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The conformation and ion-binding characteristics of a cyclic octapeptide, cyclo (Ala-Leu-Pro-Gly)2, in a liphophilic solvent, acetonitrile, have been studied using CD and nmr spectroscopy. The peptide binds preferentially to divalent cations such as calcium, magnesium, and barium. The conformations of the free cyclic peptide and its calcium complex are very similar with well-defined beta- and gamma-turns. The cyclic peptide readily forms equimolar and possibly 2:1 (peptide:cation) complexes with divalent cations.

Kinetic constants are reported for alpha-chymotrypsin-, Streptomyces griseus protease 3 (SGP3)-, and elastase-catalyzed hydrolysis of a number of peptides. SGP3, like alpha-chymotrypsine, hydrolyzes most readily amide bonds whose immediate acyl group (P1) is a large, hydrophobic, amino acid residue. SGP3, however, has a broader specificity for P1 residues than does alpha-chymotrypsin, primarily because the most important interactions between SGP3 and residue P1 of the substrate involve the Cbeta and Cgamma groups of the P1 side chain. For substrates of all three proteases, the amino acid residue contributing the amino group of the scissile bond (P1') is less important than P1 in determining kcat/Km for the hydrolysis reaction. Each enzyme interacts favorably with a hydrophobic P1' side chain. A substate with a large P1' side chain is bound more strongly, but is hydrolyzed less rapidly, than that with P1' Ala. The observation that strong binding of P1' is not necessarily conducive to rapid hydrolysis is consistent with the idea that parts of P1' must undergo a considerable displacement during substrate hydrolysis.

An approach to the study of protein receptor sites in protein mixtures or supramolecular assemblies by using fluorescence spectroscopy is described. This approach, fluorescent photoaffinity labeling, combines the merits of photoaffinity labeling to attain site-directed reactivity with the probing power of fluorescent ligands. A fluorescent photoaffinity label for cyclic AMP receptor sites of cyclic AMP-dependent protein kinases was synthesized in both unlabeled and radioactive forms. The probe, 8-azido-1,N(6)-ethenoadenosine 3',5'-cyclic monophosphate, mimics cyclic AMP in its ability to stimulate the phosphotransferase activity of the protein kinases and strongly competes with cyclic AMP for its binding sites in all preparations so far tested. Photolysis, after equilibration of protein kinase and 8-azido-1,N(6)-ethenoadenosine 3',5'-cyclic monophosphate in the dark, effects binding of the intermediate nitrene irreversibly and specifically to the cyclic AMP sites with the development of fluorescence. Excess reagent and low molecular weight photolytic products are removable by dialysis. Studies of a crude beef heart preparation containing cyclic AMP-dependent protein kinase suggest that the cyclic AMP binding sites are hydrophobic in nature and strongly immobilize the adenine moiety of the cyclic nucleotide.

Two related homodetic bicyclic nonapeptides (cyclo Glu-X-Pro-Gly-Lys-X-Pro-Gly)-cyclo (l gamma----5 epsilon), X = Ala(BCP2), X = Leu(BCP3) have been synthesized using conventional solution phase methods involving mixed anhydride coupling reactions starting with appropriately protected naturally occurring amino acids. The conformation and ion binding properties of BCP2 have been studied by nuclear magnetic resonance and circular dichroism techniques. The results of these studies have been compared to those of BCP3. The presence of Ala caused both Ala-Pro bonds to be trans in free BCP2. This characteristic imparted subtle differences to the ion-binding properties of BCP2 as compared to free BCP3 which has one cis Leu-Pro bond and one trans Leu-Pro bond.

The membrane conformation of the peptide ionophore gramicidin A is shown by 19F NMR to be described by the N-terminal to N-terminal beta LD helical dimer model proposed by Urry [Urry, D.W. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 672-676]. Fully active analogues of gramicidin with 19F labels at both the N- and C-termini are prepared synthetically. Labeled peptides are incorporated into small unilamellar vesicles of dimyristoylphosphatidylcholine. Measurements of the accessibility of the labels to either aqueous or lipophilic paramagnetic probes show that the N-terminus of gramicidin is located in the membrane interior and the C-terminus is at the membrane surface. Of the specific models proposed for the structure of gramicidin, these data are consistent only with that of Urry. The C-terminal 19F NMR peak in vesicles actually consists of three overlapping peaks. Experiments with the aqueous shift reagent Tm3+ show that C-terminal 19F nuclei in the inner and in the outer leaflets of vesicles resonate at different frequencies. The outer leaflet peak in turn consists of two overlapping peaks, possibly due to a local rearrangement of the C-terminal label.

