End-group modified retro-inverso isomers of tripeptide oxytocin analogues: binding to neurophysin II and enhancement of its self-association properties

The importance of peptide backbone structure in peptide/protein recognition events has been tested evaluating the binding properties of end-group modified retro-inverso isomers of MYF and LYF amides, tripeptides able to mimic oxytocin in binding neurophysin II and in potentiating its self-association. The isomers, topochemically related to their parent peptides, have been prepared respectively from all-D N-acetyl-FYM and N-acetyl-FYL amides via the Hofmann-type rearrangement mediated by iodobenzene bis-trifluoroacetate. Retro-inverso isomers recognised neurophysin II with similar affinity as the parent peptides, as determined by analytical affinity chromatography on columns prepared immobilising neurophysin II on preactivated supports. In addition, their effect on neurophysin II self-association was similar to the tripeptide oxytocin analogues, potentiating neurophysin II dimerization to the same extent, as evaluated by solid-phase binding assays on microtiter plates coated with neurophysin II. Recognition specificity of retro-inverso isomers was further demonstrated by their inhibitory effect on the interaction between neurophysin II and oxytocin tripeptide analogues. Results suggest that only the proper orientation of the α-amino group and of the side chains plays a dominant role in the binding of tripeptide analogues to neurophysin II and potentiation of its self-association, while the peptide backbone topology has little influence on the recognition process.     

STRUCTURAL STUDIES ON THE SUCCINYLATED BOVINE SERUM ALBUMIN

An analysis of succinylated bovine serum albumin showed that all of the 62 α-amino groups of lysine residues, 13 hydroxyamino acid residues and 12 tyrosin residues were succinylated, but the succinylated tyrosines were later deacylated. Structural properties of the modified protein have been studied with circular dichroism and sedimentation velocity. At neutral pH (pH 7.60) and in a salt-free aqueous solution the modified protein is in an expanded form and its helical content is only 30% of that of unmodified protein. The increase of ionic strength restores the original conformation of the protein, whereas the increase of pH further disorganizes the structure of the protein. The results suggest that the electrostatic force alone is responsible for the compact structure of the protein molecule. The same mechanism is believed to underlie the effect of H₃O+ and the effect of succinylation of side chain groups on the conformation of bovine serum albumin.     

IDENTIFICATION AND OBSERVATION OF ALKYL PROTON RESONANCES OF THE AMINO-TERMINAL RESIDUES OF BOVINE NEUROPHYSINS

Analysis of the 220 MHz proton magnetic resonance spectra of bovine neurophysins-I and -II and of the effects of pH and succinylation on these spectra has allowed identification of the -CH₃ proton resonances of the amino-terminal alanine of both proteins and of the -CH₃ resonance of methionine-2 of neurophysin-II. The alanine -CH₃ resonance of neurophysin-I is a sharp doublet at all pH values between 1 and 10.5 indicating relatively few restrictions on its mobility. By contrast, the -CH₃ resonances of the amino-terminal alanine and methionine-2 of neurophysin-II undergo pH-dependent changes in broadening compatible with the formation of an intramolecular salt-bridge at neutral pH between the protonated α-amino and an unprotonated side chain carboxyl. The results suggest that differences in the properties of the two proteins are partially mediated by conformational differences involving their amino-terminal sequences. The potential usefulness of the amino-terminal resonances as n.m.r. ‘reporter’ signals is additionally demonstrated by studies of the effects of spin labels on the neurophysin-I amino-terminal alanine resonance; these studies place the amino-terminus of neurophysin-I approximately 14 Å from residue 3 of peptides bound to the strong neurophysin hormone-binding site.     

STOPPED-FLOW INVESTIGATION OF NITRATED BOVINE NEUROPHYSIN MONOMER BINDING TO OXYTOCIN

By use of stopped-flow kinetic data, we have measured the kinetics of mononitrated neurophysin I monomer binding to oxytocin. The association rate constant was 1.3(±0.3) × 10⁵ M^-1 s^-1 and the dissociation rate constant was 2.0 (± 0.5)s^-1 for protonated oxytocin binding. Both rates are significantly slower than those observed for neurophysin dimer. These data suggest that the binding process by which the monomer binds oxytocin is not identical to that of dimer.     

