Circular dichroic and fluorometric studies on the acid-induced isomerization of bovine plasma albumin--1 -anilino-8-naphthalenesulfonate complex

The acid-induced isomerization (the N-F transition) and expansion of bovine plasma albumin--1-anilino-8-naphthalenesulfonate complex, BPA-ANS1.0 complex (molar ratio of added ANS to BPA = 1.0) were studied by measuring fluorescence and induced CD spectra of ANS. Decrease in the reciprocal of fluorescence polarization, increase in fluorescence intensity and blue shift of fluorescence of ANS in BPA-ANS1.0 complex were correlated with the initial part of the N-F transition and/or the N-F1 transition. Induced CD spectra of ANS showed positive bands at 250-258 and 320-350 nm and one negative band at 280 nm. Most of changes (decreases) in -[theta]280 were also correlated with the initial part of the N-F transition and/or the N-F1 transition. Changes in fluorescence parameters and induced CD spectra of ANS (-[theta]280) might indicate the conformational changes around a strong ANS binding site in the N-terminal domain (Reed et al. (1975), Jonas & Weber (1970) and Brown & Shockley (1982].     

Sodium dodecyl sulfate-bovine plasma albumin complex. Structural transition from native to relatively compact globule forms with mobile side chains in acidic region--N-F transition

The acid-induced isomerization (the N-F transition) and expansion of sodium dodecyl sulfate-bovine plasma albumin complex (ADm; m, molar ratio of added sodium dodecyl sulfate to bovine plasma albumin; O less than or equal to m less than or equal to 12) were studied by measuring CD-resolved secondary structure, fluorescence polarization and life-time of tryptophyl fluorophors, acid-titration with the electrostatic correction for the surface potential, 1H-n.m.r. spectra and cross relaxation time between irradiated and observed protons. The immobilization of tryptophyl fluorophors observed in the F-form of AD0 was suppressed in the F-form of AD10. The acidtitration analysis of AD12 showed non salt-bonding between carboxylate groups and cationic side chains in the F-form, as in the case of AD0, indicating charged side chains being presumably mobile. 1H-n.m.r. spectra and cross relaxation times between irradiated and observed protons in the F-form of AD10 indicated the increase in the local motion. On the other hand, AD10 and AD12 did not show any significant change in the CD-resolved secondary structure in the N-F transition region. The F-form of AD10 or AD12 may therefore be the moltenglobule state which has secondary structure similar to the N-form of the complexes with fluctuating tertiary structure (side chains).     

Fluorescence polarization studies on the conformational transition of bovine plasma albumin in acidic solutions.

The acid-induced isomerization (the N-F transition) and expansion of bovine plasma albumin were studied by measuring fluorescence polarization and lifetime of the excited state of tryptophyl fluorophors. Most of the changes (decreases) in the reciprocal of fluorescence polarization and lifetime of the excited state correlated exactly with the N-F1 transition and/or the initial part of the N-F transition. These findings suggest that though the N-F transition is the cooperative pH-dependent conformational transition, the N-F transition clearly involves an intermediate step, such as the N-F1 and F1-F2 transitions. Rotational relaxation times for the N- and F-forms obtained by Perrin plot of tryptophyl fluorescence polarization were approximately 75 and 120-180 ns, respectively. The unexpected short rotational relaxation time of 75 ns of the N-form might be due to the rotational freedom of the tryptophyl side chain itself and/or of small flexible loci where tryptophyl fluorophors attach.     

Circular dichroic and 1H-NMR studies on the aged form of bovine plasma albumin.

