摘要:

AbstractWe have obtained the UV excited resonance Raman spectra of five mononucleotides bound to cisplatin and to carboplatin using excitation in resonance with the first electronic absorption bands of the nucleotide bases. Substantial changes in the spectra are observed following interaction with both platinum drugs, indicating modifications to nucleotide structure. Pt (II) binds to base portions of the nucleotide molecules, altering their normal modes of vibration significantly. We present comparative data of cisplatin and carboplatin, and discuss the implications of these results. The kinetics of the drug/nucleotide reactions differ, but final products are found to be similar. © 1993 John Wiley&Sons, Inc

ISSN:0006-3525    

DOI:10.1002/bip.360331102

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe aggregation properties of zinc‐free insulin have been studied using static and dynamic light scattering. The aggregation has been investigated as a function of three parameters, the concentration of sodium chloride (in the range 10–100 mM), the pH value (in the range pH 7.5–10.5), and the insulin concentration (1.8–13.4 mg/mL). The measured homodyne autocorrelation function was used to determine the apparent mean hydrodynamic diameter as well as the apparent weight‐averaged molar mass of the insulin species in solution. A method of data analysis was employed, which allows the separation of light scattering contributions from the insulin oligomers and from irrelevant macromolecules and possible impurities present in the sample solutions. Also, a simple phenomenological equilibrium model describing the association of oligomers of insulin is presented. One aspect of this model is that it makes it possible to determine weight average molar masses corrected for virial effects on the Rayleigh ratio. This was necessary because virial effects cannot be isolated and corrected for by dilution since this would change the equilibrium distribution of oligomers. The basis of the model is a positive contribution to Gibbs free energy from charge repulsion depending on the protein charge and the number of monomers in the oligomers, and an assumed constant negative contribution to Gibbs free energy arising from either an entropic gain or hydrogen bonding upon association. The equilibrium model gives a good description of both the apparent weight average molar masses and the apparent hydrodynamic diameters, when the effect of the insulin concentration is taken into account by including virial effects arising from charge–charge repulsion (Donnan effect). The result shows that the association of insulin as a function of pH and ionic strength can be described by an effective charge equal to the charge derived from proton titration reduced by the number of sodium ions binding to insulin. At the lowest pH and highest salt concentration (pH 7.5, 100 mMNaCl, 12 mg/mL insulin), the weight average molar mass is close to that of the hexamer, and at the highest pH and lowest salt concentration (pH 10.5, 10 mMNaCl, 1.9 mg/mL), the weight average molar mass is close to that of the monomer. In all cases, however, a distribution of oligomers is present with a relative Gaussian width of about 30%. Neglecting the positive term in Gibbs free energy, an upper bound to the association constant for insulin can be calculated: The negative term in Gibbs free energy corresponds to an association constant of (0.8 ± 0.3)·105M−1, which is in agreement with published values for the monomer‐to‐monomer association. The satisfactory agreement between theory and experiments for the weight average molar mass suggests that it should be possible to predict the aggregational properties of mutant forms of insulin. © 1993

ISSN:0006-3525    

DOI:10.1002/bip.360331103

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe solution structure of toxin γ (60 residues, 4 disulfides) fromNaja nigricolliswas determined by proton nmr and molecular modeling with DIANA and X‐PLOR. The structures were calculated using 489 distance and 81 dihedral angle constraints. The average atomic rms deviation between the nine refined structures and the average structure is 0.118 nm for the backbone atoms. Toxin γ has an overall folding consisting of three loops stabilized by the four disulfides and forming a two‐ and a three‐stranded β‐sheet (loop I and loops II, III, respectively). The same type of folding has been observed for two homologous cardiotoxins. The very close similarity of the solution structure of toxin γ and the crystal structure of toxin V II4includes details of the topological arrangement of numerous side chains. Among these are the conserved residues K12, K18, K35, and Y22, known to be critical for the cytolytic activity of toxin γ. A cluster of hydrophobic side chains organized around Y22 is found on one side of the three‐stranded β‐sheet and is spatially close to a group of three lysines (K12, K18, K35). The side chains of these lysines form a cationic site that can accommodate the binding of a phosphate ion as found in the crystal structure of toxin V II4. The hydrophobic cluster constitutes a possible binding site for the hydrophobic moiety of phospholipids. Together with the complementary cationic site, this hydrophobic surface can form a conserved site by which cardiotoxins bind to membrane phospholipids. © 199

