Mid-infrared study of deoxyadenosine at high pressures: evidence of phase transitions

Room temperature mid-infrared experiments between 500 and 1800 cm(-1) have been performed on crystalline deoxyadenosine as a function of pressure up to about 10 GPa. Discontinuities observed near 2 and 4 GPa indicate that two separate phase transitions occur at these pressures. Changes in the spectra suggest that both transitions involve a rearrangement of the pucker of the deoxyribose moiety. The wavenumbers of the vibrational modes shift to higher values with applied pressure. Our results for deoxyadenosine are compared to similar measurements on adenosine. Assignments for the observed modes are made on the basis of work published in the literature.     

Raman and infrared studies of nucleosides at high pressures: I. Adenosine.

Room temperature Raman and infrared (IR) spectra of crystalline adenosine at pressures between 1 atm and 10 GPa are reported. Vibrational modes were identified through assignments in the literature. Many modes were found to increase in frequency with pressure; however, some irregularities were observed. Discontinuities were observed in numerous Raman and IR modes near 2.5 GPa, indicating a phase transition. The modes associated with the glycosidic bond shift significantly down in frequency near this pressure, suggesting a weakening of the associated bond. The IR modes associated with hydrogen-stretching motions were found to decrease in frequency with pressure.     

Raman and Infrared Studies of Nucleosides at High Pressures: I. Adenosine

Abstract

Room temperature Raman and infrared (IR) spectra of crystalline adenosine at pressures between 1 atm and 10 GPa are reported. Vibrational modes were identified through assignments in the literature. Many modes were found to increase in frequency with pressure; however, some irregularities were observed. Discontinuities were observed in numerous Raman and IR modes near 2.5 GPa, indicating a phase transition. The modes associated with the glycosidic bond shift significantly down in frequency near this pressure, suggesting a weakening of the associated bond. The IR modes associated with hydrogen-stretching motions were found to decrease in frequency with pressure.     

Observation of terahertz vibrations in Pyrococcus furiosus rubredoxin via impulsive coherent vibrational spectroscopy and nuclear resonance vibrational spectroscopy--interpretation by molecular mechanics

We have used impulsive coherent vibrational spectroscopy (ICVS) to study the Fe(S-Cys)(4) site in oxidized rubredoxin (Rd) from Pyrococcus furiosus (Pf). In this experiment, a 15 fs visible laser pulse is used to coherently pump the sample to an excited electronic state, and a second <10 fs pulse is used to probe the change in transmission as a function of the time delay. PfRd was observed to relax to the ground state by a single exponential decay with time constants of approximately 255-275 fs. Superimposed on this relaxation are oscillations caused by coherent excitation of vibrational modes in both excited and ground electronic states. Fourier transformation reveals the frequencies of these modes. The strongest ICV mode with 570 nm excitation is the symmetric Fe-S stretching mode near 310 cm(-1), compared to 313 cm(-1) in the low temperature resonance Raman. If the rubredoxin is pumped at 520 nm, a set of strong bands occurs between 20 and 110 cm(-1). Finally, there is a mode at approximately 500 cm(-1) which is similar to features near 508 cm(-1) in blue Cu proteins that have been attributed to excited state vibrations. Normal mode analysis using 488 protein atoms and 558 waters gave calculated spectra that are in good agreement with previous nuclear resonance vibrational spectra (NRVS) results. The lowest frequency normal modes are identified as collective motions of the entire protein or large segments of polypeptide. Motion in these modes may affect the polar environment of the redox site and thus tune the electron transfer functions in rubredoxins.     

Mid-Infrared Study of Deoxyadenosine at High Pressures: Evidence of Phase Transitions

Abstract

Room temperature mid-infrared experiments between 500 and 1800 cm^?1 have been performed on crystalline deoxyadenosine as a function of pressure up to about 10 GPa. Discontinuities observed near 2 and 4 GPa indicate that two separate phase transitions occur at these pressures. Changes in the spectra suggest that both transitions involve a rearrangement of the pucker of the deoxyribose moiety. The wavenumbers of the vibrational modes shift to higher values with applied pressure. Our results for deoxyadenosine are compared to similar measurements on adenosine. Assignments for the observed modes are made on the basis of work published in the literature.     