Bovine plasma albumin (BPA) has 17 disulfide bonds and approximately one SH group at Cys-34 which catalyzes the intramolecular SH, S-S exchange reaction in the alkaline region at low ionic strength, resulting in the formation of the aged form (A-form). 1) Fractions of alpha-helix (f alpha) and beta-form (f beta) of iodoacetamide-blocked non-aged BPA (IA-BPA) at pH 6.5 (the N-form) and 9.0 (the B-form) in the absence of added salt were 0.70, 0.12 and 0.62, 0.18, respectively (Era et al. (1990]. However, there were no changes in f alpha and f beta of the iodoacetamide-blocked A-form (IA-A-form) over the pH range from 5.5 to 9.1 in the absence of added salt or in 0.10 M KCl (f alpha approximately 0.60, f beta approximately 0.20), indicating that the secondary structure of the IA-A-form might be similar to that of non-aged IA-BPA at pH 9.0 (B-form) in the absence of added salt, that is, the frozen B-form, stabilized covalently by the repairing of disulfide bonds. 2) The rigidity of the A- and IA-A-forms, as monitored by cross-relaxation times between irradiated and observed protein protons, was similar to or slightly higher than that of non-aged IA-BPA or BMA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unfolding/refolding studies of smooth muscle tropomyosin. Evidence for a chain exchange mechanism in the preferential assembly of the native heterodimer

The thermal and the urea-induced unfolding profiles of the coiled-coil alpha-helix of native and refolded tropomyosin from chicken gizzard were studied by circular dichroism. Refolding of tropomyosin at low temperature from alpha + beta subunits, dissociated by guanidinium chloride, urea, or high temperature, predominantly produced alpha alpha + beta beta homodimers in agreement with earlier studies of refolding from guanidinium chloride (Graceffa, P. (1989) Biochemistry 28, 1282-1287). The presence of two unfolding transitions in low salt solutions with about equal helix loss verified the composition with the first unfolding transition of the homodimer mixture originating from alpha alpha. In contrast, refolding by equilibrating at temperatures close to physiological, however, produced the native alpha beta heterodimer, which unfolded in a single transition. The refolding kinetics of dissociated alpha + beta subunits indicated that beta beta homodimers form first, leading to alpha alpha homodimers both of which are relatively stable against chain exchange below approximately 25 degrees C. Equilibrating the homodimer mixture at 37-40 degrees C for long times, however, produced the native alpha beta molecule via chain exchange. The equilibria involved indicate that the free energy of formation from subunits of alpha beta is much less than that of (alpha alpha + beta beta)/2. In vivo folding of alpha beta from the two separate alpha and beta gene products is, therefore, thermodynamically favored over the formation of homodimers and biological factors need not be considered to explain the native preferred alpha beta composition.     

High-frequency ventilation of ducks and geese

We studied gas exchange in anesthetized ducks and geese artificially ventilated at normal tidal volumes (VT) and respiratory frequencies (fR) with a Harvard respirator (control ventilation, CV) or at low VT-high fR using an oscillating pump across a bias flow with mean airway opening pressure regulated at 0 cmH2O (high-frequency ventilation, HFV). VT was normalized to anatomic plus instrument dead space (VT/VD) for analysis. Arterial PCO2 was maintained at or below CV levels by HFV with VT/VD less than 0.5 and fR = 9 and 12 s-1 but not at fR = 6 s-1. For 0.4 less than or equal to VT/VD less than or equal to 0.85 and 3 s-1. less than or equal to fR less than or equal to 12 s-1, increased VT/VD was twice as effective as increased fR at decreasing arterial PCO2, consistent with oscillatory dispersion in a branching network being an important gas transport mechanism in birds on HFV. Ventilation of proximal exchange units with fresh gas due to laminar flow is not the necessary mechanism supporting gas exchange in HFV, since exchange could be maintained with VT/VD less than 0.5. Interclavicular and posterior thoracic air sac ventilation measured by helium washout did not change as much as expired minute ventilation during HFV. PCO2 was equal in both air sacs during HFV. These results could be explained by alterations in aerodynamic valving and flow patterns with HFV. Ventilation-perfusion distributions measured by the multiple inert gas elimination technique show increased inhomogeneity with HFV. Elimination of soluble gases was also enhanced in HFV as reported for mammals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Infrared dichroism from the X-ray structure of bacteriorhodopsin