ISSN:0006-3525    

DOI:10.1002/bip.360331104

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe Z form of poly[d(G‐m5C)2], in presence of Mg2+ion, is found to be transformed into B form upon interaction with 4′,6‐diamidino‐2‐phenylindole (DAPI). The Z → B transformation is complete at a mixing ratio of about 0.07 DAPI per DNA base pairs, i.e., each DAPI molecule may be related to the conversion of 6–7 base pairs. An interaction between DAPI and poly[d(G‐m5C)2] in its Z form at low drug: DNA ratios is suggested from optical dichroism and time‐resolved luminescence anisotropy results. The spectroscopic behaviour of DAPI indicates that the Z conformation of DNA does not provide normal binding sites for DAPI, such as groove or intercalation sites, but that the initial association may be of external nature. © 1993 Jo

ISSN:0006-3525    

DOI:10.1002/bip.360331105

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractCD, electron spin resonance, and fluorescence spectroscopy have been utilized to study the adenine binding site of ricin and its toxic A‐subunit. At acidic (4.5) and physiological (7.3) pH, adenine or a spin‐labeled analogue of adenine, N6‐(2,2,6,6‐tetramethyl‐1‐oxypiperidin‐4‐yl) adenine, alters the near uv CD spectra of the ricin A‐chain as well as intact ricin, whereas the far uv CD spectra of all proteins remain unchanged. Electron spin resonance data show that the adenine spin‐labeled analogue interacts strongly with the A‐chain both at pH 4.5 and 7.3, but no or very weak binding is observed for the intact ricin or the isolated B‐chain. The adenine spin label gets highly immobilized (2AII= 65.5G) by the A‐chain. The apparent dissociation constantKdfor the toxic A‐chain ligand complex is 1.55 × 10−4Mand 5.6 × 10−5Mat pH 7.3 and 4.5, respectively. Fluorescence intensity of ricin A‐chain bound 1,8‐anilinonaphthalenesulfonic acid (ANS) decreases by ∼55% at pH 4.5 with the addition of the spin‐labeled analogue of adenine, implying that both the ANS and adenine spin label (ADSL) bind to the hydrophobic domain of the A‐chain. Fluorescence of the only intrinsic tryptophan probe of the A‐chain is also efficiently quenched by ADSL, indicating that the tryptophan residue and the hydrophobic adenine binding site are closely located. All spectroscopic measurements indicate that adenine or its spin‐labeled analogue has a single binding site adjacent to the TRP211 residue in the A‐chain. Expansion of the A‐chain globule and subsequent exposure of the hydrophobic binding site seem to be responsible for the increased bindin

ISSN:0006-3525    

DOI:10.1002/bip.360331106

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractA generally applicable solid‐phase methodology has been developed for the synthesis of triple‐helical polypeptides incorporating native collagen sequences. Three nascent peptide chains are C‐terminal linked through one Nα‐amino and two Nε‐amino groups of Lys, while repeating Gly‐Pro‐Hyp triplets induce triple helicity. Different protecting group strategies, including several three‐dimensionally orthogonal schemes, have been utilized for the synthesis of four homotrimeric triple‐helical polypeptides (THPs) of 79–124 residues, three of which incorporate native type IV collagen sequences. Highly efficient assemblies were achieved by 9‐fluorenylmethoxycarbonyl (Fmoc) Nα‐amino group protection, in situ 2‐(1H‐benzotriazole‐l‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate mediated couplings, and 1,8‐diazabicyclo [5.4.0] undec‐7‐ene mediated Fmoc group removal. THPs were characterized by Edman degradation sequencing, size‐exclusion chromatography, mass spectrometry, reversed‐phase high performance liquid chromatography, and CD spectroscopy. THP thermal stabilities ranged from 35 to 59°C, with chain length and Hyp content being the influential factors. Melting temperatures and van't Hoff enthalpies for peptide triple‐helical denaturation could be correlated well to Hyp content. The THP synthetic protocol developed here will allow for the study of both structure and biological activity of specific collagen sequences in homotrimeric an

ISSN:0006-3525    

DOI:10.1002/bip.360331107

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractA preliminary127I‐NMR relaxation study of the binding of the I−ion to κ‐carrageenan is presented. Signal loss caused by motional correlation times out of extreme narrowing makes it difficult to analyze line widths. Longitudinal relaxation times are more suitable for a comprehensive relaxation study. From the observation of nonextreme motional narrowing, the lower limit of the residence time at the binding site is estimated to be on the order of 10 ns. © 1993 John Wiley&So

