Sequences that adopt non-B-DNA conformation in form V DNA as probed by enzymic methylation

pBR322 form V DNA is a highly torsionally strained molecule with a linking number of zero. We have used sequence- specific DNA methylases as probes for B-DNA in this molecule, exploiting the inability of methylases to methylate single-stranded DNA and Z-DNA, both of which are known to occur in form V DNA. Some sequences in form V DNA were shown to be totally in the B-form, others were totally in an altered, unmethylatable conformation, while still other sites appeared to exist partly in altered and partly in normal B-conformation. Some potential Z-forming sequences (alternating pyrimidine/purine) of less than seven base-pairs were not in the Z conformation in form V DNA, whereas others did adopt an altered structure, indicating a modulating influence of flanking sequences. Furthermore, regions of imperfect alternating pyrimidine/purine structure were sometimes capable of adopting an altered structure. In addition, some regions of altered structure had no apparent Z-forming sequences, nor were they in polypurine stretches, which have also been proposed to form left-handed DNA. These non-B-DNA conformations may represent novel left-handed helical structures or sequences that become single stranded under torsional strain. Long regions of either altered (unmethylatable) DNA or B-DNA were not always observed. In fact, one region showed three transitions between B-like DNA and altered structure within 26 base-pairs.

Hydrogen- 1 and Carbon- 13 Magnetic Resonance Studies of Nonactin-Calcium Complex

For an understanding of the cation selectivity and general binding characteristics of macrotetralide antibiotic nonactin (NA) with ions of different sizes and charges, the nature of binding of divalent cation, Ca2+, to NA and conformation of the NA-Ca2+ complex have been studied by use of 270-MHz proton nuclear magnetic resonance ('H NMR) and carbon-13 nuclear magnetic resonance (13C NMR). The calcium ion induced significantly large changes in chemical shifts for H7, H2, H3, and H5 protons of NA and relatively small changes for H18 and H2' protons. Changes in I3C chemical shift were quite large for carbonyl carbon, C,; it is noteworthy that in the NA-K+ complex, H2 and H2' protons practically do not show any change during complexation and carbonyl carbon shows a much smaller chemical shift change.

A conformational approach to the study of the dynamics of enzyme inhibition: studies on thermolysin

The preferred conformations of β-phenylpropionyl-Image -phenylalanine (β-PPP) and N-carbobenzoxy-L-phenylalanine (Cbz-Phe), two inhibitors of thermolysin, have been determined by computing potential energy using empirial potential energy functions. Of the 15 to 20 conformations that are favoured for each of these inhibitors only a few have the right conformation to reach the active site of the enzyme. The conformer of β-PPP that initiates binding with the enzyme is different from the bound one, while for Cbz-Phe the bound and initiating conformers are quite similar. Thus, β-PPP favours the ‘induced fit’ model while Cbz-Phe follows the ‘lock and key’ model of binding. The inhibitors differ in their alignment at the active site.

Effect of configuration of the inhibitors on the mode of binding to the enzyme, thermolysin

Preferred conformations of the competitive inhibitors glycyl-L-phenylalanine and glycyl-D-phenylalanine and their mode of binding to thermolysin have been studied. The difference in configuration is shown to affect significantly the mode of binding to thermolysin. Gly-D-Phe prefers to enter the active site in the global minimum conformation whereas Gly-L-Phe may enter in a higher energy conformation. Moreover, D-enantiomer is shown to have a better fit than the L-counterpart in the active site.

The modes of binding methyl-alpha (and beta)-D-glucopyranosides and some of their derivatives to concanavalin A--a theoretical approach

The probable modes of binding of Methyl--alpha (and beta)-D-glucopyranosides and some of their derivatives to concanavalin A have been proposed from theoretical studies. Theory predicts that beta-MeGlcP can bind to ConA in three different modes whereas alpha-MeGlcP can bind only in one mode. beta-MeGlcP in its most favourable mode of binding differs from alpha-MeGlcP in its alignment in the active-site of the lectin where it binds in a flipped or inverted orientation. Methyl substitution at the C-2 atom of the alpha-MeGlcP does not significantly affect the possible orientations of the sugar in the active-site of the lectin. Methyl substitution at C-3 or C-4, however, affects the allowed orientations drastically leading to the poor inhibiting power of Methyl-3-O-methyl-alpha-D-glucopyranoside and the inactivity of Methyl-4-O-methyl-alpha-D-glycopyranoside. These studies suggest that the increased activity of the alpha-MeGlcP over beta-MeGlcP may be due to the possibility of formation of better hydrogen bonds and to hydrophobic interactions rather than to steric factors as suggested by earlier workers. These models explain the available NMR and other binding studies.