To demonstrate a method by which the conformation of membrane proteins may be determined spectroscopically in model membranes, we determined the structure of a hydrophobic oligopeptide, t-butyloxycarbonylprolylleucylvalylmethyl ester, in phospholipid vesicles by nuclear magnetic resonance, circular dichroism, and infrared spectroscopy. 13C nuclear magnetic resonance and circular dichroism techniques demonstrated that the conformation of this peptide in linear hydrocarbon solutions was essentially identical to its conformation in lipid vesicles. 1H nuclear magnetic resonance and infrared spectroscopy of the peptide in hydrocarbon solution then provided additional high-resolution information concerning the structure of the peptide as found in the hydrophobic portion of the lipid bilayer. The conformation of this peptide in hydrophobic media a differs from its structure in hydrophilic solvents, not only in bond angles and the proportion of cis/trans isomers about the X-proline bond, but also in its intermolecular associations.

Circular dichroism and nuclear magnetic resonance spectroscopies have been used to study the unfolding process of cobrotoxin upon addition of fluoro alcohols/or sodium dodecyl sulfate to its aqueous solution. In each final unfolded state, the protein had its disulfide bonds intact. The unfolding process has been found to be reversible in the case of fluoro alcohol/water mixtures, while no such reversibility was found in the case of sodium dodecyl sulfate. However, when hexafluoro-2-propanol is added to the sodium dodecyl sulfate unfolded protein, refolding is induced. The mechanism of unfolding is discussed in terms of the different interactions which govern the protein conformation in solution.

The intermediates of ribonuclease with one to four disulfide bonds trapped during refolding of the reduced protein have been compared to the fully reduced and native forms of the protein by gel filtration, circular dichroism, and Raman spectroscopy. Correctly refolded ribonuclease is indistinguishable from native protein, while a three-disulfide intermediate has a compact conformation which is very similar, but not identical. In contrast, all other intermediates with one to four disulfide bonds are qualitatively similar to fully reduced ribonuclease by their circular dichroism and Raman spectra, although the disulfide cross-links affect the dimensions of the polypeptide chain. The apparent absence of stable partially ordered structures in the initial disulfide intermediates implies that during refolding there are relatively few constraints on formation on disulfide bonds, although their formation is probably not entirely random. The stable conformation appears during refolding only when the three or four disulfide bonds capable of stabilizing a native-like conformation of the entire polypeptide chain occur simultaneously.

A nonapeptide Ac-His-Phe-Gly-Cys-D-Phe-Ser-Gly-Glu-Cys-NH2 (XI) cyclized through the cysteines at positions 4 and 9 is synthesized as a model active site for the enzyme alpha-chymotrypsin. A CPK model of XI indicates that the peptide will have a high probability of folding into a conformation in which the two beta-phenyls interact to form a hydrophobic site to one side of the cyclohexyl structure, and the Ser-His-Glu side chains form a hydrogen bonded triad over the plane of cyclopeptidyl structure. Substrates can then bind at the hydrophobic pocket formed by the beta-phenyls and be acted upon by the Ser-His-Glu catalytic triad, as in the enzyme. 1H. n.m.r. shows: (i) multiplet peaks for the phenyl protons in D2O that condense to a singlet in DMSO-d6, (ii) a perturbation of the phenyl protons chemical shift on proflavin association to XI, and (iii) perturbation of the His pKa to a higher value on association of proflavin to XI. These data support the existence of a hydrophobic site and a Glu-His interaction in the peptide. Furthermore, the greater than 10(2) better affinity of proflavin to XI than to AcTrp supports the existence of a hydrophobic site. However, no acceleration of p-nitrophenyl acetate or trans-cinnamoyl imidazole hydrolysis over that of imidazole is observed. The possible reasons for a lack of esterase activity in XI and other peptidyl models of serine protease active sites are discussed.

Circular dichroism spectra of the partially folded trapped intermediates were measured in order to aid in the elucidation of the conformational forces which determine a nonrandom, nonsequential pathway of disulfide bond formation upon refolding of bovine pancreatic trypsin inhibitor. Whatever conformation was responsible for the kinetic rates of the intermediates should be stabilized by the presence of their trapped disulfide bonds. The near-ultraviolet spectra provide considerable information about the environments of the aromatic and disulfide side chains. The predominant single-disulfide intermediate has significant nonrandom conformation not present in the fully reduced protein, with aromatic rings and the disulfide bond in stabilized asymmetric environments. Forming either of the two nonnative, but kinetically important, second disulfides in this intermediate does not produce unequivocably different conformations. Forming a second native, but kinetically unproductive, disulfide produces a substantial decrease in randomness, which may hinder formation of the third disulfide. The largest conformational changes occur upon disulfide rearrangement to the stable, correctly refolded, two- and three-disulfide species. Interpretation of the far-ultraviolet spectra in terms of the secondary structure of the intermediates is uncertain, due to the atypical spectra of the folded forms of the protein. Consequently, we are unable to determine unambiguously the secondary structure of the intermediates. However, all the spectra show that nonrandom conformations of the polypeptide chain gradually appear as disulfide bond formation progresses, as expected from the nonrandom pathway of the latter.