Direct infrared detection of the covalently ring linked His-Tyr structure in the active site of the heme-copper oxidases

Infrared spectroscopy, isotopic labeling ([(15)N(delta,epsilon)]histidine and ring-deuterated tyrosine), synthetic model studies, and normal mode calculations are employed to search for the spectroscopic signatures of the unique, covalently linked (His N(epsilon)-C(epsilon) Tyr) biring structure in the heme-copper oxidases. The specific enzyme examined is the cytochrome bo(3) quinol oxidase of E. coli. Infrared features of histidine and tyrosine are identified in the frequency regions of imidazole and phenol ring stretching modes (1350-1650 cm(-1)) and C-H and N-H stretching modes as well as overtones and combinations (>3000 cm(-1)). Two of these, at ca. 1480 and 1550 cm(-1), and their combination tones between 3010 and 3040 cm(-1), are definitively identified with the biring structure involving H284 and Y288 in the E. coli enzyme. Studies of a synthetic analogue of the H-Y structure, 4-methylimidazole covalently linked to p-cresol, show that a feature near 1540 cm(-1) is unique to the biring structure and is absent from the infrared spectrum of 4-methylimidazole or p-cresol alone. This feature is readily detectable by infrared difference techniques, and offers a direct spectroscopic probe for potential radical production involving the H-Y structure in the O(2) reduction cycle of the oxidases.     

Raman and infrared studies of nucleosides at high pressures: II. Cytidine

Raman and mid-infrared (MIR) spectra have been recorded for crystalline cytidine at pressures up to 10 GPa at room temperature. Broadening and positive wavenumber shifts are observed for most of the Raman and MIR peaks with increasing pressure. However, some of the MIR peaks associated with hydrogen-stretching modes display a negative wavenumber shift as a result of charge transfer effects. Evidence of a phase transition near 4 GPa is presented and attributed to a change in the conformation of the five membered sugar ring.     

Probing inhibitors binding to human urokinase crystals by Raman microscopy: implications for compound screening

Inhibition of urokinase activity represents a promising target for antimetastatic therapy for several types of tumor. The present study sets out to investigate the potential of Raman spectroscopy for defining the molecular details of inhibitor binding to this enzyme, with emphasis on single crystal studies. It is demonstrated that high quality Raman spectra from a series of five inhibitors bound individually to the active site of human urokinase can be obtained in situ from urokinase single crystals in hanging drops by using a Raman microscope. After recording the spectrum of the free crystal, a solution of inhibitor containing an amidine functional group on a naphthalene ring was added, and the spectrum of the crystal-inhibitor complex was obtained. The resulting difference Raman spectrum contained only vibrational modes due to bound inhibitor, originating from the protonated group, i.e., the amidinium moiety, as well as naphthalene ring modes and features from other functionalities that made up each inhibitor. The identification of the amidinium modes was placed on a quantitative basis by experimental and theoretical work on naphthamidine compounds. For the protonated group, -C-(NH2)(2)(+), the symmetric stretch occurs near 1520 cm(-1), and a less intense antisymmetric mode appears in the Raman spectra near 1680 cm(-1). The presence of vibrational modes near 1520 cm(-1) in each of the Raman difference spectra of the five complexes examined unambiguously identifies the protonated form of the amidinium group in the active site. Several advantages were found for single crystal experiments over solution studies of inhibitor-enzyme complexes, and these are discussed. The use of single crystals permits competitive binding experiments that cannot be undertaken in solution in any kind of homogeneous assay format. The Raman difference spectrum for a single crystal that had been exposed to equimolar amounts of all five inhibitors in the hanging drop showed only the Raman signature of the compound with the lowest K(i). These findings suggest that the Raman approach may offer a route in the screening of compounds in drug design applications as well as an adjunct to crystallographic analysis.     