DESIGN OF A PHOTOAFFINITY LABEL FOR THE HORMONE BINDING SITE OF NEUROPHYSIN

The photolabile peptide, L-methionyl-L-tyrosyl-p-azido-L-phenylalaninamide, was synthesized by solution methods. This peptide, as well as the analogous species containing tritiated methionine, were found to bind reversibly and specifically, in the dark, to bovine neurophysin II. The dissociation constant, stoichiometry, and pH-dependence of this noncovalent interaction are typical of those properties for hormone (oxytocin) and hormone-like ligand binding to neurophysin II. Under photolytic conditions, methionyl-tyrosyl-p-azidophenylalaninamide causes irreversible inhibition of the noncovalent ligand binding activity of neurophysin II. This inactivation was achieved to the extent of about 90%. Both the dark and light (photolytic) interactions of the photolabile peptide with neurophysin II indicate its reaction at the hormone binding site of the protein and thus its potential use to identify amino acid residues at this site by covalent photoaffinity labelling.     

Ovine prolactin and human growth hormone derivatives

The α-amino group of ovine prolactin (oPRL) and human growth hormone (hGH) was selectively modified by transamination with glyoxylic acid. No difference was found in the binding capacity of transaminated oPRL to rat liver lactogenic receptors with respect to its control, although both samples showed a decrease in its binding capacity with reference to the native hormone. This decrease was due to conformational changes caused by the reaction conditions and not by the transamination itself, as shown by the circular dichroism spectra. Transaminated hGH retained the full binding capacity of the hormone. These results suggest that the α-amino group is not relevant for the binding to lactogenic liver receptors in both lactogenic hormones.     

SYNTHESIS OF [8-α-HYDROXYISOCAPROIC ACID] OXYTOCIN

The synthesis and biological activities of [8-α-hydroxyisocaproic acid] oxytocin are reported. The in vivo uterine responses to [8-α-hydroxyisocaproic acid] oxytocin are prolonged and those to deamino-[8-α-hydroxyisocaproic acid] oxytocin (as well as those we have previously reported for deamino-oxytocin) are very prolonged as compared with those to oxytocin. Time courses of the in vivo uterine responses to deamino-[8-α-hydroxyisocaproic acid] oxytocin and deamino-oxytocin (as followed by plotting the intervals between the individual contractions of a response vs. time after peptide injection) differ from those to [8-α-hydroxyisocaproic acid] oxytocin and oxytocin.     

Studies on the peptide corresponding to residues 34–47 of bovine factor X

Calcium binding studies of a 14-residue peptide corresponding to the 37–46 sequence of bovine factor X were performed using calcium ion selective electrode titrations and equilibrium dialysis. The presence of γ-carboxyglutamic acid residues at positions 36 and 40 coupled with the assumption that the peptide would bind calcium ions also prompted an investigation of possible secondary conformational changes in the peptide by use of circular dichroism spectroscopy. Equilibrium dialysis revealed a single relatively weak calcium binding site (log K_a= 2.39); an ion selective electrode experiment confirmed this result (log K_a= 2.17). The peptide maintained a random coil conformation throughout the calcium ion titrations as measured by circular dichroism.     

Evidence for the fatty acid-induced heterogeneity of the N and B conformations of human serum albumin

The influence of oleic acid on the interaction between albumin and warfarin, oxyphenbutazone or diazepam has been studied by circular dichroism and equilibrium dialysis. The pH dependences of the molar ellipticity of the drug-albumin complexes and of the free fraction of drug are completely changed by the presence of oleic acid. This phenomenon is attributed to an oleic acid-induced conformational change in both the neutral (N) and the basic (B) conformation of albumin, a change to which the warfarin-oxyphenbutazone binding area and the diazepam binding site is sensitive. The oleic acid-induced conformational states of albumin, the so-called N* and B* conformations, show binding properties that are different from the binding properties of the N and B conformations.     