A detailed comparison with the three-dimensional protein structure provides a stringent test of the models and parameters commonly used in determining the orientation of the alpha-helices from the linear dichroism of the infrared amide bands, particularly in membranes. The order parameters of the amide vibrational transition moments are calculated for the transmembrane alpha-helices of bacteriorhodopsin by using the crystal structure determined at a resolution of 1.55 A (PDB accession number 1C3W). The dependence on the angle delta(M) that the transition moment makes with the peptide carbonyl bond is fit by the expression ((3)/(2)S(alpha) cos(2) alpha)cos(2)(delta(M) + beta) - 1/2S(alpha), where S(alpha) (0.91) is the order parameter of the alpha-helices, alpha (13 degrees ) is the angle that the peptide plane makes with the helix axis, and beta (11 degrees ) is the angle that the peptide carbonyl bond makes with the projection of the helix axis on the peptide plane. This result is fully consistent with the model of nested axial distributions commonly used in interpreting infrared linear dichroism of proteins. Comparison with experimental infrared dichroic ratios for bacteriorhodopsin yields values of Theta(A) = 33 +/- 1 degree, Theta(I) = 39.5 +/- 1 degree, and Theta(II) = 70 +/- 2 degrees for the orientation of the transition moments of the amide A, amide I, and amide II bands, respectively, relative to the helix axis. These estimates are close to those found for model alpha-helical polypeptides, indicating that side-chain heterogeneity and slight helix imperfections are unlikely to affect the reliability of infrared measurements of helix orientations.     

Titration of poly(dA-dT) . poly(dA-dT) in solution at variable NaCl concentration

CD and uv absorption data showed that high molecular weight poly(dA-dT) . poly(dA-dT), at 298 K, undergoes an acid-induced transition from B-double helix to random coil in NaCl solutions of different concentrations, ranging from 0.005 to 0.600M. Similarly, titration of the polynucleotide with a strong base causes duplex-to-single strands transition. The base- and acid-induced transitions were both reversible by back-titration (with an acid or, respectively, with a base): the apparent pKa were the same in both directions. However, the number of protons per titratable site (adenine N1) required to reach half-denaturation was in great excess over the stoichiometric value; to a much larger extent, the same effect was observed also for the deprotonation of the N3H sites of thymine. Moreover, in the basic denaturation experiments, at low salt concentrations ([NaCl]< or =0.300M) less acid than calculated was needed to back-titrate the base excess to half-denaturation. Both effects could be qualitatively justified on the basis of the counterion condensation theory of polyelectrolytes and considering the energy barrier created by the negatively charged phosphodiester groups to the penetration of the OH- ions inside the double helix and the screening effect of the Na+ ions on such charges, in the deprotonation experiments.     

Contribution of alpha helix formation in human plasma apolipoproteins to their enthalpy of association with phospholipids.

The human plasma apolipoproteins, apo-A-II and apo-C-III, assocaite with phospholipid with a concurrent increase in alpha helical structure of the apoprotein and an exothermic enthalpy of association. A linear correlation of the increase in alpha helical structure and the exothermic enthalpy of association gave a value of -1.3 kcal/mol of amino acid residues converted from a random coil to an alpha helical structure. This value is very close to that of alpha helix formation by charged polyamino acids (-1.2 kcal/mol) and suggests that amino acid side chains contribute little or no heat to the random coil leads to alpha helix transition in the plasma apolipoproteins. In the absence of a change in alpha helix in the apolipoproteins studied here, the enthalpy of association is practically nil, suggesting that alpha helix formation is a major enthalpic contribution to the total free energy of lipid-apolipoprotein association.     

