Temperature dependence of near-IR excited Raman spectra of crystalline hen egg-white lysozyme.

Near-IR excited Raman spectroscopy was applied to examine the structural change of hen egg-white lysozyme in tetragonal crystals at low temperatures. There was little difference found in the amide I and amide III regions between the spectra observed at 77 and 298 K, suggesting that the secondary structures of lysozyme were conserved in the temperature range from 77 to 298 K. Several bands arising from the protein side chains, particularly the methylene and phenylalanyl groups, showed significant changes in either intensity or bandwidth (or in both of them) on going from 77 to 298 K. Some of the spectral changes occurred gradually over the wide temperature range, and others occurred abruptly at around 200-240 K. The implications of these findings are discussed.

Solution structure of midkine, a new heparin-binding growth factor.

Midkine (MK) is a 13 kDa heparin-binding polypeptide which enhances neurite outgrowth, neuronal cell survival and plasminogen activator activity. MK is structurally divided into two domains, and most of the biological activities are located on the C-terminal domain. The solution structures of the two domains were determined by NMR. Both domains consist of three antiparallel beta-strands, but the C-terminal domain has a long flexible hairpin loop where a heparin-binding consensus sequence is located. Basic residues on the beta-sheet of the C-terminal domain form another heparin-binding site. Measurement of NMR signals in the presence of a heparin oligosaccharides verified that multiple amino acids in the two sites participated in heparin binding. The MK dimer has been shown to be the active form, giving signals to endothelial cells and probably to neuronal cells. We present a head-to-head dimer model of MK. The model was supported by the results of cross-linking experiments using transglutaminase. The dimer has a fused heparin-binding site at the dimer interface of the C-terminal domain, and the heparin-binding sites on MK fit the sulfate group clusters on heparin. These features are consistent with the proposed stronger heparin-binding activity and biological activity of the dimer.

Infrared studies of interaction between metal ions and Ca(2+)-binding proteins. Marker bands for identifying the types of coordination of the side-chain COO- groups to metal ions in pike parvalbumin (pI = 4.10).

Metal-ligand interactions in the Ca(2+)-binding sites of pike parvalbumin (pI = 4.10) have been examined by Fourier-transform infrared spectroscopy. The region of the COO- antisymmetric stretch provides useful information on the types of coordination of the COO- groups to the metal ions in the Mg(2+)-, Mn(2+)-, and Ca(2+)-bound forms. In the spectrum of the Ca(2+)-bound form, two bands are observed at 1,582 and 1,553 cm-1, whereas, in the spectra of the Mg(2+)- and Mn(2+)-bound forms, bands are observed only in the region around 1,582 cm-1 and no band is found in the region around 1,553 cm-1. The 1,553-cm-1 band of the Ca(2+)-bound form reflects the bidentate coordination of the COO- groups of both Glu-62 in the CD site and Glu-101 in the EF site to the Ca2+ ions, which has been made clear by X-ray analysis as a feature of the Ca(2+)-bound form. Absence of such a band in the spectrum of the Mn(2+)-bound form is consistent with the X-ray structure of this form where both of the two COO- groups are unidentate. These unidentate COO- groups of Glu-62 and Glu-101 in the Mn(2+)-bound form seem to give rise to a band at 1,577-1,574 cm-1. The spectrum of the Mg(2+)-bound form is also consistent with the 'pseudo-bridging' coordination of the COO- group of Glu-101 reported in the X-ray structure of a form where the Mg2+ ion occupies only the EF site, and the same spectrum is further indicative of the 'pseudo-bridging' coordination of the COO- group of Glu-62.

Infrared studies of octopus rhodopsin. Existence of a long-lived intermediate and the states of the carboxylic group of Asp-81 in rhodopsin and its photoproducts.

The infrared absorption spectra of octopus rhodopsin and its photoproducts have been observed at 282K and 210K under irradiation of blue and orange light in a neutral condition. The acid metarhodopsin-minus-rhodopsin and lumirhodopsin-minus-rhodopsin difference spectra have been obtained. A new intermediate (called transient acid metarhodopsin) with a lifetime of about 5 s has been found to exist prior to acid metarhodopsin. The present results, together with the data obtained previously, give information on the state of the carboxylic group in the side chain of Asp-81, which is the only acidic amino-acid residue in the part of opsin buried inside the membrane. This carboxylic group is protonated throughout the transformation series, but its state changes on going from transient acid metarhodopsin to acid metarhodopsin. It is probable that these two photoproducts are different from each other only in the opsin moiety.

Conformational adaptability of the terminal regions of flagellin.

