ON THE TWO FORMS OF INTERMEDIATE M OF BACTERIORHODOPSIN

Abstract— The decay time course of intermediate M of bacteriorhodopsin was investigated by flash spectrophotometry. The decay was composed of two exponentials showing the existence of two forms of intermediate absorbing around 410 nm. The two were very different in kinetic character whereas the absorption spectra were almost the same. The relative yield of the two components was a function of the intensity of the exciting flash and the slower component disappeared when the flash intensity was made very small. A model based on the trimeric cluster structure of bacteriorhodopsin is proposed.     

The detection of red pigment-concentrating hormone (RPCH) in crustacean eyestalk tissues using matrix-assisted laser desorption/ionization-Fourier transform mass spectrometry: [M + Na]+ ion formation in dried droplet tissue preparations

Red pigment-concentrating hormone (RPCH), an octapeptide found in crustaceans and insects with the sequence pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2, is an N- and C-terminally blocked uncharged peptide. These structural features are shared with many members of the larger adipokinetic hormone (AKH)/RPCH peptide family in insects. We have applied vacuum UV matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron mass spectrometry (FTMS) to the direct analysis of crustacean sinus gland tissues, using 2,5-dihydroxybenzoic acid (DHB) as the MALDI matrix, and have found that RPCH is detected in the cationized, [M + Na]+, form under conditions where other peptides in the direct tissue spectra are protonated without accompanying [M + Na]+ or [M + K]+ satellite peaks. The [M + H]+ ion for RPCH is not detected in tissue samples or for an RPCH standard, even when care is taken to eliminate metal ions. This behavior is not unprecedented; however, both direct tissue spectra and SORI-CID spectra provide no clues to suggest that the ionizing agent is a metal cation. In this communication, we characterize the MALDI-FTMS ionization and SORI-CID mass spectra of the [M + Na]+ and [M + K]+ ions from RPCH, and report on the detection of this neuropeptide in sinus gland tissues from the lobster Homarus americanus and the kelp crab Pugettia producta. We describe two strategies, an on-probe extraction procedure and a salt-doping approach, that can be applied to previously analyzed MALDI tissue samples to enhance and unmask sodiated peptides that may otherwise be mistaken for novel neuropeptides.     

Characterization of fluorescence of ANS-tear lipocalin complex: evidence for multiple-binding modes

ANS is widely used as a probe for locating binding sites of proteins and studying structural changes under various external conditions. However, the nature of ANS-binding sites in proteins and the accompanying changes in fluorescence properties are controversial. We examined the steady-state and time-resolved fluorescence of the ANS-protein complexes for tear lipocalin (TL) and its mutants in order to discern the origin of lifetime components via analysis that included the multiexponential decay and the model-free maximum entropy methods. Fluorescence lifetimes of ANS-TL complexes can be grouped into two species, 14.01-17.42 ns and 2.72-4.37 ns. The log-normal analyses of fluorescence spectral shapes reveal the heterogeneous nature of both long- and short-lifetime species. The constructed time-resolved emission, amplitude (TRES) and area normalized (TRANES), and decay-associated spectra are consistent with a model that includes heterogeneous modes of ANS binding with two separate lifetime components. The two lifetime components are not derived from solvent relaxation, but rather may represent different binding modes.     

Effects of ionizing radiation on epoxy,graphite fiber,and epoxy/graphite fiber composites. Part II: Radical types and radical decay behavior

Electron spin resonance (ESR) investigations of line shapes and radical decay behavior have been made on an epoxy based on tetraglycidyl diaminodiphenyl methane (TGDDM)/diaminodiphenyl sulfone (DDS), T-300 graphite fiber, and T-300/5208 (graphite fiber/epoxy) composites after irradiation with Co₆₀ γ-radiation or 0.5 MeV electrons. Two kinetically distinct radical species are found in the irradiated epoxy as the temperature is raised beyond 120 K following irradiation of samples at 77 K with Co₆₀ γ. One has been termed a fast-decaying species and the other a slow-decaying species. The ratio of fast-decaying/slow-decaying radicals increases as the decay temperature rises. The fast-decaying radicals at room temperature are attributed largely to alkyl type radicals residing in regions of relatively low crosslink density, while the long-lived radicals are attributed to radicals residing in the highly crosslinked regions of the epoxy. A large concentration (ca. 10₂₀ to 10₂₁ spins/g) of unpaired electrons was found in unirradiated graphite fiber which masked the ESR spectral change in irradiated composites.     