Probing the mechanism of rhodopsin-catalyzed transducin activation

An agonist-bound G protein-coupled receptor (GPCR) induces a GDP/GTP exchange on the G protein alpha-subunit (G alpha) followed by the release of G alpha GTP and G beta gamma which, subsequently, activate their targets. The C-terminal regions of G alpha subunits constitute a major receptor recognition domain. In this study, we tested the hypothesis that the GPCR-induced conformational change is communicated from the G alpha C-terminus, via the alpha 5 helix, to the nucleotide-binding beta 6/alpha 5 loop causing GDP release. Mutants of the visual G protein, transducin, with a modified junction of the C-terminus were generated and analyzed for interaction with photoexcited rhodopsin (R*). A flexible linker composed of five glycine residues or a rigid three-turn alpha-helical segment was inserted between the 11 C-terminal residues and the alpha 5 helix of G alpha(t)-like chimeric G alpha, G alpha(ti). The mutant G alpha subunits with the Gly-loop (G alpha(ti)L) and the extended alpha 5 helix (G alpha(ti)H) retained intact interactions with G beta gamma(t), and displayed modestly reduced binding to R*. G alpha(ti)H was capable of efficient activation by R*. In contrast, R* failed to activate G alpha(ti)L, suggesting that the Gly-loop absorbs a conformational change at the C-terminus and blocks G protein activation. Our results provide evidence for the role of G alpha C-terminus/alpha 5 helix/beta 6/alpha 5 loop route as a dominant channel for transmission of the GPCR-induced conformational change leading to G protein activation.     

Calorimetric and spectroscopic studies of the thermotropic phase behavior of lipid bilayer model membranes composed of a homologous series of linear saturated phosphatidylserines

The thermotropic phase behavior of lipid bilayer model membranes composed of the even-numbered, N-saturated 1,2-diacyl phosphatidylserines was studied by differential scanning calorimetry and by Fourier-transform infrared and (31)P-nuclear magnetic resonance spectroscopy. At pH 7.0, 0.1 M NaCl and in the absence of divalent cations, aqueous dispersions of these lipids, which have not been incubated at low temperature, exhibit a single calorimetrically detectable phase transition that is fully reversible, highly cooperative, and relatively energetic, and the transition temperatures and enthalpies increase progressively with increases in hydrocarbon chain length. Our spectroscopic observations confirm that this thermal event is a lamellar gel (L(beta))-to-lamellar liquid crystalline (L(alpha)) phase transition. However, after low temperature incubation, the L(beta)/L(alpha) phase transition of dilauroyl phosphatidylserine is replaced by a higher temperature, more enthalpic, and less cooperative phase transition, and an additional lower temperature, less enthalpic, and less cooperative phase transition appears in the longer chain phosphatidylserines. Our spectroscopic results indicate that this change in thermotropic phase behavior when incubated at low temperatures results from the conversion of the L(beta) phase to a highly ordered lamellar crystalline (L(c)) phase. Upon heating, the L(c) phase of dilauroyl phosphatidylserine converts directly to the L(alpha) phase at a temperature slightly higher than that of its original L(beta)/L(alpha) phase transition. Calorimetrically, this process is manifested by a less cooperative but considerably more energetic, higher-temperature phase transition, which replaces the weaker L(beta)/L(alpha) phase transition alluded to above. However, with the longer chain compounds, the L(c) phase first converts to the L(beta) phase at temperatures some 10-25 degrees C below that at which the L(beta) phase converts to the L(alpha) phase. Our results also suggest that shorter chain homologues form L(c) phases that are structurally related to, but more ordered than, those formed by the longer chain homologues, but that these L(c) phases are less ordered than those formed by other phospholipids. These studies also suggest that polar/apolar interfaces of the phosphatidylserine bilayers are more hydrated than those of other glycerolipid bilayers, possibly because of interactions between the polar headgroup and carbonyl groups of the fatty acyl chains.     