Secondary structure formation in the disordered terminal regions of flagellin were studied by circular dichroic (CD) spectroscopy, Fourier transform infrared spectroscopy, and x-ray diffraction. The terminal regions of flagellin are known to form alpha-helical bundles upon polymerization into flagellar filaments. We found from comparative CD studies of flagellin and its F40 tryptic fragment that a highly alpha-helical conformation can be induced and stabilized in the terminal regions in 2,2,2-trifluoroethanol (TFE) containing solutions, which is known to promote intra-molecular hydrogen bonding. Two oligopeptides, N(37-61) and C(470-494), each corresponding to a portion of terminal regions and predicted to have a high alpha-helix forming potential, were synthesized and studied. Both peptides were disordered in an aqueous environment, but they showed a strong tendency to assume alpha-helical structure in solutions containing TFE. On the other hand, peptides were found to form transparent gels at high concentrations (> 15 mg/ml) and all three methods confirmed that the peptides become ordered into a predominantly beta structure upon gel formation. Our results show that large segments of the disordered terminal regions of flagellin can adopt alpha-helical as well as beta structure depending on the environmental conditions. This high degree of conformational adaptability may be reflecting some unique characteristics of the flagellin termini, which are involved in self-assembly and polymorphism of flagellar filament.

In vivo states and functions of carotenoids in an aerobic photosynthetic bacterium, Erythrobacter longus.

In vivo states and functions of carotenoids in the membranes and the isolated RC-B865 pigment-protein complexes from an aerobic photosynthetic bacterium, Erythrobacter longus, are investigated by means of fluorescence excitation and resonance Raman (RR) spectra. Erythroxanthin sulfate, a dominant carotenoid species in the membranes (>70%), is found not to transfer the absorbed light energy to bacteriochlorophyll (Bchl), and its RR spectra are similar between the in vivo and in vitro states. These observations indicate that erythroxanthin sulfate does not interact with either Bchl or proteins in the membranes, and suggest that its function may be limited to photoprotection by quenching the harmful singlet oxygen. On the other hand, two other carotenoid species contained in the isolated RC-B865 complexes, zeaxanthin and bacteriorubixanthinal, have a high efficiency of energy transfer to Bchl (88±5%). The RR spectra of these two carotenoids, each of which can be selectively obtained by choosing the excitation wavelength, show some characteristics of interactions with proteins or Bchl.

Vibrational spectroscopy of excited electronic states in carotenoids in vivo. Picosecond time-resolved resonance Raman scattering.

The vibrational spectroscopy and population dynamics of excited singlet (2(1)Ag), excited triplet (3B u), and the ground (1Ag) electronic states of carotenoids in chromatophores of Chromatium vinosum (mainly spirilloxanthin and rhodopin) and of the same carotenoids in benzene solutions are examined by picosecond time-resolved resonance Raman scattering. Coherent Stokes Raman scattering from the ground states of carotenoids in chromatophores also is observed. Resonance Raman spectra of in vitro rhodopin and spirilloxanthin when compared with in vivo data demonstrate that scattering from spirilloxanthin dominates the in vivo spectrum. Comparisons of the time-dependent intensities of 2(1)Ag and 1Ag resonance Raman bands from both in vitro and in vivo carotenoids suggest that vibrationally excited levels in 1Ag are populated directly by the decay of the 2(1)Ag state and that these levels relax into a thermalized distribution in less than 50 ps. The appearance of asymmetrically broadened, ground-state resonance Raman bands supports this conclusion. Formation of the 3Bu state is observed for carotenoids in chromatophores, but not for in vitro spirilloxanthin indicating that the 3Bu state is formed by fission processes originating from the spatial organization of pigments within chromatophores. The rate at which the intensities of 2(1)Ag resonance Raman bands decay is faster for the carotenoids in vivo than for those in vitro thereby indicating that additional relaxation channels (e.g., energy transfer to bacteriochlorophylls) are present in the chromatophore. The similarity of the in vivo and in vitro 2(1)Ag resonance Raman spectra shows that no significant modifications in the vibronic coupling has been caused by the chromatophore environment.

Structural analyses of a channel-forming fragment of colicin E1 incorporated into lipid vesicles. Fourier-transform infrared and tryptophan fluorescence studies.

Structural changes upon binding to the membrane of a COOH-terminal channel-forming thermolytic fragment of colicin E1 have been studied by means of a variety of spectroscopic techniques. Circular dichroism measurements show that the thermolytic fragment predominantly takes a helical structure in aqueous and detergent solutions. Fourier transform infrared spectroscopic measurements indicate that the content of the beta-structure is significantly increased when the thermolytic fragment is bound to vesicles. On the basis of the result of tryptophan fluorescence measurements, we have concluded that each of the three tryptophan residues of the thermolytic fragment exists in different environments, i.e. one is buried in the lipid bilayer, one exists on the cis side of the vesicles, and one exists near the surface of the lipid bilayer. The Fourier transform infrared and fluorescence data have been used along with the crystal structure of colicin A, which is highly homologous to colicin E1 in structure and function, to propose a model of the thermolytic fragment bound to the lipid vesicles.

Resonance Raman studies on the structure of bacteriochlorophyll c in chlorosomes from Chloroflexus aurantiacus.