A revised mechanism of thermal decay of arylnitroso oxides

The electronic spectra were measured and the unimolecular decay kinetics of the isomeric forms (cis and trans) of 4-methoxyphenylnitroso oxide in acetonitrile, benzene, and hexane was studied using flash photolysis. The cis form absorbed in a shorter wavelength region and was more labile than the trans form. The difference between the reactivity of the two species increased on going from hexane to acetonitrile. The temperature dependences of reaction rate constants were studied for both isomeric forms. The analysis of products of flash photolysis of 4-methoxyphenyl azide in the presence of oxygen allowed for understanding the mechanism of thermal decay of nitroso oxides. It was shown that the trans nitroso oxide is converted into cis nitroso oxide. The latter undergoes an unusual ring cleavage reaction to form 4-methoxy-6-oxohexa-2,4-dienenitrile N-oxide derivative. We conclude that the nitro- and nitrosobenzenes, which are the main products of the steady-state photolysis of aromatic azides in the presence of oxygen, are formed by the photochemical transformation of the nitroso oxides.     

Structural changes during the photocycle of photoactive yellow protein monitored by ultraviolet resonance raman spectra of tyrosine and tryptophan

Photoactive yellow protein (PYP) is a bacterial blue light photoreceptor, and photoexcitation of dark-state PYP (PYP(dark)) triggers a photocycle that involves several intermediate states. We report the ultraviolet resonance Raman spectra of PYP with 225-250 nm excitations and investigate protein structural changes accompanying the formation of the putative signaling state denoted PYP(M). The PYP(M)-PYP(dark) difference spectra show several features of tyrosine and tryptophan, indicating environmental changes for these amino acid residues. The tyrosine difference signals show small upshifts with intensity changes in Y8a and Y9a bands. Although there are five tyrosine residues in PYP, Tyr42 and Tyr118 are suggested to be responsible for the difference signals on the basis of a global fitting analysis of the difference spectra at different excitation wavelengths and the crystal structure of PYP(dark). A further experiment on the Thr50-->Val mutant supports environmental changes in Tyr42. The observed upshift of the Y8a band suggests a weaker or broken hydrogen bond between Tyr42 and the chromophore in PYP(M). In addition, a reorientation of the OH group in Tyr42 is suggested from the upshift of the Y9a band. For tryptophan, the Raman bands of W3, W16, and W18 modes diminish in intensity upon formation of PYP(M). The loss of intensities is attributable to an exposure of tryptophan in PYP(M). PYP contains only one tryptophan (Trp119) that is located more than 10 A from the active site. Thus the observed changes are indicative of global conformational changes in protein during the transition from PYP(dark) to PYP(M). These results are in line with the currently proposed photocycle mechanism of PYP.     

Origins of the Intermediate Spectral Form in M100 Mutants of Photoactive Yellow Protein

Numerous single‐site mutants of photoactive yellow protein (PYP) from Halorhodospira halophila and as well as PYP homologs from other species exhibit a shoulder on the short wavelength side of the absorbance maximum in their dark‐adapted states. The structural basis for the occurrence of this shoulder, called the “intermediate spectral form,” has only been investigated in detail for the Y42F mutation. Here we explore the structural basis for occurrence of the intermediate spectral form in a M121E derivative of a circularly permuted H. halophila PYP (M121E‐cPYP). The M121 site in M121E‐cPYP corresponds to the M100 site in wild‐type H. halophila PYP. High‐resolution NMR measurements with a salt‐tolerant cryoprobe enabled identification of those residues directly affected by increasing concentrations of ammonium chloride, a salt that greatly enhances the fraction of the intermediate spectra form. Residues in the surface loop containing the M121E (M100E) mutation were found to be affected by ammonium chloride as well as a discrete set of residues that link this surface loop to the buried hydroxyl group of the chromophore via a hydrogen bond network. Localized changes in the conformational dynamics of a surface loop can thereby produce structural rearrangements near the buried hydroxyl group chromophore while leaving the large majority of residues in the protein unaffected.     