Elastic modulus and stress-transfer properties of tunicate cellulose whiskers

Experimental deformation micromechanics of natural cellulose fibers using Raman spectroscopy and X-ray diffraction have been widely reported. However, little has been published on the direct measurements of the mechanical properties, and in particular the elastic modulus, of the highly crystalline material in the native state. Here we report on measurements of the elastic modulus of tunicate cellulose using a Raman spectroscopic technique. A dispersed sample of the material is deformed using a four-point bending test, and a shift in a characteristic Raman band (located at 1095 cm(-1)) is used as an indication of the stress in the material. Relatively little intensity change of the Raman band located at 1095 cm(-1) is shown to occur for samples oriented parallel and perpendicular to the polarization direction of the laser, as compared to a highly oriented flax sample. This indicates that the tunicate sample is a two-dimensional in-plane random network of fibers. By use of this result, the Raman shift, and calibrations with strain from other materials, it is shown that the modulus of the material is very high, at about 143 GPa, and a lack of Raman band broadening is thought to be due to the fact that there is pure crystalline deformation occurring without the effect of crystalline/amorphous fractions. A strain sensitivity of the shift in the 1095-cm(-1) Raman peak for this specimen is shown to be -2.4 +/- 0.2 cm(-1)/%. A molecular mechanics approach, using computer simulation and an empirical force field, was used to predict the modulus of a highly oriented chain of the material, and this is found to be 145 GPa, which is in agreement with the experimental data. However, by use of a normal-mode analysis, it is found that a number of modes have positions close to the central positions of the experimental Raman band. One in particular is found to shift at a rate of 2.5 cm(-1)/%, but due to the complex nature of the structure, it is not entirely conclusive that this band is representative of the experimental findings.     

Redox-triggered FTIR difference spectra of FAD in aqueous solution and bound to flavoproteins

Flavin adenine dinucleotide (FAD) and three different flavoproteins in aqueous solution were subjected to redox-triggered Fourier transform infrared difference spectroscopy. The acquired vibrational spectra show a great number of positive and negative peaks, pertaining to the oxidized and reduced state of the molecule, respectively. Density functional theory calculations on the B3LYP/6-31G(d) level were employed to assign several of the observed bands to vibrational modes of the isoalloxazine moiety of the flavin cofactor in both its oxidized and, for the first time, its reduced state. Prominent modes measured for oxidized FAD include nu(C(4)=O) and nu(C(2)=O) at 1716 and 1674 cm(-1), respectively, nu(C(4a)=N(5)) at 1580 cm(-1), and nu(C(10a)=N(1)) at 1548 cm(-1). Measured modes of the reduced form of FAD include nu(C(2)=O) at 1692 cm(-1), nu(C(4)=O) at 1634 cm(-1), and nu(C(4a)=C(10a)) at 1600 cm(-1). While the overall shape of the enzyme spectra is similar to the shape of the spectrum of free FAD, there are numerous differences in detail. In particular, the nu(C=N) modes of the flavin exhibit frequency shifts in the protein-bound form, most prominently for pyruvate oxidase where nu(C(10a)=N(1)) downshifts by 14 cm(-1) to 1534 cm(-1). The significance of this shift and a possible explanation in connection with the bent conformation of the flavin cofactor in this enzyme are discussed.     

Simultaneous resonance Raman detection of the heme a3-Fe-CO and CuB-CO species in CO-bound ba3-cytochrome c oxidase from Thermus thermophilus. Evidence for a charge transfer CuB-CO transition

Understanding of the chemical nature of the dioxygen and nitric oxide moiety of ba3-cytochrome c oxidase from Thermus thermophilus is crucial for elucidation of its physiological function. In the present work, direct resonance Raman (RR) observation of the Fe-C-O stretching and bending modes and the C-O stretching mode of the CuB-CO complex unambiguously establishes the vibrational characteristics of the heme-copper moiety in ba3-oxidase. We assigned the bands at 507 and 568 cm(-1) to the Fe-CO stretching and Fe-C-O bending modes, respectively. The frequencies of these modes in conjunction with the C-O mode at 1973 cm(-1) showed, despite the extreme values of the Fe-CO and C-O stretching vibrations, the presence of the alpha-conformation in the catalytic center of the enzyme. These data, distinctly different from those observed for the caa3-oxidase, are discussed in terms of the proposed coupling of the alpha-and beta-conformations that occur in the binuclear center of heme-copper oxidases with enzymatic activity. The CuB-CO complex was identified by its nu(CO) at 2053 cm(-1) and was strongly enhanced with 413.1 nm excitation indicating the presence of a metal-to-ligand charge transfer transition state near 410 nm. These findings provide, for the first time, RR vibrational information on the EPR silent CuB(I) that is located at the O2 delivery channel and has been proposed to play a crucial role in both the catalytic and proton pumping mechanisms of heme-copper oxidases.     