Coordinate changes in fast thin filament and Z-line protein expression in the early response to chronic stimulation

The early response to chronic low frequency stimulation is characterized by coordinate changes in fast thin filament and Z-line protein expression prior to the expression of slow contractile proteins. Within the first 3 weeks of intervention there is 1) a transition from expression of the fast troponin (Tn) Ts TnT1f and TnT2f to expression of TnT3f, 2) a parallel change in Z-line protein expression in which alpha-actinin1f/s becomes predominant, and 3) a small but significant increase in the levels of the beta-tropomyosin (Tm) subunit. The timing of these changes coincides with the conversion to thick Z-lines, and the kinetics of changes in fast TnT and alpha-actinin isoforms suggests that the expression of TnT3f with alpha-actinin1f/s and a combination of alpha beta and beta 2-Tm, which we have designated the TnT3f program, is coordinated. Because fast fibers expressing TnT3f with alpha beta and beta 2-Tm, like slow fibers, exhibit a more graded response to calcium (Schachat, F.H., Diamond, M.S., and Brandt, P.W. (1987) J. Mol. Biol., 198, 551-555), this change appears to be an adaptive response, illustrating the contribution of isoform diversity to the physiological plasticity of fast skeletal muscle and indicating that expression of the TnT3f program may be an intermediate phase in the conversion from a fast to a slow molecular phenotype.     

Effects of bisphenol A on thyroid hormone-dependent up-regulation of thyroid hormone receptor alpha and beta and down-regulation of retinoid X receptor gamma in Xenopus tail culture

We investigated effects of different concentrations (10(-7) - 10(-5) M) of bisphenol A (BPA), which is known as an estrogenic and anti-thyroid hormonal endocrine disrupter, on the expression of thyroid hormone receptor (TR) alpha and beta and retinoid X receptor (RXR) gamma mRNA in tails of stage 52-54 Xenopus tadpoles in organ culture in the presence or absence of different concentrations of triiodo-thyronine (T(3)). In the absence of T(3), BPA at any concentration examined did not show remarkable effects on tail length but blocked 10(-7) M T(3)-induced tail resorption in a concentration-dependent manner. Semi-quantitative analyses of TRalpha and TRbeta mRNAs by RT-PCR in the tail specimens indicated that BPA shows an apparent antagonistic effect towards the receptors and reduced their mRNA levels relative to controls. When administered together with 10(-7) M T(3), the antagonistic effects of BPA were detected more clearly and dose-dependently. While BPA prevented the autoinduction of both TRalpha and TRbeta genes by T(3), the effect was less marked on TRalpha than on TRbeta. BPA also moderately suppressed RXRgamma gene expression. Gene expression of RXRgamma, a partner for heterodimer formation of TRs, was supressed by T(3) alone and also by BPA alone, but no additive effects were observed so far as studied. The present study indicates that a relatively low concentration of BPA, 10(-7) M, as compared with those examined previously (10(-5) to 10(-4) M) by us and other investigators, acts as an antagonist of T(3) through suppression of TRalpha and TRbeta gene expression in Xenopus tail in culture.     

Serine modulates substrate channeling in tryptophan synthase. A novel intersubunit triggering mechanism

Tryptophan synthase, an alpha 2 beta 2 complex, is a classic example of an enzyme that is thought to "channel" a metabolic intermediate (indole) from the active site of the alpha subunit to the active site of the beta subunit. We now examine the kinetics of substrate channeling by tryptophan synthase directly by chemical quench-flow and stopped-flow methods. The conversion of indole-3-glycerol phosphate (IGP) to tryptophan at the active site proceeds at a rate of 24 s-1, which is limited by the rate of cleavage of IGP to produce indole (alpha reaction). In a single turnover experiment monitoring the conversion of radiolabeled IGP to tryptophan, only a trace of indole is detectable (less than or equal to 1% of the IGP), implying that the reaction of indole to form tryptophan must be quite fast (greater than or equal to 1000 s-1). The rate of reaction of indole from solution is much too slow (40 s-1 under identical conditions) to account for the negligible accumulation of indole in a single turnover. Therefore, the indole produced at the alpha site must be rapidly channeled to the beta site, where it reacts with serine to form tryptophan: channeling and the reaction of indole to form tryptophan must each occur at rates greater than or equal to 1000 s-1. Steady-state turnover is limited by the slow rate of tryptophan release (8 s-1). In the absence of serine, the cleavage of IGP to indole is limited by a change in protein conformation at a rate of 0.16 s-1. When the alpha beta reaction is initiated by mixing enzyme with IGP and serine simultaneously, there is a lag in the cleavage IGP and formation of tryptophan. The kinetics of the lag correspond to the rate of formation of the aminoacrylate in the reaction of serine with pyridoxal phosphate at the beta site, measured by stopped-flow methods (45 s-1). There is also a change in protein fluorescence, suggestive of a change in protein conformation, occurring at the same rate. Substitution of cysteine for serine leads to a longer lag in the kinetics of IGP cleavage and a correspondingly slower rate of formation of the aminoacrylate (6 s-1). Thus, the reaction of serine at the beta site modulates the alpha reaction such that the formation of the aminoacrylate leads to a change in protein conformation that is transmitted to the alpha site to enhance the rate of IGP cleavage 150-fold.(ABSTRACT TRUNCATED AT 400 WORDS)     

