunits, but our results are probably more consistent with each unit representing the two heads of a single myosin molecule rather than larger aggregates.     

Structure-function studies on bacteriorhodopsin. IX. Substitutions of tryptophan residues affect protein-retinal interactions in bacteriorhodopsin

Bacteriorhodopsin contains 8 tryptophan residues distributed across the membrane-embedded helices. To study their possible functions, we have replaced them one at a time by phenylalanine; in addition, Trp-137 and -138 have been replaced by cysteine. The mutants were prepared by cassette mutagenesis of the synthetic bacterio-opsin gene, expression and purification of the mutant apoproteins, renaturation, and chromophore regeneration. The replacement of Trp-10, Trp-12 (helix A), Trp-80 (helix C), and Trp-138 (helix E) by phenylalanine and of Trp-137 and Trp-138 by cysteine did not significantly alter the absorption spectra or affect their proton pumping. However, substitution of the remaining tryptophans by phenylalanine had the following effects. 1) Substitution of Trp-86 (helix C) and Trp-137 gave chromophores blue-shifted by 20 nm and resulted in reduced proton pumping to about 30%. 2) As also reported previously (Hackett, N. R., Stern, L. J., Chao, B. H., Kronis, K. A., and Khorana, H. G. (1987) J. Biol. Chem. 262, 9277-9284), substitution of Trp-182 and Trp-189 (helix F) caused large blue shifts (70 and 40 nm, respectively) in the chromophore and affected proton pumping. 3) The substitution of Trp-86 and Trp-182 by phenylalanine conferred acid instability on these mutants. The spectral shifts indicate that Trp-86, Trp-182, Trp-189, and possibly Trp-137 interact with retinal. It is proposed that these tryptophans, probably along with Tyr-57 (helix B) and Tyr-185 (helix F), form a retinal binding pocket. We discuss the role of tryptophan residues that are conserved in bacteriorhodopsin, halorhodopsin, and the related family of opsin proteins.     

Ethidium ion binds more strongly to a DNA double helix with a bulged cytosine than to a regular double helix

Thermodynamic parameters for ethidium intercalation were determined for the double helices formed by the oligonucleotides dCA6G + dCT6G, which form a normal helix, and dCA3CA3G + dCT6G, which form a double helix with the middle cytosine bulged outside of the helix. Ethidium intercalation was measured by monitoring the absorbance at 260 and 283 nm as a function of temperature for a number of concentrations of ethidium. The binding to the normal helix occurs equally at all the intercalation sites, with an enthalpy of binding of -8 kcal mol-1, an entropy of binding of -6 eu, and an equilibrium constant at 25 degrees C of 2.2 X 10(4) M-1. The binding to the bulged double helix was considerably stronger and is consistent with a model in which the intercalation sites on either side of the bulged base bind 10 times stronger than the other sites. Thus, there are two strong binding sites on the perturbed helix with equilibrium constants for binding of 2 X 10(5) M-1 at 25 degrees C in addition to five normal sites. Several other binding models were tested but did not fit the data satisfactorily.     

Protein-RNA interactions during TMV assembly

A review of the structural studies of tobacco mosaic virus (TMV) is given. TMV is essentially a flat helical microcrystal with 16 1/3 subunits per turn. A single strand of RNA runs along the helix and is deeply embedded in the protein. The virus particles form oriented gels from which high-resolution X-ray fiber diffraction data can be obtained. This may be interpreted by the use of six heavy-atom derivatives to give an electron density map at 0.4 nm resolution from which the RNA configuration and the form of the inner part of the protein subunit may be determined. In addition, the protein subunits form a stable 17-fold two-layered disk which is involved in virus assembly and which crystallizes. By the use of noncrystallographic symmetry and a single heavy-atom derivative, it has been possible to solve the structure of the double disk to 0.28 nm resolution. In this structure one sees that an important structural role is played by four alpha-helices, one of which (the LR helix) appears to form the main binding site for the RNA. The main components of the binding site appear to be hydrophobic interactions with the bases, hydrogen bonds between aspartate groups and the sugars, and arginine salt bridges to the phosphate groups. The binding site is between two turns of the virus helix or between the turns of the double disk. In the disk, the region proximal to the RNA binding site is in a random coil until the RNA binds, whereupon the 24 residues involved build a well-defined structure, thereby encapsulating the RNA.     

Conformational changes of an exchangeable apolipoprotein, apolipophorin III from Locusta migratoria, at low pH: correlation with lipid binding.

Locusta migratoria apolipophorin III (apoLp-III) is a helix bundle exchangeable apolipoprotein that reversibly binds to lipoprotein surfaces. Structural reorganization of its five amphipathic alpha-helices enables the transition from the lipid-free to lipid-bound state. ApoLp-III-induced transformation of dimyristoylphosphatidylcholine (DMPC) bilayer vesicles into smaller discoidal complexes is enhanced as a function of decreasing pH, with maximal transformation occurring at pH 3.5. Over the entire pH range studied, apoLp-III retains nearly all of its secondary structure content. Whereas no changes in fluorescence emission maximum of the two Trp residues in apoLp-III were observed in the pH range from 7.0 to 4.0, a further decrease in pH resulted in a strong red shift. Near-UV circular dichroism spectra of apoLp-III showed well-defined extrema (at 286 and 292 nm) between pH 7.0 and pH 4.0, which were attributed to Trp115. Below pH 4.0, these extrema collapsed, indicating a less rigid environment for Trp115. Similarly, the fluorescence intensity of 8-anilinonaphthalene-1-sulfonate in the presence of apoLp-III increased 4-fold below pH 4.0, indicating exposure of hydrophobic sites in the protein in this pH range. Taken together, the data suggest two conformational states of the protein. In the first state between pH 7.0 and pH 4.0, apoLp-III retains a nativelike helix bundle structure. The second state, found between pH 3.0 and pH 4.0, is reminiscent of a molten globule, wherein tertiary structure contacts are disrupted without a significant loss of secondary structure content. In both states DMPC vesicle transformation is enhanced by lowering the solution pH, reaching an optimum in the second state. The correlation between tertiary structure and lipid binding activity suggests that helix bundle organization is a determinant of apoLp-III lipid binding activity.     

