Circular dichroism studies on synthetic peptides corresponding to the cleavage site region of precursor proteins

The conformations of synthetic peptides which span the region in which the precursor part of proteins (signal sequences) destined for export are cleaved by signal peptidases, were investigated by circular dichroism spectroscopy. Pentapeptides comprising amino acids only from the carboxy-terminus of signal sequences or the amino terminus of the mature protein do not have any preferred conformation in a variety of solvents. Octa- and nonapeptides containing amino acids from the carboxy-terminal protion of signal sequences and the amino-terminus of the mature portions of precursor proteins tend to adopt beta-turn conformations in trifluoroethanol and micelles of sodium dodecylsulphate. Hence, in addition to the distribution of amino acids with small side chains at the carboxy terminus of signal sequences, it is conceivable that signal peptidases also recognize a beta-turn conformation in the cleavage site region of precursor proteins.     

Circular dichroism studies on synthetic signal peptides indicate beta-conformation as a common structural feature in highly hydrophobic environment

The conformations of synthetic peptides corresponding to signal sequences of chicken lysozyme and Escherichia coli proteins alkaline phosphatase and lipoprotein (wild-type) and their "variants" with a charged amino acid in the hydrophobic region, have been studied by circular dichroism spectroscopy in trifluoroethanol and micelles of sodium dodecyl sulfate, Brij 35, and sodium deoxycholate. In trifluoroethanol and aqueous mixtures of trifluoroethanol, the "wild-type" and variant signal sequences show similar conformational behavior. The wild-type signal peptides show increasing amounts of beta-structure going from sodium dodecyl sulfate to deoxycholate micelles (i.e. increasing order of hydrophobicity). The variant signal sequences, however, are largely unordered in micelles. The absence of beta-structure in variant signal sequences which do not initiate protein translocation across membranes, strongly suggests that the ability of signal sequences to adopt beta-structure in a highly hydrophobic environment is important for function.     

Circular dichroism of bradykinin and related peptides

1. The circular dichroism of bradykinin and a number of its analogues and homologues was measured over the spectral range 200-300nm. All of the biologically active peptides showed maxima at 220nm and minima at 235nm. The spectra were independent of solvent and temperature. The vibronic transitions of phenylalanyl residues in the 250-280nm range showed no evidence of intra- or inter-molecular interactions. We take this as evidence that bradykinin and its biologically active analogues and homologues exist in solution as disordered chains. 2. None of the analogues with spectra unlike bradykinin possessed biological activity. However, peptides such as retro-bradykinin, des-6-serine-bradykinin, des-1-arginine-bradykinin and des-9-arginine-bradykinin produced spectra like that of bradykinin but were devoid of biological activity. Although we could not identify spectral features that were clearly correlated with biological activity, it appears unlikely that highly ordered peptides of the same amino acid composition as bradykinin would possess bradykinin-like effects.     

Phenylalanine oligopeptides: Circular dichroism studies in solution

The conformations of a series of l-phenylalanine oligomers having the general formula BOC-(Phe)_n-OMe (n = 1–9) were investigated by circular dichroism in a number of solvent systems. These studies indicate that in trifluoroethanol and in hexafluoroisopropanol these oligomers probably form β-associated conformations beginning at the hexamer.     

Circular dichroism studies of ampullosporin-A analogues.

Ampullosporin A (AmpA), a 15mer peptalbol containing seven Aib residues is able to induce pigmentation on Phoma destructiva and hypothermia in mice, as well as to exhibit a neuroleptic effect. A circular dichroism study of ampullosporin A and its analogues was carried out in organic solvents with different polarities and detergent micelles to determine the relationship between their conformational flexibility and biological activities. The analogues were obtained by modifying the N- and C-termini of ampullosporin A. Furthermore, Gln and Leu were systematically substituted by Ala and Aib residues were replaced by Ala and/or Ac6c. To estimate the helicity of the analogues, the CD spectrum of AmpA recorded in acetonitrile was correlated to its crystal structure. All analogues displayed similar CD curve shapes in organic solvents with the ratio between two negative band intensities R = [theta]n-pi*/[theta]pi-pi* < 1. In acetonitrile, most of the analogues adopted a 70%-85% helical structure, which was higher than the average of 40%-60% obtained in TFE. In detergent micelles, the analogues were distinguishable by their CD profiles. For most of the biologically active analogues, the CD spectra in detergent micelles were characterized by a R ratio > 1 and increased helicity compared with those recorded in TFE, suggesting that the interaction of the peptides with the membrane and peptide association was necessary for their hypothermic effect.     