Resonance Raman spectra of chlorosomes isolated from the thermophilic green photosynthetic bacterium Chloroflexus aurantiacus have been obtained with several excitation wavelengths from 441.6 to 514.5 nm. Resonance Raman spectra of bacteriochlorophyll (BChl) c isolated from C. aurantiacus cells have also been observed. The C=C stretching frequencies of BChl c in the chlorosomes were found to be at 1,556 (strong) and 1,544 (shoulder) cm-1, which correspond to those expected for the 5-coordinated BChl c. The C-9 carbonyl resonance Raman frequency was found at 1,642 cm-1, indicating that this group is either hydrogen-bonded to an Mg-coordinated hydroxyl group or coordinated to the Mg ion.

Raman spectroscopic study of the interaction between sulfate anion and an imidazolium ring in ribonuclease A.

Raman spectra of ribonuclease A in D2O solution at various pD values have been studied with special attention to the N-deuterated imidazolium ring vibration at 1408 cm-1, the SO4(2-) symmetric stretching vibration at 984 cm-1, the amide I' band, and the tyrosine doublet. Concomitant decrease and increase in the intensities of the 1408- and 984-cm-1 bands in the pD range between 5 and 7 indicate that a sulfate anion is actually hydrogen bonded to an imidazolium ring of a histidine residue located in the interior of the molecule. The mechanism of the sulfate desorption has been compared with that on heat denaturation.

Nuclear magnetic relaxation study on the interaction of glycyl-L-tyrosine with manganese-carboxypeptidase A in solution.

The spin-lattice and spin-spin relaxation rates were measured of the Gly C alpha and Tyr aryl protons of glycyl-L-tyrosine (Gly-Tyr) bound to manganese(II)-substituted carboxypeptidase A (MnCPA) in aqueous solution. The temperature and frequency dependences of the relaxation rates were analyzed using the Solomon-Bloembergen-Morgan equations. The binding modes of MnCPA with Gly-Tyr in solution are different from that of ZnCPA in crystals. 1. Mn(II)-coordinated water of MnCPA is not excluded by the binding of Gly-Tyr substrate molecules. 2. The Gly carbonyl group does not coordinate tightly to the metal ion of MnCPA. The Gly C alpha protons of Gly-Tyr in the productive binding site are appreciably mobile. 3. A non-productive loose binding of another Gly-Tyr molecule is suggested by simulation of the temperature and frequency dependences of the proton relaxation rates.

Nuclear-magnetic-resonance study of aggregations and conformations of melanostatin and related peptides.

The 1H and 13C nuclear magnetic resonance spectra of melanostatin (Pro-Leu-Gly-NH2) and related peptides (Pro-Leu-Gly, Z-Pro-Leu-Gly, Z-Pro-Leu-Gly-NH2 and Z-Pro-Leu-Gly-OCH3, where Z = benzyloxycarbonyl) were analysed in a variety of solvents. At physiological pH, the melanostatin molecule is N-protonated in aqueous solution. The concentration dependences of the chemical shifts of amide-proton and carbonyl-carbon resonances and of proton spin-lattice relaxation times were observed in relation to molecular aggregations. In dimethylsulfoxide solution, aggregations were observed for N-protonated melanostatin and Pro-Leu-Gly prepared with HCl and for the Na salt of Z-Pro-Leu-Gly but not for N-protonated melanostatin prepared with HClO4 or HNO3, unprotonated melanostatin, Z-Pro-Leu-Gly-NH2, or Z-Pro-Leu-Gly-OCH3. The leucine NH and glycine CO groups of N-protonated melanostatin are involved in the intermolecular hydrogen bonds of aggregates. The leucine NH group of N-protonated Pro-Leu-Gly also forms the intermolecular hydrogen bond. The solvent and temperature dependences of the chemical shifts of amide-proton and carbonyl-carbon resonances were measured to determine intramolecular hydrogen bonding. In dimethylsulfoxide solution, N-protonated melanostatin molecules in part take the beta-turn structure and the trans carboxamide NH proton and carbonyl oxygen of the proline residue form an intramolecular hydrogen bond.

The Protein Data Bank. A computer-based archival file for macromolecular structures.

The Protein Data Bank is a computer-based archival file for macromolecular structures. The Bank stores in a uniform format atomic co-ordinates and partial bond connectivities, as derived from crystallographic studies. Text included in each data entry gives pertinent information for the structure at hand (e.g. species from which the molecule has been obtained, resolution of diffraction data, literature citations and specifications of secondary structure). In addition to atomic co-ordinates and connectivities, the Protein Data Bank stores structure factors and phases, although these latter data are not placed in any uniform format. Input of data to the Bank and general maintenance functions are carried out at Brookhaven National Laboratory. All data stored in the Bank are available on magnetic tape for public distribution, from Brookhaven (to laboratories in the Americas), Tokyo (Japan), and Cambridge (Europe and worldwide). A master file is maintained at Brookhaven and duplicate copies are stored in Cambridge and Tokyo. In the future, it is hoped to expand the scope of the Protein Data Bank to make available co-ordinates for standard structural types (e.g. alpha-helix, RNA double-stranded helix) and representative computer programs of utility in the study and interpretation of macromolecular structures.