A 270-MHz 1H nuclear magnetic resonance investigation of an ion-binding cyclic peptide analogue of valinomycin, cyclo(L-Val-Gly-Gly-L-Pro)3, and its cation complexes is reported. In CD2Cl2 and CDCl3, the peptide is proposed to occur in a C3-symmetric conformer with the N--H's of all six glycine residues intramolecularly hydrogen bonded. This conformation is different from the familiar valinomycin bracelet structure and lacks any "cavity". Cations do not bind, or bind only weakly, to the peptide in these solvents. Uncomplexed cyclo(L-Val-Gly-Gly-L-Pro)3 in acetonitrile appears to be averaging among several conformations with no evidence found for any preferred intramolecular hydrogen bonds. The strong 1:1 complexes of cyclo(L-Val-Gly-Gly-L-Pro)3 with K+ ANd Ba2+ in acetonitrile are structurally analogous to the bracelet conformation of valinomycin and involve the N--H's of the Val residues and of the Gly's preceding Pro in intramolecular hydrogen bonding. Tl+ was also found to form strong 1:1 complexes with the dodecapeptide.

The solvent surface accessibilities of the aromatic amino acids of bovine pancreatic trypsin inhibitor have been examined after the protein has been trapped at various stages of unfolding and refolding. Two types of near-ultraviolet difference spectroscopy were used in making these measurements. One type compares the near-ultraviolet spectrum of each protein with the spectrum of the native inhibitor; the other is solvent perturbation spectroscopy. The two types of difference spectroscopy utilized are compared and found to be equivalent measures of tyrosine solvent exposure if the perturbation spectra are corrected for a probable contribution by buried residues. The experimental values for tyrosine solvent exposure in the native inhibitor are in agreement with those calculated from its crystal structure. The results of these studies identify an order in which the four tyrosines and the four phenylalanines of bovine pancreatic trypsin inhibitor are removed from solvent as refolding proceeds. The relative solvent accessibilities of the aromatic residues suggest an ordering in which the protein chain obtains compact globular structures.

We have determined the conformation of the channel-forming polypeptide antibiotic gramicidin A in phosphatidylcholine vesicles by using 13C and 19F NMR spectroscopy. The models previously proposed for the conformation of the dimer channel differ in the surface localization of the NH2 and COOH termini. We have incorporated specific 13C and 19F nuclei at both the NH2, and COOH termini of gramicidin and have used 13C and 19F chemical shifts and spin lattice relaxation time measurements to determine the accessibility of these labels to three paramagnetic NMR probes--two in aqueous solution and one attached to the phosphatidylcholine fatty acid chain9 all of our results indicate that the COOH terminus of gramicidin in the channel is located near the surface of the membrane and the NH2 terminus is buried deep within the lipid bilayer. These findings strongly favor an NH2-terminal to NH2-terminal helical dimer as the major conformation for the gramicidin channel in phosphatidylcholine vesicles.

Gramicidins A, B, and C are a family of poly-peptide antibiotics which facilitate the passive diffusion of alkali cations and protons through lipid bilayer membranes. It is clear that gramicidin forms a multimeric transmembrane channel and it has been suggested that the channel is an io-conducting dimer in equilibrium on the membrane with non-conducting monomer. We describe the preparation and purification of a derivative of gramicidin C in which the phenolic hydroxyl of the tyrosine at position 11 has been esterified to 8-dimethylaminonaphthalene-1-sulfonate (dansyl). This derivative fluoresces strongly in the visible with an emission maximun in dioxane of 530 nm, an emission lifetime of 16 ns, and a quantum yield of 0.8. Veatch et al. ((1975),J. Mol. Biol. 99, 75) have shown this 0-dansyltyrosine gamicidin C to be a fully active analogue of gramicidin A in artificial lipid bilayer membranes. We here utilize this derivative to further characterize the state of aggregation and rotational mobility of the four interconvertible conformational species formed by gramicidin in nonpolar organic solvents (Veatch et al. (1974), Biochemsitry 13, 5249; Veatch and Blout (1974), Biochemistry 13, 5257). Fluorescence energy transfer from the tryptophans of gramicidin A to the 0-dansyltyrosine of this derivatives supports the conclusion that all of these gramicidin isolated species are aggregates. Decay of fluorescence polarization anisotropy measurements yield a rotational correlation time of 1 ns for the 0-dansyltyrosine chromophore in ethanol in good agreement with the more detailed information previously obtained by 13C-nuclear magnetic resonance for the monomer in dimethyl sulfoxide (Fossel et al. (1974), Biochemistry 13, 5264). However, it is likely that the chromophore has much more rotational mobility than the rest of the gramicidin molecule in the aggregated comformational states.