Circular dichroism of adenine and thymine containing synthetic polynucleotides

Circular dichroism (CD) spectra of poly(dA), poly(dT), poly(dA)·poly(dT), and poly[d(A-T)]·poly[d(T-A)] have been measured as a function of temperature. From these data difference spectra have been calculated by subtracting the spectrum measured at low temperature from the spectra measured at higher temperatures. The CD difference spectra obtained upon melting of the two double-stranded polymers are very similar. From a comparison of these difference spectra with calculated ones it is shown that optical transitions near 272 nm (on A) and 288 nm (most probably on T) are present. The premelting changes of the CD spectrum of poly[d(A-t)]·poly[d(T-A)] are due to a change in conformation in which the secondary structure goes from a C- to B-type spectrum by increasing the A-type nature of the polymer. Such a change is not observed for poly(dA)·poly(dT). Instead, a transition between two different B-type geometries occurs.     

Circular dichroism studies of some arginine-vasopressin analogues

CD spectra of arginine-vasopressin (AVP) and of its analogues substituted in position 1 and/or 7 were measured in aqueous solution at different pH values. The shapes of the CD spectra of AVP analogues substituted in position 1 are strongly influenced by the type of group attached to the beta-carbon of residue 1. The substitution of the proline residues in position 7 by N-methylalanine also leads to a change in conformation of the peptide. The differences in the CD spectra are interpreted in terms of conformational changes, which are due to the interaction of the tyrosine side chain with neighbouring residues (for 1-substituted analogues of AVP), or to that between the hexapeptide ring and acyclic tripeptide chain (for 7-substituted analogues).     

Circular dichroism studies on human chorionic somatomammotropin

The conformation of human chorionic somatomammotropin has been studied by means of circular dichroism spectra. The protein appears to contain about 45% α-helix in the native state. Circular dichroism bands in the region of side chain absorption have been assigned to phenylalanine and tryptophan residues. Tentative assignments has also been made to bands probably arising mostly from tyrosine residues. The stability of the native structure has been assessed by challenging the protein with four perturbing solvents. With the exception of 0.1 n NaOH which produced permanent denaturation, all conformational changes produced by the perturbants were fully reversible. In addition, the monomer molecular weight has been evaluated by gel filtration and osmotic pressure measurements. A value of 21,600 ± 900 was found by osmotic pressure at pH 8.4. The results have been compared with similar findings on human pituitary growth hormone and ovine pituitary lactogenic hormone.     

Circular dichroism of beta turns in peptides and proteins.

Circular dichroism (CD) spectra are reported for two groups of cyclic hexapeptides having beta turns whose geometry can be firmly established by X-ray crystallography and by NMR spectroscopy. One series contains the sequence L-Pro-D-Phe in the geometry of the classical type II beta turn, while the second group has the sequence D-Phe-L-Pro in the closely related geometry of the gramicidin S turn. CD data on the hydrogenated peptides show that in neither series do Cotton effects due to the aromatic phenylalanyl chromophore make a significant contribution to the spectra in the 195--240-nm region. In spite of the close geometric similarity of the beta turns of these two groups of peptides, their CD spectra are quite distinct. Furthermore, comparison of our data with the CD spectra of published models for beta-turn structures suggests that it may not be possible to characterize the contribution of all beta turns to the CD spectra of proteins by a single model curve. the CD spectra of model beta turns will be more useful in characterizing the folding of oligopeptides and sequence polypeptides, where a single type of turn is present.     

Circular dichroism studies of cobalt substituted lentil lectin

A recent method has been developed to effect metal ion substitution at the Mn2+ site in the lentil lectin (Bhattacharyya et al. (1984) Biochem. Biophys. Res. Commun. 124, 857-862). We report here the preparation of cobalt substituted lentil lectin, containing Co2+ at the S1 site and Ca2+ at the S2 site. The cobalt derivative possesses full saccharide binding activity and can be used for spectroscopic studies. The near UV and visible CD spectra of the derivative are shown, and its spectral properties are compared with various cobalt complexes of concanavalin A.     

Circular dichroism studies of isoleucine oligopeptides in solution

A conformational analysis was carried out on a series of L -isoleucine oligomers having the general formula BOC-(Ileu)_n-OMe (n = 2–8). These oligopeptides were examined in trifluoroethanol, trifluoroethanol-acid and mixed organic-water media. The results indicate that these oligomers form β-conformations beginning at the heptamer. The stability of the β-conformations was found to be greater than those formed by oligopeptides derived from L -alanine.     

Circular dichroism studies of sheep beta-lipotropic hormone.