The Photobiological Differences of Gilvocarcins V and M Are not Related to their Transient Intermediates and Triplet Yields

Abstract— The transient absorption spectra of the intermediates produced by the 355 nm laser excitation of gilvocarcin derivatives have been investigated in various solvents. The spectra consist of a triplet-triplet absorption in the visible region and a residual absorption observed between 340 and 700 nm due to a long-lived species, assigned to the radical cation. A broad-fast decaying band with a maximum at around 700 nm attributed to the solvated electron is also seen in solutions containing a low DMSO/water volume ratio and at 266 nm irradiation of a 50% methanol/water solvent mixture. The molar absorption coefficient of the triplet state of gilvocarcin V (GV) and gilvocarcin M (GM), determined by the energy transfer method, is independent of the solvent properties and has a value of 3.0 × 10⁴/ M cm. The triplet decay rate constants for both drugs are between 1 and 5 × 10⁴/s. A similar initial yield and triplet decay rate constant of GV were observed in the presence of 3.4 m M thymine. Thus, a quenching rate constant of the GV's triplet state by thymine is estimated to be lower than 10⁶/Ms. The triplet quantum yields of both antibiotics determined by using the comparative method are higher in dimethylsulfoxide (DMSO) (0.18) than are those corresponding to 25% DMSO/water (0.06). The decrease in φ_T in the presence of water could be attributed to an enhanced internal conversion rate constant from the S₁ state or to an increase in the photoionization yield. The similarity of the transient intermediates and their yields for GV and GM suggest that their photobiological differences are due to other factors such as DNA binding constants, preferential localization of the drugs in the cell or the enhanced reactivity of the vinyl group toward cellular components.     

A DETAILED RESONANCE RAMAN STUDY OF THE M412 INTERMEDIATE IN THE BACTERIORHODOPSIN PHOTOCYCLE

Abstract— Resonance Raman spectra of various M₄₁₂ species associated with the bacteriorhodopsin photocycle have been obtained. These correspond to the two forms observed during the formation of M₄1₂ and the two forms that are observed during its decay in absorption experimeents. We do not see any significant difference between the Raman spectra of any of these forms. We therefore conclude that the differences in these species are due to the differences in the protein structure and not in the chromophore.     

The Atomic Structure of Cu(II) Acetate-Bipyridine Under Thermal Decomposition: An X-Ray Spectroscopy Study

A number of experimental techniques were used to study the changes in the atomic structure of copper(II) acetate-bipyridine under thermal decomposition between 25°C and 700°C to form copper-containing nanoparticles. The dynamics of structural changes during decomposition in a thermogravimetric chamber is compared with X-ray absorption spectra, IR spectra, and diffraction patterns for a sequence of annealing temperatures. The experimental results were used to construct theoretical structural models of the complex under thermal decomposition.     

A 13C NMR spectroscopic study of the phase transitions of alkane dicarboxylic acids in the solid state

¹³C NMR spectra of a few odd-member alkane dicarboxylic acids such as glutaric and pimelic acids have been studied across their phase transitions. The spectra provide clear evidence for the structural changes accompanying the transitions. In the case of malonic acid, the study establishes that the two carboxyl units are equivalent in the high-temperature phase unlike in the other odd-member dicarboxylic acids where they are non-equivalent.     

The lifetimes of Pharaonis phoborhodopsin signaling states depend on the rates of proton transfers--effects of hydrostatic pressure and stopped flow experiments