ISSN:0006-3525    

DOI:10.1002/bip.360331108

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractTwo crystals d(C2GCG2) and d(C5GCG5) have been studied under microscope by Fourier transform ir spectroscopy and Raman spectroscopy. The x‐ray diffraction study of the latter crystal had shown that the d(C5GCG5) sequence is the first DNA dodecamer known to adopt a canonical A conformation [N. Verdaguer, J. Aymami, D. Fernandez‐Forner, I. Fita, M. Coll, T. Huynh‐Dinh, J. Igolen, and J. A. Subirana (1991)Journal of Molecular Biology, Vol. 221, pp. 623–635]. Characteristic ir marker bands and Raman marker peaks of the A conformation have thus been obtained and are compared with previously proposed assignments correlated to fiber diffraction x‐ray results obtained on polymers. The d(C2GCG2) sequence crystal had previously been studied in an intermediate form between B and Z [L. Urpi, J. P. Ridoux, J. Liquier, N. Verdagner, I. Fita, J. A. Subirana, F. Iglesias, T. Huynh‐Dinh, J. Igolen, and E. Taillandier (1989)Nucleic Acids Research, Vol. 17, pp. 6669–6679]. In this paper we present results obtained from a crystal with this oligonucleotide in Z conformation. The effect of the crystallization conditions on the geometry of the obtained oligomer helix is discussed. The influence of the addition, to the central tetramer CGCG, of dCnstretches (at the 5′ end) and dGnstretches (at the 3′ end) of different lengths, on the conformational flexibility of the nucleic acid, is considered. © 1993 Jo

ISSN:0006-3525    

DOI:10.1002/bip.360331109

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe effects of inserting 16 base pair (bp) of alternating CG[(CG)8] near the middle of a much longer restriction fragment (1097 bp) are investigated by measuring various properties that are sensitive to secondary and tertiary structure. Results for this fragment are compared with those for a control fragment (1089 bp) with the identical sequence except at the insert. Another fragment (1382 bp), which contains a 296‐bp extension at the 5′‐end of the 1089‐bp control fragment, is also used as a secondary control in some experiments. When the 1097‐bp (CG)8insert fragment is compared with the control fragments in 0.1MNaCl buffer, the (CG)8insert is found to induce disproportionately large relative changes in the molar ellipticity at 273 nm ([θ)273], the torsion constant (α) measured by fluorescence polarization anisotropy, the optical melting profile, and the susceptibility to S1 nuclease. Estimates of theminimumdistance over which the (CG)8insert alters the secondary structure range from 330 to 550 bp. With increasing NaCl concentration, the 1097‐bp insert fragment undergoes a structural transition between 2.0 and 2.5Mthat is manifested in the apparent diffusion coefficient (Dplat) from dynamic light scattering at large scattering vector. This transition, which is not exhibited by the control DNAs, is presumed to involve formation of Z‐helix at the insert. However, the observeddecreasein (Dplat) is attributed to anincreasein bending rigidity, which perforce must be globally distributed far beyond the (CG)8insertper se.In 4.25MNaCl (but not in 0.1MNaCl), the addition of 1 ethidium dye per 300 bp induces an extensive structural transition in the 1097 bp (CG)8insert fragment. This transition, which also is not exhibited by the control DNAs, significantly decreases the bending rigidity, doubles [θ]273, and takes place on a time scale of a few days. Removal of ethidium and salt by dialysis vs 0.1MNaCl buffer restores the original properties of the 1097‐bp (CG)8insert fragment. The present results are consistent with a (fluctuating, long‐range) description of the secondary structure in which a given short sequence transiently fluctuates among two or more distinct secondary structures that extend over much larger domains of variable position and size, and whose relative stabilities depend on distant as well as close‐lying base pairs. © 19

ISSN:0006-3525    

DOI:10.1002/bip.360331110

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY

摘要:

AbstractThe urea‐induced unfolding of 16S RNA at low ionic strength has been studied by dynamic light scattering, uv spectroscopy, and some hydrodynamic methods. Three components could be resolved in the photon correlation spectra of scattered light, using the inverse Laplace transform SIPP program [G. R. Danovich and I. N. Serdyuk (1983) inPhoton Correlation Techniques in Fluid Mechanics, vol. B38, E. O. Schulz‐Dubois, Ed., Springer, Berlin/Heidelberg, New York, p. 315]. One component is assigned to the center‐of‐mass translation of the RNA, another one to a combination of translational and internal motion, and the last to diffusion of urea clusters. The hydrodynamic dimensions of RNA increase strongly upon transition from 4 to 6Murea. We conclude that up to 2Murea, 16S RNA is highly elongated, and coiled above 4Murea, with a great increase of the hydrodynamic dimensions of RNA being observed upon transition from 4 to 6Murea. A scheme for RNA unfolding is proposed. © 1993 John Wiley&S

ISSN:0006-3525    

DOI:10.1002/bip.360331111

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1993

数据来源: WILEY