N.M.R. AND EQUILIBRIUM DIALYSIS STUDIES OF THE INTERACTION OF BOVINE NEUROPHYSIN-I WITH VASOPRESSIN AND SMALL PEPTIDES

The binding to bovine neurophysin of lysine-vasopressin and of lysine-vasopressin selectively deuterated at the protons ortho to the tyrosine hydroxyl was studied by proton n.m.r. and equilibrium dialysis. The principal object of these studies was to investigate reports that, at standard salt concentrations, neurophysin contained a second site specific for vasopressin. At pH 6, the effects of neurophysin-I on the line-width, longitudinal relaxation rate and nuclear Overhauser properties of the lysine-vasopressin tyrosine ring protons were interpretable in terms of a slow-exchange 1:1 interaction between lysine-vasopressin and neurophysin. Additionally, n.m.r. competition studies between lysine-vasopressin and L-phenylalanyl-L-tyrosinamide suggested 1:1 competition for a single binding site on neurophysin. No evidence pointing to a significant second lysine-vasopressin-binding site was obtained from the n.m.r. studies. The lack of a moderately strong second binding site for lysine-vasopressin at neutral pH was also indicated by equilibrium dialysis studies at relatively high free hormone concentrations. These studies demonstrated only a single thermodynamically significant site for either oxytocin or vasopressin and failed to confirm a reported effect of LiCl on the number of sites available to oxytocin. It is suggested that secondary sites for the hormones are probably markedly weaker and less specific than reported elsewhere.     

Effect of divalent cations on structure-function relationships of the antitumor protein α-sarcin

α-Sarcin binds one Zn(II) cation per protein molecule, with a K_d value of 0.9 mM, determined by equilibrium dialysis experiments. Ca(II), Mg(II), and Mn(II) do not bind to α-sarcin. Cd(II) and Co(II) also behave as Zn(II). The binding produces local modifications on the protein conformation affecting the microenvironment of tryptophan residues. The three cations modify the fluorescence emission of the protein. The near-u. v. circular dichroism spectrum of the protein is also altered. The binding of Zn(II) and related cations does not modify the secondary structure of the protein. The ribonucleolytic activity of a-sarcin is inhibited upon Zn(II) binding, but no alteration of the ability of the protein to aggregate phospholipid vesicles has been observed.     

POLY (δ-L-ORNITHINE)

Poly (δ-L-Orn) is an example of an iso-polypeptide (i.e. variant of the usual poly α-peptide chain), where the α-carboxyl and δ-amino groups of ornithine are used in polymerization while the α-amino groups form the side chain. A procedure for the synthesis of this iso-polypeptide is described. Circular dichroism studies of poly (δ-L-Orn) and its Nα-Boc derivative suggest that these polymers might adopt a conformation in solution similar to the β-pleated sheet.     

Retinoic acid binding properties of the lipocalin member beta-lactoglobulin studied by circular dichroism,electronic absorption spectroscopy and molecular modeling methods

Interaction between the Vitamin A derivative all-trans retinoic acid and the lipocalin member bovine beta-lactoglobulin (BLG) was studied by circular dichroism (CD) and electronic absorption spectroscopy at different pH values. In neutral and alkaline solutions achiral retinoic acid forms a non-covalent complex with the protein as indicated by the appearance of a negative Cotton effect around 347 nm associated to the narrowed and red shifted pi-pi(*) absorption band of the ligand. The induced optical activity is attributed to the helical distortion of the conjugated chain caused by the chiral protein binding environment. As the disappearing CD activity showed in the course of CD-pH titration experiment, retinoic acid molecules dissociate from BLG upon acidification but this release is completely reversible as proved by the reconstitution of the CD and absorption spectra after setting the pH back to neutral. This unique behavior of the complex is explained by the conformational change of BLG (Tanford transition) which involves a movement of the EF loop at the entrance of the central cavity from open to closed conformation in the course of pH lowering. From these results it was inferred that retinoic acid binds within the hydrophobic calyx of the beta-barrel.     