Pharaonis phoborhodopsin (ppR), a negative phototaxis receptor of Natronomonas pharaonis, undergoes photocycle similar to the light-driven proton pump bacteriorhodopsin (BR), but the turnover rate is much slower due to much longer lifetimes of the M and O intermediates. The M decay was shown to become as fast as it is in BR in the L40T/F86D mutant. We examined the effects of hydrostatic pressure on the decay of these intermediates. For BR, pressure decelerated M decay but slightly affected O decay. In contrast, with ppR and with its L40T/F86D mutant, pressure slightly affected M decay but accelerated O decay. Clearly, the pressure-dependent factors for M and O decay are different in BR and ppR. In order to examine the deprotonation of Asp75 in unphotolyzed ppR we performed stopped flow experiments. The pH jump-induced deprotonation of Asp75 occurred with 60 ms, which is at least 20 times slower than deprotonation of the equivalent Asp85 in BR and about 10-fold faster than the O decay of ppR. These data suggest that proton transfer is slowed not only in the cytoplasmic channel but also in the extracellular channel of ppR and that the light-induced structural changes in the O intermediate of ppR additionally decrease this rate.     

Compositional dependence of negative thermal expansion in the Prussian Blue analogues M(II)Pt(IV)(CN)6 (M = Mn, Fe, Co, Ni, Cu, Zn, Cd)

The effect of M(II) substitution on the magnitude of the negative thermal expansion (NTE) behavior within a series of Prussian Blue analogues, M(II)Pt(IV)(CN)(6) for M(II) = Mn, Fe, Co, Ni, Cu, Zn, Cd, has been investigated using variable-temperature powder X-ray diffraction (100-400 K). The NTE behavior varies widely with M(II) substitution, from near zero thermal expansion in NiPt(CN)(6) (alpha = dl/l dT = -1.02(11) x 10(-)(6) K(-)(1)) up to a maximum in CdPt(CN)(6) (alpha = -10.02(11) x 10(-)(6) K(-)(1)). The trend in the magnitude of the NTE behavior, with increasing atomic number (Z) of the M(II) ion, follows the order Mn(II) > Fe(II) > Co(II) > Ni(II) < Cu(II) < Zn(II) < Cd(II), which correlates with the trends for M(II) cation size, the lattice parameter, and structural flexibility as indicated by the temperature-dependent structural refinements and Raman spectroscopy. Analysis of the temperature dependence of the average structures suggests that the differences in the thermal expansion are due principally to the different strengths of the metal-cyanide binding interaction and, accordingly, the different energies of transverse vibration of the cyanide bridge, with enhanced NTE behavior for more flexible lattices.     

Solid-state carbon-13 NMR Studies of vulcanized elastomers. V. Observation of new structures in sulfur-vulcanized natural rubber

The complementary application of the NMR inversion recovery measurements and the computer fitting of the overlapping spectral region is found to be a useful method for structural analysis of vulcanized natural rubber in the solid state. Since the linewidths in ¹³C-NMR spectra of solids are relatively broad compared with the differences between chemical shifts, some weak signals are completely obscured in the resulting spectra. If the resonances have sufficiently different relaxation times, such as methyl and methylene carbons, it is possible to detect neighboring heavily overlapped signals by using the inversion recovery delay τ value at which the interfering strong resonance has null intensity. The 20 resonances observed in the spectra of crosslinked rubbers are tentatively assigned to the structural units formed during the vulcanization process. It is found that vulcanizates containing smaller amounts of sulfur (1 and 3%) show insignificant changes in the NMR spectra for curing times of 30 and 90 min. Structural modifications in rubbers cured with 10% sulfur continuously increase with the increasing curing time up to 120 min, indicating a significant loss in double bonds in the later stages of the reaction.     

Solvent effects on the vibrational activity and photodynamics of the green fluorescent protein chromophore: a quantum-chemical study

Vibrational activities in the Raman and resonance Raman spectra of the cationic, neutral, and anionic forms of 4'-hydroxybenzylidene-2,3-dimethyl-imidazolinone, a model compound for the green fluorescent protein chromophore, have been obtained from quantum-chemical calculations in vacuo and with the inclusion of solvent effects through the polarizable continuum model. It is found that inclusion of solvent effects improves slightly the agreement with experimental data for the cationic and neutral forms, whose spectra are qualitatively well-described already by calculations in vacuo. In contrast, inclusion of solvent effects is crucial to reproduce correctly the activities of the anionic form. The structural effects of solvation are remarkable both in the ground and in the lowest excited state of the anionic chromophore and influence not only the vibrational activity but also the photodynamics of the lowest excited state. CASPT2//CASSCF photoreaction paths, computed by including solvent effects at the CASSCF level, indicate a facile torsional deformation around both exocyclic CC bonds. Rotation around the exocyclic CC double bond is shown to lead to a favored radiationless decay channel, more efficient than that in gas phase, and which explains the ultrafast fluorescence decay and ground-state recovery observed in solution. Conversely, rotation around the exocyclic CC single bond accounts for the bottleneck observed in the ground-state recovery cycle. It is also speculated that the ultrafast radiationless decay channel would be hampered in protein for unfavorable electrostatic interactions and steric reasons.     