Theoretical investigations of the two-bond proton-carbon-13 coupling constants. Angular variations of the couplings involving carboxyl carbon

A formulation has been developed using perturbation theory to evaluate the π-contribution to the nuclear spin coupling constants involving nuclei at least one of which is an unsaturated center. This fromulation accounts for the π-contribution in terms of the core polarization and one-center exchange at the π-center. The formulation developed together with the Dirac vector model and Penney-Dirac bond-order formalisms was employed to calculate the geminal (two-bond) proton coupling constants of carboxyl carbons in α-disubstituted acetic acids. The calculated coupling constants were found to have an orientational dependence. The results of the calculation are in good agreement with the experimental values.

Computer simulation of polytypes

Polytypes have been simulated, treating them as analogues of a one-dimensional spin-half Ising chain with competing short-range and infinite-range interactions. Short-range interactions are treated as random variables to approximate conditions of growth from melt as well as from vapour. Besides ordered polytypes up to 12R, short stretches of long-period polytypes (up to 33R) have been observed. Such long-period sequences could be of significance in the context of Frank's theory of polytypism. The form of short-range interactions employed in the study has been justified by carrying out model potential calculations.

Binding of N-dansylgalactosamine to winged-bean tuber lectin: studies by fluorescence quenching titrations

The winged-bean tuber lectin binds to N-dansyl(5-dimethylaminonaphthalene-1-sulphonic acid)galactosamine, leading to a 12.5-fold increase in dansyl fluorescence with a concomitant 25 nm blue-shift in the emission maximum. The enhancement of fluorescence intensity was completely reversed by the addition of methyl α-galactopyranoside. The lectin has two binding sites per molecule for this fluorescent sugar and an association constant of 2.59 · 105 M−1 at 25° C. The binding of N-dansylgalactosamine to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of d-galactose. Studies with other sugars indicate that a hydrophobic substituent with α-conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are also critical for sugar binding to this lectin.

A detailed study of Li-DNA fibres at various salt concentrations reveals a non-helical B-DNA and a possible similarity of solution and solid state structures

A thorough investigation of salt concentration dependence of lithium DNA fibres is made using X-ray diffraction. While for low salt the C-form pattern is obtained, crystalline B-type diffraction patterns result on increasing the salt concentration. The salt content in the gel (from which fibres are drawn) is estimated by equilibrium dialysis using the Donnan equilibrium principle. The salt range giving the best crystalline B pattern is determined. It is found that in this range meridional reflections occur on the fourth and sixth layer lines. In addition, the tenth layer meridian is absent at a particular salt concentration. These results strongly suggest the presence of non-helical features in the DNA molecule. Preliminary analysis of the diffraction patterns indicates a structural variability within the B-form itself. Further, the possibility of the structural parameters of DNA being similar in solid state and in solution is discussed.

Structural effects of a dimer interface mutation on catalytic activity of triosephosphate isomerase.The role of conserved residues and complementary mutations

The active site of triosephosphate isomerase (TIM, EC: 5.3.1.1), a dimeric enzyme, lies very close to the subunit interface. Attempts to engineer monomeric enzymes have yielded well-folded proteins with dramatically reduced activity. The role of dimer interface residues in the stability and activity of the Plasmodium falciparum enzyme, PfTIM, has been probed by analysis of mutational effects at residue 74. The PfTIM triple mutant W11F/W168F/Y74W (Y74W*) has been shown to dissociate at low protein concentrations, and exhibits considerably reduced stability in the presence of denaturants, urea and guanidinium chloride. The Y74W* mutant exhibits concentration-dependent activity, with an approximately 22-fold enhancement of kcat over a concentration range of 2.5–40 μm, suggesting that dimerization is obligatory for enzyme activity. The Y74W* mutant shows an approximately 20-fold reduction in activity compared to the control enzyme (PfTIM WT*, W11F/W168F). Careful inspection of the available crystal structures of the enzyme, together with 412 unique protein sequences, revealed the importance of conserved residues in the vicinity of the active site that serve to position the functional K12 residue. The network of key interactions spans the interacting subunits. The Y74W* mutation can perturb orientations of the active site residues, due to steric clashes with proximal aromatic residues in PfTIM. The available crystal structures of the enzyme from Giardia lamblia, which contains a Trp residue at the structurally equivalent position, establishes the need for complementary mutations and maintenance of weak interactions in order to accommodate the bulky side chain and preserve active site integrity.