Kinetic properties and inhibition of human T lymphoblast deoxycytidine kinase

The kinetic properties of 50,000-fold purified cultured human T lymphoblast (MOLT-4) deoxycytidine kinase were examined. The reaction velocity had an absolute requirement for magnesium. Maximal activity was observed at pH 6.5-7.0 with Mg:ATP for 1:1. High concentrations of free Mg2+ or free ATP were inhibitory. Double reciprocal plots of initial velocity studies yielded intersecting lines for both deoxycytidine and MgATP2-. dCMP was a competitive inhibitor with respect to deoxycytidine and ATP. ADP was a competitive inhibitor with respect to ATP and a mixed inhibitor with respect to deoxycytidine. dCTP, an important end product, is a very potent inhibitor and was a competitive inhibitor with respect to deoxycytidine and a non-competitive inhibitor with respect to ATP. TTP reversed dCTP inhibition. The data suggest that (a) MgATP2- is the true substrate of deoxycytidine kinase; (b) the kinetic mechanism of deoxycytidine kinase is consistent with rapid equilibrium random Bi Bi; (c) deoxycytidine kinase may be regulated by its product ADP and its end product dCTP as well as the availability of deoxycytidine. While many different nucleotides potently inhibit deoxycytidine kinase, their low intracellular concentrations make their regulatory role less important.     

Vibrational modes of ubiquinone in cytochrome bo(3) from Escherichia coli identified by Fourier transform infrared difference spectroscopy and specific (13)C labeling.

In this study we present the infrared spectroscopic characterization of the bound ubiquinone in cytochrome bo(3) from Escherichia coli. Electrochemically induced Fourier transform infrared (FTIR) difference spectra of DeltaUbiA (an oxidase devoid of bound ubiquinone) and DeltaUbiA reconstituted with ubiquinone 2 and with isotopically labeled ubiquinone 2, where (13)C was introduced either at the 1- or at the 4-position of the ring (C=O groups), have been obtained. The vibrational modes of the quinone bound to the discussed high-affinity binding site (Q(H)) are compared to those from the synthetic quinones in solution, leading to the assignment of the C=O modes to a split signal at 1658/1668 cm(-)(1), with both carbonyls similarly contributing. The FTIR spectra of DeltaUbiA reconstituted with the labeled quinones indicate an essentially symmetrical and weak hydrogen bonding of the two C=O groups from the neutral quinone with the protein and distinct conformations of the 2- and 3-methoxy groups. Perturbations of the vibrational modes of the 5-methyl side groups are discussed for a signal at 1452 cm(-)(1). Only negligible shifts of the aromatic ring modes can be reported for the reduced and the protonated form of the quinone. Alterations of the protein upon quinone binding are reflected in the electrochemically induced FTIR difference spectra. In particular, difference signals at 1640-1633 cm(-)(1) and 1700-1670 cm(-)(1) indicate variations of beta-sheet secondary structure elements and loops, bands at 1706 and 1678 cm(-)(1) are tentatively attributed to individual amino acids, and a difference signal a 1540 cm(-)(1) is discussed to reflect an influence on C=C modes of the porphyrin ring or on deprotonated propionate groups of the hemes. Further tentative assignments are presented and discussed. The (13)C labeling experiments allow the assignment of the vibrational modes of a bound ubiquinone 8 in the electrochemically induced FTIR difference spectra of wild-type bo(3).     

