Solution structure of the 45-residue MgATP-binding peptide of adenylate kinase as examined by 2-D NMR, FTIR, and CD spectroscopy.

The structure of a synthetic peptide corresponding to residues 1-45 of rabbit muscle adenylate kinase has been studied in aqueous solution by two-dimensional NMR, FTIR, and CD spectroscopy. This peptide, which binds MgATP and is believed to represent most of the MgATP-binding site of the enzyme [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694], appears to maintain a conformation similar to that of residues 1-45 in the X-ray structure of intact porcine adenylate kinase [Sachsenheimer, W., & Schulz, G.E. (1977) J. Mol. Biol. 114, 23-26], with 42% of the residues of the peptide showing NOEs indicative of phi and psi angles corresponding to those found in the protein. The NMR studies suggest that the peptide is composed of two helical regions of residues 4-7 and 23-29, and three stretches of beta-strand at residues 8-15, 30-32, and 35-40, yielding an overall secondary structure consisting of 24% alpha-helix, 38% beta-structure, and 38% aperiodic. Although the resolution-enhanced amide I band of the peptide FTIR spectrum is broad and rather featureless, possibly due to disorder, it can be fit by using methods developed on well-characterized globular proteins. On this basis, the peptide consists of 35 +/- 10% beta-structure, 60 +/- 12% turns and aperiodic structure, and not more than 10% alpha-helix. The CD spectrum is best fit by assuming the presence of at most 13% alpha-helix in the peptide, 24 +/- 2% beta-structure, and 66 +/- 4% aperiodic. The inability of the high-frequency FTIR and CD methods to detect helices in the amount found by NMR may result from the short helical lengths as well as from static and dynamic disorder in the peptide. Upon binding of MgATP, numerous conformational changes in the backbone of the peptide are detected by NMR, with smaller alterations in the overall secondary structure as assessed by CD. Detailed assignments of resonances in the peptide spectrum and intermolecular NOEs between protons of bound MgATP and those of the peptide, as well as chemical shifts of peptide resonances induced by the binding of MgATP, are consistent with the previously proposed binding site for MgATP on adenylate kinase.

Fourier transform infrared study of proteins with parallel beta-chains.

Deconvolved and second derivative Fourier transform infrared spectra of the proteins flavodoxin and triosephosphate isomerase have been obtained in the 1600 to 1700 cm-1 (amide I) region. To our knowledge these results provide the first experimental infrared data on proteins with parallel beta-chains. Characteristic absorption bands for the parallel beta-segments are observed at 1626-1639 cm-1 (strong) and close to 1675 cm-1 (weak). Previous theoretical studies based on hypothetical models with large, regular beta-sheets had suggested bands close to 1650 and 1666 cm-1. Our new assignments were confirmed by band area measurements, which yield conformational information in good agreement with results from X-ray diffraction data. The spectra were compared with corresponding spectra of concanavalin A and carboxypeptidase A. The first contains only antiparallel beta-segments, the second "mixed" beta-segments, with some strands lying antiparallel and others parallel. None of the observed amide I band frequencies assigned to parallel beta-chains occurs in the 1650 cm-1 region associated with helical segments.

Estimation of beta-structure content of proteins by means of deconvolved FTIR spectra.

Fourier self-deconvolution was applied to the infrared spectra of five globular proteins with a high beta-structure content and to the essentially alpha-helical protein hemoglobin. The featureless amide I' bands around 1650 cm-1 were thereby resolved into six to nine components, depending on the protein. Specific components were assigned to the beta-structure segments in each protein. The frequencies and the number of 'beta-bands' differ from one protein to another. The areas of the components were evaluated by means of a Gauss-Newton iteration procedure. It appears that the total area of the beta-bands, as a fraction of the total amide I' band area, reflects the relative beta-structure content of each protein studied.

Solvent denaturation of proteins as observed by resolution-enhanced Fourier transform infrared spectroscopy.

Fourier self-deconvolution of Fourier transform infrared (FTIR) spectra and second derivative FTIR spectroscopy were applied to study solvent-induced conformational changes in globular proteins. For beta-lactoglobulin a total of three different denatured forms were identified in alkaline solution and in aqueous methanol-d1 and isopropanol-d1. In isopropanol-d1 solution a new conformation was identified which appears to resemble, but is not identical with, the beta-structure of native proteins. This conformation is characterized by absorption bands around 1615-1618 and 1684-1688 cm-1, and is also observed for concanavalin A and chymotrypsinogen A in aqueous isopropanol-d1 solution.

Protein structure by Fourier transform infrared spectroscopy: second derivative spectra.

Second derivative Fourier transform infrared spectra of the proteins ribonuclease A, hemoglobin, and beta-lactoglobulin A (native and denatured) have been obtained in deuterium oxide solution from 1350 to 1800 cm-1. The relationship of the original spectra to their second derivatives is briefly discussed. In the second derivative spectra, clearly resolved peaks are observed which can be associated with the alpha-helix, beta-strands, and turns. No protein spectra with such resolution have heretofore been reported. Tentative assignments are proposed, and the observed peaks are related to the secondary structure of the proteins studied. The data appear to present the first direct spectroscopic evidence of turns in a native protein.

Laser-Raman investigation of phospholipid-polypeptide interactions in model membranes.

The interaction of aqueous dimyristoyl-phosphatidylcholine liposomes with the polypeptides gramicidin A, poly-L-lysine, valinomycin, and gramicidin S was investigated by means of laser-Raman spectroscopy. Auxiliary data were obtained with differential scanning calorimetry. Studies were carried out over the temperature range of 0--50 degrees C, encompassing the gel phase, the transition region, and the liquid crystalline phase of the liposomes. Conformational changes in the phospholipid molecules were investigated by measuring the intensity of the 1062-cm-1 Raman band which is assigned to C-C stretching vibrations of trans segments. Three different types of phospholipid-polypeptide interactions were indicated by the observed Raman data. They are interpreted as (a) orderly penetration of the phospholipid bilayer by a hydrophobic polypeptide; (b) polar interactions involving primarily the head groups of the phospholipid; and (c) disorderly hydrophobic binding between a polypeptide and the hydrocarbon domain of the phospholipid.