Analysis of proteins with the `hot dog' fold: Prediction of function and identification of catalytic residues of hypothetical proteins

Background: The hot dog fold has been found in more than sixty proteins since the first report of its existence about a decade ago. The fold appears to have a strong association with fatty acid biosynthesis, its regulation and metabolism, as the proteins with this fold are predominantly coenzyme A-binding enzymes with a variety of substrates located at their active sites. Results: We have analyzed the structural features and sequences of proteins having the hot dog fold. This study reveals that though the basic architecture of the fold is well conserved in these proteins, significant differences exist in their sequence, nature of substrate and oligomerization. Segments with certain conserved sequence motifs seem to play crucial structural and functional roles in various classes of these proteins. Conclusion: The analysis led to predictions regarding the functional classification and identification of possible catalytic residues of a number of hot dog fold-containing hypothetical proteins whose structures were determined in high throughput structural genomics projects.

Biochemical and structural characterization of residue 96 mutants of Plasmodium falciparum triosephosphate isomerase: active-site loop conformation, hydration and identification of a dimer-interface ligand-binding site

Plasmodium falciparum TIM (PfTIM) is unique in possessing a Phe residue at position 96 in place of the conserved Ser that is found in TIMs from the majority of other organisms. In order to probe the role of residue 96, three PfTIM mutants, F96S, F96H and F96W, have been biochemically and structurally characterized. The three mutants exhibited reduced catalytic efficiency and a decrease in substrate-binding affinity, with the most pronounced effects being observed for F96S and F96H. The k(cat) values and K-m values are (2.54 +/- 0.19) x 10(5) min(-1) and 0.39 +/- 0.049 mM, respectively, for the wild type; (3.72 +/- 0.28) x 10(3) min(-1) and 2.18 +/- 0.028 mM, respectively, for the F96S mutant;(1.11 +/- 0.03) x 10(4) min(-1) and 2.62 +/- 0.042 mM, respectively, for the F96H mutant; and (1.48 +/- 0.05) x 10(5) min(-1) and 1.20 +/- 0.056 mM, respectively, for the F96W mutant. Unliganded and 3-phosphoglycerate (3PG) complexed structures are reported for the wild-type enzyme and the mutants. The ligand binds to the active sites of the wild-type enzyme (wtPfTIM) and the F96W mutant, with a loop-open state in the former and both open and closed states in the latter. In contrast, no density for the ligand could be detected at the active sites of the F96S and F96H mutants under identical conditions. The decrease in ligand affinity could be a consequence of differences in the water network connecting residue 96 to Ser73 in the vicinity of the active site. Soaking of crystals of wtPfTIM and the F96S and F96H mutants resulted in the binding of 3PG at a dimer-interface site. In addition, loop closure at the liganded active site was observed for wtPfTIM. The dimer-interface site in PfTIM shows strong electrostatic anchoring of the phosphate group involving the Arg98 and Lys112 residues of PfTIM.

Solid state and solution conformation of Boc-L-Met-Aib-L-Phe-OMe. Beta-turn conformation of a sequence related to an active chemotactic peptide analog

The conformation of the peptide Boc-L-Met-Aib-L-Phe-OMe has been studied in the solid state and solution by X-ray diffraction and 1H n.m.r., respectively. The peptide differs only in the N-terminal protecting group from the biologically active chemotactic peptide analog formyl-L-Met-Aib-L-Phe-OMe. The molecules adopt a type-II beta-turn in the solid state with Met and Aib as the corner residues (phi Met = -51.8 degrees, psi Met = 139.5 degrees, phi Aib = 58.1 degrees, psi Aib = 37.0 degrees). A single, weak 4----1 intramolecular hydrogen bond is observed between the Boc CO and Phe NH groups (N---O 3.25 A, N-H---O 128.4 degrees). 1H n.m.r. studies, using solvent and temperature dependencies of NH chemical shifts and paramagnetic radical induced line broadening of NH resonances, suggest that the Phe NH is solvent shielded in CDCl3 and (CD3)2SO. Nuclear Overhauser effects observed between Met C alpha H and Aib NH protons provide evidence of the occurrence of Met-Aib type-II beta-turns in these solvents.

Identification and analysis of extended strands and beta-sheets in globular proteins

A method to identify β-sheets in globular proteins from extended strands, using only α-carbon positions, has been developed. The strands that form β-sheets are picked up by means of simple distance criteria. The method has been tested by applying it to three proteins with accurately known secondary structures. It has also been applied to ten other proteins wherein only α-carbon coordinates are available, and the list of β-sheets obtained. The following points are worth noting: (i) The sheets identified by the algorithm are found to agree satisfactorily with the reported ones based on backbone hydrogen bonding, wherever this information is available. (ii) β-Strands that do not form parts of any sheet are a common feature of protein structures. (iii) Such isolated β-strands tend to be short. (iv) The conformation corresponding to the preferred right-handed twist of the sheet is overwhelmingly observed in both the sheet-forming and isolated β-strands.