As part of a conformational study of the pathway of unfolding and refolding of bovine pancreatic trypsin inhibitor that accompanies breakage and formation of its three disulfide bonds, circular dichroism spectra have been measured for several limiting conformational states: native and refolded, with the three correct disulfide bonds; the (30--51, 5--55) two-disulfide species trapped during unfolding and refolding, which have a stable nativelike conformation; the fully reduced protein, with no disulfide bonds. Refolded protein with the three correct disulfide bonds has been found to be slightly different from the native protein; this conformational difference could be removed by gently heating the refolded protein. The same difference appears to be present between the two-disulfide intermediates, lacking the 14--38 disulfide bond, produced during unfolding and refolding. The conformational difference appear to be introduced at an early stage of refolding. The fully reduced protein, with no disulfides, exists as a flexible polypeptide chain with no detectable fixed conformation. The near-ultraviolet portions of the spectra are resolved into probable contributions by tyrosine, disulfide, and phenylalanine side-chain electronic transitions. The probable contributions to the native protein spectrum by tyrosines were also elucidated by observing the spectral shifts caused by their ionization at pH 12.5, where the folded conformation is maintained. The rotational strengths of the isolated transitions provide a measure of conformational flexibilities for the chromophores. Resolution of the far-ultraviolet spectrum of the native protein into contributions of its known secondary structures was not successful.

Circular dichroism spectroscopy has been used to study the unfolding-refolding process of a cardiotoxin from Taiwan cobra (Naja naja atra) venom upon addition of fluoroalcohols or sodium dodecyl sulfate (SDS) to its aqueous solution. In these experiments, the disulfide bridges remained intact. The unfolding process has been found to be reversible both for fluoroalcohols and for SDS unfolding. The reversibility of the unfolding-refolding process of cardiotoxin in aqueous mixtures of fluoroalcohols was dependent on the volume per volume ratio of alcohol to water. SDS did not unfold the secondary structures of cardiotoxin whereas its tertiary structure was affected. If the SDS concentration in aqueous solution exceeded the critical micelle concentration value of SDS, a quasi-refolded state of cardiotoxin was observed. The mechanism of unfolding-refolding is discussed in terms of molecular interactions which might govern the protein conformation in solution.

Kinetic constants are reported for alpha-chymotrypsin- and Streptomyces griseus protease 3 (SGP3)-catalyzed amide hydrolysis of a number of peptide amides of varying substrate chain length. alpha-Chymotrypsin, but not SGP3, will hydrolyze rapidly specific acetyl amino acid amides. SGP3-catalyzed, but not alpha-chymotrypsin-catalyzed, hydrolysis is greatly stimulated by the presence of up to four amino acid residues N-terminal to the scissile bond of the substrate. The enzyme-substrate interactions utilized to promote hydrolysis, therefore, differ in these two enzymes, which, in other respects, show marked similarities. alpha-Chymotrypsin depends mainly on primary enzyme-substrate contacts, those with the amino acid residue (P1) whose carbonyl group forms part of the scissile bond, whereas SGP3 depends mainly on "secondary" enzyme-substrate contacts with amino acid residues (P2-P4) more remote from the scissile bond. A comparison with porcine elastase, a related serine protease, indicates that there is an inverse relation between the importance of primary and secondary enzyme substrate interactions in this family of enzymes. A rationale is proposed for this effect based on the observation that both types of enzyme-substrate interaction predominantly affect the rate constant for the acylation step of substrate hydrolysis.

The human erythrocyte contains two types of cyclic AMP-dependent protein kinase. The membrane-associated protein kinase holoenzyme can be released intact by hypotonic treatment at alkaline pH. Chromatography on DEAE-cellulose and molecular weight determinations demonstrate that this enzyme is a type I cyclic AMP-dependent protein kinase, while the predominant cyclic AMP-dependent protein kinase found in the cytoplasm is type II. The catalytic subunits of the two types of kinase found in the erythrocyte are identical, but the regulatory subunits, which distinguish the two types of kinase, determine their localization within the cell. It is proposed that the regulatory subunit of the type I enzyme, in addition to binding the catalytic subunit, interacts specifically with one or more membrane proteins and that this interaction may serve to position the kinase in preferential proximity to protein substrates.

Studies of the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in membranes of normal, transformed, and revertant 3T3 cells allowed estimation of microviscosities (eta) of the lipid bilayers of these cells. Fluorescence polarization values observed with normal cells were significantly lower than those observed with cells transformed either by polyoma virus or by simian virus 40. The values of fluorescence polarization obtained with revertant Py6R1 cells were lower than those obtained with the normal cells. The calculated microviscosities (eta) show a 50% increase upon transformation. Possible correlations between the bilayer viscosity and the mobility of the receptors in the membrane are discussed.