The far ultraviolet circular dichroism spectra of sheep beta-lipotropic hormone (beta-LPH) were recorded under different conditions of pH, temperature, salt concentration, and solvent composition. Results confirm the stability of the hormone in strong basic or acidic solutions; moreover, temperatures up to 50 degrees C do not seem to affect noticeably the conformation of beta-LPH. However, increasing the NaC1 concentration or addition of dioxane in the solution brings about a conformational transition of the chain, interpreted as an increase in the helical content. The method of Yang (Chen, Y.H., Yang, J. T. & Martinez, H. M. (1972) Biochemistry 11, 4120-4131) was used to compute the proportion of helical, beta, and unordered forms of the hormone chain. The proportions are compared with those obtained from Fasman's predictive method (Chou, P. Y & Fasman, G. D. (1974) Biochemistry 13, 211-221 and Chou, P. Y. & Fasman, G. D. (1974) Biochemistry 13, 222-245) based on the known amino acid sequence of beta-LPH.     

Circular dichroism and cross-linking studies of bacteriorhodopsin mutants

Summary. Circular dichroism (CD) spectroscopy was employed for native (wild type, WT) bacteriorhodopsin (bR) and several mutant derivatives: R134K, R134H, R82Q, S35C, L66C, and R134C/E194C. Comparative analysis of the CD spectra in visible range shows that only R134C/E194C exhibits biphasic CD, typical for native bR, the other mutants demonstrate CD spectra with significantly smaller or absent negative band. Since the biphasic CD is a feature of hexagonal lattice structure composed by bR trimers in the purple membrane, these mutants and WT were examined by cross-linking studies, which confirmed the same trend towards trimeric organization. Therefore, a single amino acid substitution may lead to drastically different CD spectra without disruption of bR trimeric organization. Thus, although disruption of bR trimeric crystalline lattice structure (e.g., solubilization with detergents) directly results in the disappearance of characteristic bilobe in visible CD, the lack of the bilobe in the CD alone does not predict the absence of trimers.     

Circular dichroism and spin-label studies of carp hemoglobin

Circular dichroism (c.d.) spectra were obtained for deoxy, oxy, carboxy, nitrosyl, aquomet and azidomet derivatives of carp hemoglobin. The spectra of the hemolysate and its two major components are virtually identical. Binding of diatomic ligands induces large changes in the 287 nm ellipticity. In the case of oxygen binding this change appears to be proportional to the free energy of co-operation. The changes of L-band ellipticity and Soret rotational strength with ligation reflect tertiary structural alterations and bear no relationship to quaternary transitions. The c.d. results indicate that carp deoxyhemoglobin has very similar tertiary and quaternary structures between pH 6·4 and 8·0, whereas the oxyhemoglobin undergoes continuous conformational adjustment in response to pH changes. The effect of inositol hexaphosphate on c.d. spectra is much smaller than it is on the functional properties. Electron paramagnetic resonance spectra of iodoacetamide nitroxide label are sensitive to ligation, the label is probably attached to Cys142β.     

Circular dichroism studies of myoglobin and leghemoglobin.

The circular dichroism spectra of leghemoglobin a from the root nodules of soybean have been compared with those for sperm whale myoglobin in the fat- and near-ultraviolet and the Soret and visible regions of the spectrum. Circular dichroism spectra in the far-ultraviolet show that the leghemoglobins all have a high alpha-helix content (soybean leghemoglobin a, 55%) regardless of the nature of bound ligands and oxidation or spin state of the heme iron. The known sequence homologies with mammalian hemoglobins may therefore be reflected in conformational homologies as suggested by the x-ray studies of Vainshtein et al. ((1975) Nature (London) 254, 163-164) on lupin leghemoglobin. Removal of the heme moiety decreases helicity by only 9% for leghemoglobins, compared with 23% for myoglobin. This, the much smaller heme contribution to the near-ultraviolet circular dichroism than in myoglobin, and the greater accessibility of the heme moiety to aqueous solvent (Nicola et al. (1974), Proc. Aust. Biochem. Soc. 7, 21) suggest that the association between heme and protein is much weaker in leghemoglobins than in myoglobin. The aromatic Soret and visible circular dichroism spectra for all derivatives of leghemoglobin are opposite in sense to those for myoglobin, showing that the patterns of protein side chain contacts with the heme are different in the two classes of heme proteins. There is strong evidence that one of the two tryptophans whose identity and structural role in myoglobin is known, is present also in plant leghemoglobins, hydrogen-bonded and in a similar nonpolar environment whether heme is present or not. The above findings help to explain the remarkably high oxygen affinity and some other ligand-binding properties of leghemoglobins which differ from those of myoglobin.