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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.

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.

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.

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.

Large increases in the esterase, amidase, and peptidase activities of elastase are observed on increasing the length of peptide substrates. The results suggest that at least five contiguous peptide units of the substrate (four N-terminal and one C-terminal to the scissile bond) can interact concurrently with the enzyme. When analyzed in terms of an acyl-enzyme mechanism, the increased effectiveness of the enzyme with larger substrates appears mainly in the acylation reaction and, to a lesser extent, in the binding and deacylation steps.

A cyclic hexapeptide, cyclo(Pro-Gly-Pro-Gly-Pro-Gly), has been synthesized; its solution conformations were examined by 220-MHz nuclear magnetic resonance spectroscopy. The solution structures have been deduced, and shown to vary as a function of solvent polarity. In addition, it has been found that this cyclic peptide binds alkali metal cations. While the predominant conformation of this cyclic peptide is 3-fold symmetric in the apolar solvent methylene chloride, an asymmetric structure is preferred in some polar solvents (water, dimethylsulfoxide). However, addition of alkali metal salts, such as sodium thiocyanate, to dimethylsulfoxide solutions of the peptide shifts the conformational equilibrium in favor of a second type of C(3)-symmetric structure, presumably the result of the formation of a stable peptide-metal ion complex. Nuclear magnetic resonance data suggest that the peptide in methylene chloride solution takes up a conformation containing three cis' Pro C(alpha)-C=O bonds and three cis Gly-Pro peptide bonds; that water and dimethylsulfoxide stabilize a conformer in which one (or two) sets of such bonds of a given Pro-Gly unit have undergone interconversion to trans'/trans forms; and that alkali metal cations complex the cyclic peptide in a C(3)-symmetric all-trans'/trans structure.

An investigation has been made of the optical rotatory dispersion in the region 226 to 366 mmu of tobacco mosaic virus (TMV), the protein subunits isolated therefrom, the rods synthesized from the protein subunits, and the ribonucleic acid (RNA) isolated from TMV. Both TMV and the protein rods show anomalous rotatory dispersion. The RNA shows a Cotton effect with an inflection point around 260 mmu, which is shifted to 272 mmu in concentrated urea solution. A suggested interpretation of the RNA rotatory dispersion is given. The rotatory dispersion of the protein subunits shows an incipient Cotton effect with an inflection point around 293 mmu and the beginnings of a large negative Cotton effect with a trough at 232 mmu. The dispersion data from the protein subunits can be interpreted to indicate that they contain between 25 and 35 per cent alpha-helix. On the basis of recent sequence investigations and the relationship between amino acid composition and polypeptide structure, the helical portion of the protein subunits can be located in the central section of the protein chain.