Thermal sensitivity of the red absorption tail of the photosystem II reaction center complex.

The red tail of the absorption spectrum of the D1-D2-cytb559 complex, defined as the absorption signal not described by the two Gaussian sub-bands associated with the intense electronic transitions at 680 and 683 nm, exhibits anomalous temperature behavior. This tail was analyzed in the temperature interval between 80 and 300 K in terms of the mean square deviation (sigma2) of the total Qy absorption band and by Gaussian sub-band decomposition. The value of the average optical reorganization energy (Snum) obtained from the temperature dependence of sigma2 for the whole absorption band was 32 cm(-1), and changed to 16-20 cm(-1) after subtraction of the sub-bands describing the red tail. This latter value is in agreement with the hole burning literature data for chlorophyll bound to proteins, and indicates that the rather high value for the apparent optical reorganization energy obtained by analysis of the total Qy band of the D1-D2-cytb559 complex is determined by the temperature sensitivity of the red tail. This suggests that the long wavelength absorption tail might be due to vibrational transitions associated with vibrational modes in the range of 80-150 cm(-1) which are thermally accessible and give rise to an absorption signal on the low-energy side of the (0,0) transition. On the basis of this assumption, the electron-phonon coupling strength (S) for these modes is estimated to be in the range 0.028-0.18. This interpretation furthermore supports the idea that the electronic transition near 683 nm is that of a monomer chlorophyll.     

Vibrational and electronic couplings in ultraviolet resonance Raman spectra of cyclic peptides

Ultraviolet resonance Raman (UVRR) spectra are reported for a series of cyclic amides. 2-Azacyclotridecone, which has a 13-membered ring, shows a classic trans-amide UVRR spectral pattern, with comparable enhancement of the amide modes II, III and S. When the ring is diminished to eight (epsilon -caprolactam) or seven (2-azacyclooctanone) members, this pattern is replaced by a single strong band near 1497 cm(-1), characteristic of the Cz-N stretch of a cis-amide vibration (amide IIc). Further shrinkage of the ring decreases the amide IIc frequency. It is lowered over 100 cm(-1) to 1389 cm(-1) in the case of a 4-membered ring (2-azaidine), reflecting diminution of the Cz-N bond order due to ring strain and pyramidalization of the C and N atoms. At the same time the amide Ic (Cz=O stretching) frequency increases, reflecting the localization of the Cz=O double bond. Also the sensitivity to hydrogen-deuterium exchange reverses for amide Ic and IIc modes as ring size decreases. The UV absorption maximum, which is red-shifted for cis-relative to trans-amides, shifts increasingly to the blue as the ring size decreases, again reflecting localization of the pi bonding. In the case of amides with 5- and 6-membered rings (2-pyrrolidinone and delta-eloctam) multiple UVRR bands are seen in the amide IIc region, whose relative intensities are temperature-dependent. These are assigned to conformers in which different members of the ring are out of the mean plane, resulting in variable perturbations of the amide bond. The cyclic dipeptides cyclo(Gly-Gly) and cyclo(Gly-Pro) have perturbed amide IIc frequencies, reflecting the kinematic mixing of the amide coordinates into in- and out-of-phase modes. Excitation profiles reveal electronic mixing as well, with the transition dipoles adding for the in-phase and cancelling for the out-of-phase modes.     

Modeling crystal and molecular deformation in regenerated cellulose fibers

Experimental deformation micromechanics of regenerated cellulose fibers using Raman spectroscopy have been widely reported. Here we report on computer modeling simulations of Raman band shifts in modes close to the experimentally observed 1095 cm(-1) band, which has previously been shown to shift toward a lower wavenumber upon application of external fiber deformation. A molecular mechanics approach is employed using a previously published model structure of cellulose II. Changing the equilibrium c-spacing of this structure and then performing a minimization routine mimics tensile deformation. Normal-mode analysis is then performed on the minimized structure to predict the Raman-intensive vibrations. By using a dot-product analysis on the predicted eigenvectors it is shown that some Raman active modes close to the 1095 cm(-1) band interchange at certain strain levels. Nevertheless, when this is taken into account it is shown that it is possible to find reasonable agreement between theory and experiment. The effect of the experimentally observed broadening of the Raman bands is discussed in terms of crystalline and amorphous regions of cellulose, and this is compared to the lack of X-ray broadening to explain why discrepancies between theory and experiment are present. A hybrid model structure with a series-parallel arrangement of amorphous and misaligned amorphous-crystalline domains is proposed which is shown to agree with what is observed experimentally. Finally, the theoretical crystal modulus for cellulose II is reported as 98 GPa, which is shown to be in agreement with other studies and with an experimental measurement using synchrotron X-ray diffraction.     