EFFECT OF MELITTIN ON PHOTOCYCLE AND PHOTOPOTENTIAL OF PURPLE MEMBRANE: SITES OF INTERACTION BETWEEN BACTERIORHODOPSIN AND MELITTIN

Abstract— Purple membrane (PM) suspension and artificial bilayer lipid membranes (BLM) containing PM sheets were treated with melittin. Both the decaying of the photocycle intermediate M412 and proton translocation were inhibited by melittin: The yields and rate of the slow-decaying component of M412 (M412s) together with the proton release and its uptake rate were significantly decreased, but the rate of the fast-decaying component of M412 (M4120 had only slight changes. Relatively high concentrations of melittin could cause aggregation in PM suspensions. Addition of melittin to a BLM solution increased the continuous photopotential signal but decreased the transient signal. We suggest that there might exist strong interactions between melittin and bacteriorhodopsin in addition to the melittin–lipid action. On the other hand, the results also indicate that proton translocation was more likely to be coupled with M412s and both were more sensitive to the changes caused by the melittin–PM interaction than was M412f.     

Synthesis and Structure of Bismuth(III)-Containing Noncentrosymmetric Phosphates, Cs(3)KBi(2)M(4)(PO(4))(6)Cl (M = Mn, Fe). Monoclinic (Cc) and Tetragonal (P4(3)) Polymorphs Templated by Chlorine-Centered Cl(Bi(2)Cs) Acentric Units

Single crystals of three new noncentrosymmetric (NCS) phosphates, α (1) and β (2) forms of Cs(3)KBi(2)Mn(4)(PO(4))(6)Cl and α-Cs(3)KBi(2)Fe(4)(PO(4))(6)Cl (3), were grown in a reactive CsCl/KCl molten-salt media. Their structures were determined by single-crystal X-ray diffraction methods showing that the α form crystallizes in the space group Cc (No. 9), which is in one of the 10 NCS polar crystal classes, m (2/m) while the β form crystallizes in P4(3) (No. 78) of another polar class, 4 (4/m). The unit cell parameters of the α form can be approximately correlated with that of the β form via the 3 × 3 orientation matrix [0.5, 0.5, 0; -0.5, 0.5, 0; 0, 0, 2 sin β]. The structures of these otherwise complicated phosphates exhibit two types of channels with circular and elliptical windows where the Cl-centered Cl(Bi(2)Cs) acentric unit is located. The neighboring acentric units are arranged in a parallel fashion in the α form, resulting in the monoclinic (Cc) lattice, but "antiparallel" in the β form, thus giving the tetragonal (P4(3)) unit cell. 1-3 feature the compatible M-O-P unit that contains four crystallographically independent MO(x) (x = 4, 5) polyhedra, which are connected to the Cl(Bi(2)Cs) acentric unit through one short and one long M(II)···Cl bond. The compositions of 1 and 2 consist of three Mn(2+) (d(5)) and one Mn(3+) (d(4)) per formula unit and that of 3 has three Fe(2+) (d(6)) and one Fe(3+) (d(5)). Bond valence sums reveal that, in the α phase, the trivalent site adopts distorted tetrahedral M(1)(3+)O(4) coordination and, in the β phase, distorted trigonal-bipyramidal M(4)(3+)O(5). Thus far, the iron phase has only been isolated in the α form presumably because of little extra stabilization energy gain if the Fe(2+) d(6) ion were to occupy the M(1)O(4) site. The possible origins pertaining to the structural differences in the α and β forms are discussed.     