Interactions between DNA and benzo- and tetrahydrobenzofurocoumarins: thermodynamic and molecular modeling studies

The non-covalent interaction of a series of new water-soluble benzo- and tetrahydrobenzofurocoumarins with salmon testes DNA has been studied using flow linear dichroism, circular dichroism, contact fluorescence energy transfer and ethidium bromide displacement assay. The new derivatives are characterised by having an alkyl amino side chain protonated at physiological pH; this fact strongly enhances the solubility in aqueous media and the affinity for the macromolecule. The results show significant difference in the affinity and the mode of binding among the examined compounds depending on the nature of the fourth condensed ring and the position of the alkylamino side chain. Benzofurocoumarins derivatives bind DNA by undergoing intercalation inside the duplex macromolecule, whereas tetrahydrobenzofurocoumarins derivatives show a substantial tilt relative to the base planes. Molecular modeling studies have been performed to characterise in detail the intercalation mechanism of these benzofurocoumarins to DNA.     

Drug-biomolecule interactions: proton magnetic resonance studies of complex formation between bovine neurophysins and oxytocin at molecular level.

Proton magnetic resonance spectroscopy was used to monitor individual amino acid residues in bovine neurophysin, in the nonapeptide hormone oxytocin, and in the complex formed between them. For neurophysin I alone, a normal titration curve for the C-2 proton resonance of the lone histidine residue was obtained with an apparent ionization constant of 6.9 addition of oxytocin to a solution of neurophysin I at pH 6.5 resulted in several changes in the spectrum. The effect on the histidine C-2 proton resonance signal indicated a slow exchange process between two states, probably representing a conformational change in the protein. The apparent pK of the histidine residue in the hormonal complex was shifted to 6.7, indicating a slightly more positive (less electron dense) environment for the histidine residue. Resonances of the single tyrosine residue of oxytocin were observed to broaden significantly, but not to shift appreciably, on the addition of neurophysin II. These observations may indicate involvement of the tyrosyl residue of oxytocin in the hormone-"carrier protein" interaction.     

Studies on the binding of benzodiazepines to human serum albumin by circular dichroism measurements

The binding of twenty-one different benzodiazepine derivatives to human serum albumin (HSA) has been studied by circular dichroism (CD) measurements in 0.1 M KCl and 0.005 M phosphate buffer at pH 7.4 and 25°. The binding has been related to the qualitative changes of the CD spectra of HSA between 250 and 350 nm and, in some representative benzodiazepine derivatives, to the electron distribution as calculated by the CNDO/2-method. The binding has also been quantitatively studied with a continuous CD titration technique. The data were numerically analyzed with computer programs based on one-site, two-sites and three-sites models. It is concluded that most derivatives will bind primarily to one site on HSA. It is moreover concluded that variations of the C₂-amino side chains will not influence the binding properties. Oxygens at C₂ or C₃ will increase the binding, while oxygens in both positions will decrease the binding. A derivative with a C₇-amino group will show only weak affinity for HSA, which might be explained by the positive character of the hydrogens in the amino group according to the CNDO/2-calculation. A C₇-nitro group will also impair the binding, as well as large substituents at N₁.     