Random coil structures in bacterial proteins. Relationships of their amino acid compositions to flanking structures and corresponding genic base compositions

In this study we classified regions of random coil into four types: coil between alpha helix and beta strand, coil between beta strand and alpha helix, coil between two alpha helices and coil between two beta strands. This classification may be considered as natural. We used 610 3D structures of proteins collected from the Protein Data Bank from bacteria with low, average and high genomic GC-content. Relatively short regions of coil are not random: certain amino acid residues are more or less frequent in each of the types of coil. Namely, hydrophobic amino acids with branched side chains (Ile, Val and Leu) are rare in coil between two beta strands, unlike some acrophilic amino acids (Asp, Asn and Gly). In contrast, coil between two alpha helices is enriched by Leu. Regions of coil between alpha helix and beta strand are enriched by positively charged amino acids (Arg and Lys), while the usage of residues with side chains possessing hydroxyl group (Ser and Thr) is low in them, in contrast to the regions of coil between beta strand and alpha helix. Regions of coil between beta strand and alpha helix are significantly enriched by Cys residues. The response to the symmetric mutational pressure (AT-pressure or GC-pressure) is also quite different for four types of coil. The most conserved regions of coil are “connecting bridges” between beta strand and alpha helix, since their amino acid content shows less strong dependence on GC-content of genes than amino acid contents of other three types of coil. Possible causes and consequences of the described differences in amino acid content distribution between different types of random coil have been discussed.     

Acid titrations of poly(dG-dC).poly(dG-dC) in aqueous solution and in a w/o microemulsion

The model polynucleotide poly(dG-dC).poly(dG-dC) (polyGC) was titrated with a strong acid (HCl) in aqueous unbuffered solutions and in the quaternary w/o microemulsion CTAB/n-pentanol/n-hexane/water. The titrations, performed at several concentrations of NaCl in the range 0.005 to 0.600 M, were followed by recording the modifications of the electronic absorption and of the CD spectra (210< or = lambda < or =350 nm) upon addition of the acid. In solution, the polynucleotide undergoes two acid-induced transitions, neither of which corresponds to denaturation of the duplex to single coil. The first transition leads to the Hoogsteen type synG.C+ duplex, while the second leads to the C+.C duplex. The initial B-form of polyGC was recovered by back-titration with NaOH. The apparent pKa values were obtained for both steps of the titration, at all salt concentrations. A reasonably linear dependence of pKa1 and pKa2 from p[NaCl] was obtained, with both pKa values decreasing with increasing ionic strength. In microemulsion, at salt concentrations < or = 0.300 M, an acid-induced transition was observed, matching the first conformational transition recorded also in solution. However, further addition of acid led to denaturation of the protonated duplex. Renaturation of polyGC was obtained by back-titration with NaOH. At salt concentrations > 0.300 M, polyGC is present as a mixture of B-form and psi- aggregates, that slowly separate from the microemulsion. The acid titration induces at first a conformational transition similar to the one observed at low salt or in solution, then denaturation occurs, which is however preceded by the appearance of a transient conformation, that has been tentatively classified as a left-handed Z double helix.     