Analyis of structure/property relationships in silkworm (Bombyx mori) and spider dragline (Nephila edulis) silks using Raman spectroscopy

The molecular deformation of both silkworm (Bombyx mori) and spider dragline (Nephila edulis) silks has been studied using a combination of mechanical deformation and Raman spectroscopy. The stress/strain curves for both kinds of silk showed elastic behavior followed by plastic deformation. It was found that both materials have well-defined Raman spectra and that some of the bands in the spectra shift to lower frequency under the action of tensile stress or strain. The band shift was linearly dependent upon stress for both types of silk fiber. This observation provides a unique insight into the effect of tensile deformation upon molecular structure and the relationship between structure and mechanical properties. Two similar bands in the Raman spectra of both types of silk in the region of 1000-1300 cm(-1) had significant identical rates of Raman band shift of about 7 cm(-1)/GPa and 14 cm(-1)/GPa demonstrating the similarity between the silk fibers from two different animals.     

Dynamics of the [4Fe-4S] cluster in Pyrococcus furiosus D14C ferredoxin via nuclear resonance vibrational and resonance Raman spectroscopies, force field simulations, and density functional theory calculations

We have used (57)Fe nuclear resonance vibrational spectroscopy (NRVS) to study oxidized and reduced forms of the [4Fe-4S] cluster in the D14C variant ferredoxin from Pyrococcus furiosus (Pf D14C Fd). To assist the normal-mode assignments, we conducted NRVS with D14C ferredoxin samples with (36)S substituted into the [4Fe-4S] cluster bridging sulfide positions, and a model compound without ligand side chains, (Ph(4)P)(2)[Fe(4)S(4)Cl(4)]. Several distinct regions of NRVS intensity are identified, ranging from "protein" and torsional modes below 100 cm(-1), through bending and breathing modes near 150 cm(-1), to strong bands from Fe-S stretching modes between 250 and ～400 cm(-1). The oxidized ferredoxin samples were also investigated by resonance Raman (RR) spectroscopy. We found good agreement between NRVS and RR frequencies, but because of different selection rules, the intensities vary dramatically between the two types of spectra. The (57)Fe partial vibrational densities of states for the oxidized samples were interpreted by normal-mode analysis with optimization of Urey-Bradley force fields for local models of the [4Fe-4S] clusters. Full protein model calculations were also conducted using a supplemented CHARMM force field, and these calculations revealed low-frequency modes that may be relevant to electron transfer with Pf Fd partners. Density functional theory (DFT) calculations complemented these empirical analyses, and DFT was used to estimate the reorganization energy associated with the [Fe(4)S(4)](2+/+) redox cycle. Overall, the NRVS technique demonstrates great promise for the observation and quantitative interpretation of the dynamical properties of Fe-S proteins.     

Two-dimensional near-IR correlation spectroscopy study of molten globule-like state of ovalbumin in acidic pH region: simultaneous changes in hydration and secondary structure