Structure of complexes between a major protein of heterogeneous nuclear ribonucleoprotein particles and polyribonucleotides

We have investigated the structure of complexes formed between a series of poly(A)n (n = 30 to 480) and HD40 (helix-destabilizing protein, molecular weight of 40,000), the major protein component of 30 S heterogeneous nuclear ribonucleoprotein particles (hnRNP) from the brine shrimp Artemia salina. Protein HD40 is similar to corresponding hnRNP proteins from higher eukaryotes and the complexes it forms with single-stranded nucleic acids are strikingly similar to the native "beads-on-a-string" structure of hnRNP. Using analytical ultracentrifugation and electron microscopy we find: (1) complexes formed between HD40 and long ribohomopolymers also have a beads-on-a-string structure, showing that the ability to form this structure is an inherent property of HD40, and is not dependent on any structural features of natural RNA; (2) complexes between HD40 and poly(A)160 form disks that are about 3 nm high by 18 nm in diameter and contain 20 HD40 molecules; (3) complexes of HD40 with poly(A)n with fewer than 160 nucleotides form sectors of a disk: 40 nucleotides give rise to a quarter of a disk, 80 nucleotides, half a disk, etc. The molecular weights increase with the size of poly(A)n at the rate of 5300 per nucleotide, a stoichiometry of eight nucleotides per HD40; (4) as the size of the poly(A)n increases beyond 160 nucleotides, the additional nucleoprotein elements may either initiate the formation of a second disk adjacent to the first or stack on top of the first disk to form a 6 nm high helix with a diameter of 18 nm. Based on these results, we propose that the existence of lateral protein-protein interactions that produce the basic 3 nm X 18 nm disk, combined with the marginal stability of the helix result in (a) interruptions of the helix that give rise to the beads-on-a-string appearance of the complexes, and (b) inherent heterogeneity of individual "beads" which may contain one or more turns of the helix. From measurements of HD40 complexes with coliphage MS2 RNA, phi X174 viral DNA as well as with the homopolymers, a bead is estimated to contain an average of approximately 300 nucleotides; approximately 1 X 8 turns of the helix.     

Molecular modeling of xanthan: galactomannan interactions

X-ray diffraction patterns from stretched fibers of xanthan, guaran and the complex between the two are indicative of good orientation and reasonable crystallinity. The ordered structures of xanthan and guaran appear to be a 5-fold helix of pitch 47.4Aoand a 2-fold helix of pitch 10.3Ao, respectively. The diffraction pattern of the complex is a hybrid of those of the individual components. Both xanthan and guaran in the complex may adopt cellulose-like helices having a slightly longer pitch of 10.5Aoand form a non-coaxial duplex. Alternately, the complex may adopt a xanthan-like, coaxial, 5-fold, double helix in which one strand is xanthan and the other is guaran. The association of a pair of these hybrid helices can take place by direct cross linking of the car☐ylate groups in the side chains of xanthan by divalent ions. The morphologies of these arrangements have now been visualized by computer modeling.     

Circular dichroism of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus

Circular dichroism (CD) spectra of catechol 1,2-dioxygenase from Acinetobacter calcoaceticus exhibit three positive ellipticity bands between 240 and 300 nm (250, 283, and 292 nm), two negative bands at 327 and 480 nm, and a low-intensity positive band at 390 nm. The fractions of helix β-form, and unordered form of the enzyme are 8, 38, and 54%, respectively. The circular dichroic bands at 327 and 480 nm and a part of the positive bands at 292 and 390 nm are associated with enzyme activity. Significant changes in absorption and CD spectra of the enzyme were observed when the temperature of the enzyme preparation was increased to 47°C, coinciding with the sharp decrease in enzyme activity observed at this temperature.     

Molecular structure of an A-DNA decamer d(ACCGGCCGGT)

The molecular structure of the DNA decamer d(ACCGGCCGGT) has been solved and refined by single-crystal X-ray-diffraction analysis at 0.20 nm to a final R-factor of 18.0%. The decamer crystallizes as an A-DNA double helical fragment with unit-cell dimensions of a = b = 3.923 nm and c = 7.80 nm in the space group P6(1)22. The overall conformation of this A-DNA decamer is very similar to that of the fiber model for A-DNA which has a large average base-pair tilt and hence a wide and shallow minor groove. This structure is in contrast to that of several A-DNA octamers in which the molecules all have low base-pair-tilt angles (8-12 degrees) resulting in an appearance intermediate between B-DNA and A-DNA. The average helical parameters of this decamer are typical of A-DNA with 10.9 base pairs/turn of helix, an average helical twist angle of 33.1 degrees, and a base-pair-tilt angle of 18.2 degrees. However, the CpG step in this molecule has a low local-twist angle of 24.5 degrees, similar to that seen in other A-DNA oligomers, and therefore appears to be an intrinsic stacking pattern for this step. The molecules pack in the crystal using a recurring binding motif, namely, the terminal base pair of one helix abuts the surface of the shallow minor groove of another helix. In addition, the GC base pairs have large propeller-twist angles, unlike those found most other A-DNA structures.