Exploring the correlation between network structure and electron binding energy in the (H(2)O)(7)(-) cluster through isomer-photoselected vibrational predissociation spectroscopy and ab initio calculations: addressing complexity beyond types I-III

We report a combined photoelectron and vibrational spectroscopy study of the (H(2)O)(7)(-) cluster anions in order to correlate structural changes with the observed differences in electron binding energies of the various isomers. Photoelectron spectra of the (H(2)O)(7)(-) . Ar(m) clusters are obtained over the range of m=0-10. These spectra reveal the formation of a new isomer (I') for m>5, the electron binding energy of which is about 0.15 eV higher than that of the type I form previously reported to be the highest binding energy species [Coe et al., J. Chem. Phys. 92, 3980 (1990)]. Isomer-selective vibrational predissociation spectra are obtained using both the Ar dependence of the isomer distribution and photochemical depopulation of the more weakly (electron) binding isomers. The likely structures of the isomers at play are identified with the aid of electronic structure calculations, and the electron binding energies, as well as harmonic vibrational spectra, are calculated for 28 low-lying forms for comparison with the experimental results. The HOH bending spectrum of the low binding type II form is dominated by a band that is moderately redshifted relative to the bending origin of the bare water molecule. Calculations trace this feature primarily to the bending vibration localized on a water molecule in which a dangling H atom points toward the electron cloud. Both higher binding forms (I and I') display the characteristic patterns in the bending and OH stretching regions signaling electron attachment primarily to a water molecule in an AA binding site, a persistent motif found in non-isomer-selective spectra of the clusters up to (H(2)O)(50)(-).     

Polarized IR spectra of the hydrogen bond in two different oxindole polymorphs with cyclic dimers in their lattices

This article focuses on the problem of remarkably strong changes in the fine structure patterns of the ν(N-H) and ν(N-D) bands attributed to the hydrogen and deuterium bonds accompanying the phase transition, which occurs between two polymorphic forms of oxindole. The lattices of these two different crystals contain hydrogen-bonded cyclic dimers differ in their geometry parameters. The source of these differences in the polymorph spectral properties results from the geometry relations concerning the dimers constituting the lattice structural units. In the case of the "alpha" phase, the hydrogen bond lengths of the dimers differ by 0.18 ?. This leads to the "off-resonance exciton coupling" weakly involving the dimer hydrogen bonds. For the "beta" phase, with practically symmetric dimers in the lattice, the spectra become typical for centrosymmetric hydrogen bond systems due to the full resonance of the proton or deuteron vibrations.     

Photoisomerization of azobenzene derivatives confined in gold nanoparticle aggregates

Photoisomerization is an important reaction that confers photoresponsive functionality on nanoparticles. Although photoisomerization of molecules forming self-assembled monolayers on two-dimensional surfaces or three-dimensional clusters has been studied, a detailed picture of interactions of molecules undergoing isomerization with nanoparticles is not available. In this paper, we report on the photoisomerization of azobenzene derivatives spatially confined in gold nanoparticle (AuNP) aggregates. AuNP aggregates allow us to simultaneously probe the structural changes of molecules via surface-enhanced Raman spectroscopy (SERS) and the accompanying changes in interparticle interactions via surface plasmon couplings. AuNP aggregates are formed by the adsorption of synthesized azobenzene-derivatized sulfides (Az) onto the surfaces of AuNPs. The photoisomerization of the adsorbed Az from trans to cis by excitation at 365 nm causes the AuNPs to move close to each other in the aggregates, leading to a redshift of the surface plasmon coupling band in the UV-vis spectra and a concomitant rise in SERS intensity. SERS spectra reveal that the vibrational modes containing the N=N stretching character redshift upon irradiation, suggesting that the N=N bond is significantly weakened when Az is in the cis form in the AuNP aggregates. The weakening of the N=N bond is attributed to the interaction of the N=N bond, which is more exposed to the outside in the cis conformation, with the nearby AuNPs that have come closer by the isomerization of adsorbed Az. We find that backisomerization from cis to trans occurs much faster in the AuNP aggregates (k = 1.9 × 10(-2) min(-1)) than in solution (k = 1.3 × 10(-3) min(-1)) because of the reduced N=N bond order of cis-Az in the aggregates.