The molten globule-like states of ovalbumin (OVA) in acid aqueous solutions are investigated by generalized two-dimensional (2D) Fourier transform near-IR (FT-NIR) correlation spectroscopy. This new method allows us to explore the changes in hydration and the secondary structure simultaneously. FT-NIR spectra are measured for OVA aqueous solutions with concentrations of 1, 2, 3, 4, and 5 wt % over a pH range of 2.4-5.4. Concentration-perturbed 2D correlation spectra are calculated for the spectra in the 4850-4200 and 7500-5350 cm(-1) regions at different pH values. The 2D NIR synchronous spectrum in the 4850-4200 cm(-1) region shows a significant change upon going from pH 5.4 to 3.6. An autopeak at 4265 cm(-1) that is due to a combination of a symmetric CH(2) stretching mode and a CH(2) bending mode of side chains seen at pH 5.0 disappears completely in the synchronous spectrum at pH 3.6. This suggests that some amino acid residues of OVA are subjected to microenvironmental changes with decreasing pH. More remarkable changes are observed in the synchronous spectra at pHs below 2.8. A band near 4600 cm(-1) arising from a combination of amide B and amide II modes (amide B/II) shifts downward with considerable broadening between pH 3.0 and 2.4, suggesting that the strength of the hydrogen bonds of amide groups of OVA changes significantly. The synchronous and asynchronous spectra in the 4850-4200 cm(-1) region show that the intensities of the bands attributable to amide groups and side chains of OVA and that of the band near 4800 cm(-1) arising from water change in phase with the increase in the concentration above pH 2.8, but they vary out of phase below pH 2.8. The 2D synchronous map in the 7500-5350 cm(-1) region also shows marked changes upon going from pH 2.8 to 2.6. A broad autopeak at around 6950 cm(-1) assigned to free water and bound water with weak hydrogen bonds becomes very weak in the synchronous spectrum at pH 2.6, while broad autopeaks around 6450 cm(-1) suddenly appear that are due to bound water with several hydrogen bonds and the first overtone of an NH stretching mode of the amide groups of OVA. Therefore, it is very likely that protein hydration and the hydrogen bonds of amide groups change simultaneously in a narrow pH region of 2.8-2.6. It is probably that below pH 2.6 the protein assumes a molten globule-like state in which the whole molecule is very flexible, and side chains (but not the backbone chain) fluctuate significantly.     

Structural changes in bacteriorhodopsin following retinal photoisomerization from the 13-cis form

Bacteriorhodopsin (BR), a light-driven proton pump in Halobacterium salinarum, accommodates two resting forms of the retinylidene chromophore, the all-trans form (AT-BR) and the 13-cis,15-syn form (13C-BR). Both isomers are present in thermal equilibrium in the dark, but only the all-trans form has proton-pump activity. In this study, we applied low-temperature Fourier-transform infrared (FTIR) spectroscopy to 13C-BR at 77 K and compared the local structure around the chromophore before and after photoisomerization with that in AT-BR. Strong hydrogen-out-of-plane (HOOP) vibrations were observed at 964 and 958 cm(-)(1) for the K state of 13C-BR (13C-BR(K)) versus a vibration at 957 cm(-)(1) for the K state of AT-BR (AT-BR(K)). In AT-BR(K), but not in 13C-BR(K), the HOOP modes exhibit isotope shifts upon deuteration of the retinylidene at C15 and at the Schiff base nitrogen. Whereas the HOOP modes of AT-BR(K) were significantly affected by the mutation of Thr89, this was not the case for the HOOP modes of 13C-BR(K). These observations imply that, while the chromophore distortion is localized near the Schiff base in AT-BR(K), it is located elsewhere in 13C-BR(K). By use of [zeta-(15)N]lysine-labeled BR, we identified the N-D stretching vibrations of the 13C-BR Schiff base (in D(2)O) at 2173 and 2056 cm(-)(1), close in frequency to those of AT-BR. These frequencies indicate strong hydrogen bonding of the Schiff base in 13C-BR, presumably with a water molecule as in AT-BR. In contrast, the N-D stretching vibration appears at 2332 and 2276 cm(-)(1) in 13C-BR(K) versus values of 2495 and 2468 cm(-)(1) for AT-BR(K), suggesting that the rupture of the Schiff base hydrogen bond that occurs in AT-BR(K) does not occur in 13C-BR(K). Rotational motion of the Schiff base upon retinal isomerization is probably smaller in magnitude for 13C-BR than for AT-BR. These differences in the primary step are possibly related to the absence of light-driven proton pumping by 13C-BR.