Sulfated oligosaccharides isolated from the deamination products of heparins

Porcine heparin, whale heparin, and a solvolyzed porcine heparin were deaminated, and sulfated oligosaccharides, compounds 3f, 4f, 3s, 4s, 5, 6, 7s, 10, 11f, 11s, and 13 were isolated from the deamination products by Dowex 1 x 2 (Cl- form) column chromatography and high voltage paper electrophoresis and/or gel filtration on Sephadex G-25. Based on the results of chemical, 1H and 13C NMR spectral analyses, and of Smith degradation, together with previous observations, the structures of these sulfated oligosaccharides are proposed to be as follows: compound 3f, IdUA(2S)alpha 1 leads to GlcNAc alpha 1 leads to 4GlcUA; compound 4f, IdUA alpha 1 leads to 4GlcNAc(6S) alpha 1 leads to 4GlcUA; compound 3s, IdUA(2S) alpha 1 leads to 4GlcNAc alpha 1 leads to 4 GlcUA beta 1 leads to 4a Man; compound 4s, IdUA alpha 1 leads to 4Glc NAc(6S) alpha 1 leads to 4 GlcUA beta 1 leads to 4aMan; compound 5, IdUA(2S) alpha 1 leads to 4aMan; compound 6, GlcUA beta 1 leads to aMan(6S); compound 7s, IdUA alpha 1 leads to 4aMan(6S); compound 10, IdUA(2S)alpha 1 leads 4GlcNAc(6S)alpha 1 leads to 4 GlcUA beta 1 leads to 4aMan; compound 11f, IdUA(2S) alpha 1 leads 4GlcNAc alpha 1 leads to 4GlcUA beta 1 leads to 4a Man (6S); compound 11s, IdUA alpha 1 leads to GlcNAc(6S) alpha 1 leads to 4GlcUA beta 1 leads to 4aMan(6S); compound 13, IdUA(2S) alpha 1 leads to 4aMan(6S). For ths sulfated disaccharides, the same results as those reported in our previous papers were obtained. On the other hand, the proportion of total sulfated tri- and tetrasaccharides from whale heparin was 1.9 times higher than that from porcine heparine, reflecting a higher content of GlcNAc in the former. Also, the yields of compound 11s from these two heparins were comparable to their anticoagulant activities. In addition, certain 2-O-sulfates on IdUA flanked with GlcNS(6X) (X=H or S) in the heparin molecule are suggested to be important for the activity.     

Design and synthesis of sequence-specific DNA-binding peptides

Design, synthesis and DNA binding activities of two peptides containing 32 and 102 residues are reported. A nonlinear 102-residue peptide contains four modified alpha helix-turn-alpha helix motifs of 434 cro protein. These four units are linked covalently to a carboxyterminal crosslinker containing four arms each ending with an aliphatic amino group. From CD studies we have found that in aqueous buffer in the presence of 20% trifluoroethanol the peptide residues assume alpha-helical, beta-sheet and random-coiled conformations with the alpha-helical content of about 16% at room temperature. Upon complex formation between peptide and DNA, a change in the peptide conformation takes place which is consistent with an alpha - beta transition in the DNA binding alpha helix-turn-alpha helix units of the peptide. Similar conformation changes are observed upon complex formation with the synthetic operator of a linear peptide containing residues 7-37 of 434 cro repressor. Evidently, in the complex, residues present in helices alpha 2 and alpha 3 of the two helix motif form a beta-hairpin which is inserted in the minor DNA groove. The last inference is supported by our observations that the two peptides can displace the minor groove-binding antibiotic distamycin A from poly(dA).poly(dT) and synthetic operator DNA. As revealed from DNase digestion studies, the nonlinear peptide binds more strongly to a pseudooperator Op1, located in the cro gene, than to the operator OR3. A difference in the specificity shown by the non-linear peptide and wild-type cro could be attributed to a flexibility of the linker chains between the DNA-binding domains in the peptide molecule as well as to a replacement of Thr-Ala in the peptide alpha 2-helices. Removal of two residues from the N-terminus of helix alpha 2 in each of the four DNA-binding domains of the peptide leads to a loss of binding specificity.