PURIFICATION OF SEVERAL PROTEOLYTIC ENZYMES BY TOSYL- AND CARBOBENZOXY-TRIETHYLENETETRAMINE-SEPHAROSES

Tosyl-triethylenetetramine-Sepharose (Tos-T*-Sepharose) and carbobenzoxy-triethylenetetramine-Sepharose (Z-T-Sepharose) were found to be adsorbents utilizable in the purification of several microbial and animal proteases. The former Sepharose derivative adsorbed α-chymotrypsin, trypsin, subtilisin, thermolysin and neutral subtilopeptidase at neutral pH range, and acid proteases such as pepsin and Rhizopus niveus protease at pH 3.5–6.5. α-Chymotrypsin and trypsin were eluted with 0.1 N acetic acid and Rhizopus protease with 0.5 N acetic acid, thermolysin with 1 M guanidine ˙ HCI or 33% ethyleneglycol, whilst pepsin was recovered by elution with 2 M guanidine ˙ HCl at pH 3.5. The binding of neutral subtilopeptidase and subtilisin to this adsorbent was comparatively weak and both the enzymes were recovered by elution with 0.5 I M Nacl at neutral pH. On the other hand, Z-T-Sepharose was found to bind tightly to these proteolytic enzymes except neutral subtilopeptidase. Trypsin and α-chymotrypsin were released from the adsorbent column with 1 M p-toluenesulfonate, and subtilisin with 1 M guanidine ˙ HCl or 33% ethyleneglycol at neutral pH region. By these chromatographic procedures, the specific activities of these proteolytic enzymes increased effectively. Comparison of the binding abilities of acetyl-, benzoyl-, tosyl- and carbobenzoxy-T-Sepharoses to these enzymes suggests that hydrophobicity of tosyl and carbobenzoxy groups plays an important role in the enzyme-adsorbent interaction.     

Purification and conformation of ribosomal protein L25 from E. coli ribosome

Ribosomal protein L25 from the large subunit of E. coli ribosomes has been purified using a new procedure involving a 2 m LiCl extraction followed by phosphocellulose chromatography in 6 m urea elution buffer. The conformation of purified L25 was studied employing circular dichroism and ultraviolet absorption spectroscopy in reconstitution buffer. The analysis of the far u.v. circular dichroism spectrum of L25 indicates L25 contains ～ 16%α-helix and ～ 19%β-structure. The conformation of L25 was also studied using the predictive methods of Chou & Fasman and Maxfield & Scheraga. Both of these methods predict approximately three times the percent α-helix present in L25 as compared with that determined from the analysis of the circular dichroism spectrum. A structure for L25 is predicted which contains two positively charged binding domains and is consistent with published binding data on the interaction of 5S RNA and L25. The large difference in the %α-helix as determined from the analysis of the circular dichroism spectrum and the predictive techniques is suggested to result from the denaturing effects of 6 m urea used in the preparation of ribosomal proteins.     

Influence of neurophysin residues 1–8 on the optical activity of neurophysin-peptide complexes Direct evidence that the 1–8 sequence alters the environment of bound peptide

Circular dichroism was used to compare the environment of peptides bound to native and des 1-8 neurophysin in order to further elucidate the role of the neurophysin 1-8 sequence in peptide-binding. A very large positive ellipticity (～6000 degcm²dmol^?1), shown earlier to be induced in tyrosine at position 2 of peptides bound to the native protein, was determined by the present study to be paralleled by similar induced changes in tyrosine at peptide position 1. Deletion of the neurophysin 1-8 sequence led to loss of half of the induced optical activity at peptide positions 1 and 2 and changes in binding-induced optical activity in the protein, the latter partially assignable to protein disulfides. In the rnononitrated native and des 1-8 proteins, the optical activity of neurophysin Tyr-49, a residue at the peptide-binding site, was reduced by 80% in complexes of the des 1-8 protein relative to those of the native protein. The results suggest a role for neurophysin Arg-8 in modulating the optical activity at the binding site by directly placing a charge proximal to the binding site and/or by altering binding site conformation. The data provide the first unambiguous evidence of a difference in the environment of bound peptide between the native and des 1-8 proteins.     

Conformational characteristics of homo-oligopeptides of O-benzyl-L-tyrosine+

Conformational studies of X [-L-Tyr(Bzl)-]_n-series bound to polyethyleneglycol (X = H₂ Nps; n = 3–8) in the solid state and in solvents of different polarities and capabilities of forming hydrogen bonds are reported. By using i.r. absorption, the occurrence of the β-structure in the higher oligomers in the solid state was established. By means of i.r. absorption and CD the onset of that ordered conformation in solution was assessed as a function of chain length. The effects induced by the presence of the N-protecting group and added base, and by changing the nature of solvent on the conformational preferences of the [-L-Tyr(Bzl)-]_n homo-peptides were also examined. The 2-nitrophenylsulphenyl chromophoric derivative of the α-amino group is proposed as a circular dichroism sensor for β-structure in peptides.