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.     

The vibrational normal modes of beta-barrels in an IgG antibody molecule.

Based on the quasi-continuity model, and using the method of group theory, we studied the normal vibrations of the VL- and the CHL-beta-barrels in an IgG molecule. We put emphasis on the Raman- and the infrared-active normal modes. The Raman modes we obtained include both the breathing motion mode (or the dominant low-frequency mode) which corresponds to the maximum peak in the Raman spectrum, and the normal modes that correspond to the lower peaks. Our calculated vibration frequencies are found to be in good agreement with the experimental results observed by Painter et al. (Biopolymers 20 (1981) 243). The method and work presented in this paper may improve Chou's quasi-continuity theory in calculating the vibrational modes of a beta-barrel protein.     

Raman microscope studies on the primary photochemistry of vertebrate visual pigments with absorption maxima from 430 to 502 nm

Raman microscope vibrational spectra have been recorded from single photoreceptor cells frozen at 77 K. Spectra of photostationary steady-state mixtures of visual pigments and their primary photoproducts were obtained from toad red rods (lambda max 502 nm), angelfish rods (lambda max 500 nm), gecko blue rods (lambda max 467 nm), and bullfrog green rods (lambda max 430 nm). All four photoproducts have enhanced low-wavenumber Raman lines at approximately 850, 875, and 915 cm-1 and show the anomalous decoupling of the 11- and 12-hydrogen out-of-plane (HOOP) wagging vibrations, as is observed in the bovine primary photoproduct. The low-wavenumber lines are enhanced in the resonance Raman spectrum by conformational distortion, and the uncoupling of the 11- and 12-hydrogen wags is caused by additional protein perturbations. The similarity of the HOOP modes in all four photoproducts indicates that the protein perturbations that uncouple the 11- and 12-hydrogen wags and that enhance the HOOP modes are very similar. Thus, these perturbations of the photoproduct Raman spectrum cannot be caused by the same protein-chromophore interactions that are responsible for wavelength regulation in these pigments.     

Resonance Raman spectroscopy of amicyanin, a blue copper protein from Paracoccus denitrificans

The copper binding site of amicyanin from Paracoccus denitrificans has been examined by resonance Raman spectroscopy. The pattern of vibrational modes is clearly similar to those of the blue copper proteins azurin and plastocyanin. Intense resonance-enhanced peaks are observed at 377, 392, and 430 cm-1 as well as weaker overtones and combination bands in the high frequency region. Most of the peaks below 500 cm-1 shift 0.5-1.5 cm-1 to lower energy when the protein is exposed to D2O. Based on the pattern of conserved amino acids, the axial type EPR spectrum, and the resonance Raman spectrum, it is proposed that the copper binding site in amicyanin contains a Cu(II) ion in a distorted trigonal planar geometry with one cysteine and two histidine ligands and an axial methionine ligand at a considerably longer distance. Furthermore, the presence of multiple intense Raman peaks in the 400 cm-1 region which are sensitive to deuterium substitution leads to the conclusion that the Cu-S stretch is coupled with internal ligand vibrational modes and that the sulfur of the cysteine ligand is likely to be hydrogen-bonded to the polypeptide backbone.     

Primary donor structure and interactions in bacterial reaction centers from near-infrared Fourier transform resonance Raman spectroscopy

Preresonance Raman and resonance Raman spectra of the primary donor (P) from reaction centers of the Rhodobacter (Rb.) sphaeroides R26 carotenoidless strain in the P and P+ states, respectively, were obtained at room temperature with 1064-nm excitation and a Fourier transform spectrometer. These spectra clearly indicate that the chromophore modes are observable over those of the protein with no signs of interference below 1800 cm-1. The chromophore modes are dominated by those of the bacteriochlorophylls (BChl a), and it is estimated that, in the P state, ca. 65% of the Raman intensity of the BChl a modes arises from the primary donor. This permits the direct observation of a vibrational spectrum of the primary donor at preresonance with the excitonic 865-nm band. The Raman spectrum of oxidized reaction centers in the presence of ferricyanide clearly exhibits bands arising from a BChl a+ species. The magnitude of the frequency shift of a keto carbonyl of neutral P from 1691 to 1717 cm-1 upon P+ formation strongly suggests that one BChl molecule in P+ carries nearly the full +1 charge. Our results indicate that the unpaired electron in P.+ does not share a molecular orbital common to the two components of the dimer on the time scale of the resonance Raman effect (ca. 10(-13) s).     

Resonance Raman spectra of copper(II)-substituted liver alcohol dehydrogenase: a type 1 copper analogue

Liver alcohol dehydrogenase (LADH) with copper in place of the catalytic zinc has recently been proposed to contain a type 1 site analogous to that in "blue" copper proteins. Resonance Raman spectra for the copper-substituted enzyme, Cu(II) X LADH, and its binary complexes with reduced nicotinamide adenine dinucleotide (NADH) and pyrazole support this viewpoint. These spectra have two dominant features: a sharp peak at approximately 415 cm-1, which is believed to be associated with vibration of the single histidine ligand, and a broader, asymmetric band at approximately 350 cm-1, whose components are assigned predominantly to vibrational modes of the two cysteinate ligands. The high frequency of these transitions, which is reminiscent of the blue copper proteins, is ascribed to the tetrahedral nature of the metal site that produces unusually short Cu-S bonds and coupled vibrational modes. Solvent exchange with H218O reveals no contribution to the resonance Raman spectrum of the water molecule, which is a metal ligand in free Cu(II) X LADH; however, the spectrum of the binary complex with pyrazole has several new peaks attributable, in part, to pyrazole ligation. The strong similarity among the vibrational spectra demonstrates that the Cu(II) environment in alcohol dehydrogenase maintains its near-tetrahedral geometry in the various enzyme derivatives. The resonance Raman spectrum of Ni(II) X LADH is close to that of Cu(II) X LADH and suggests a similar tetrahedral site. The Raman spectra presented here together with available optical and EPR data indicate that Cu(II) X LADH belongs to the type 1 copper classification and, thus, can provide new insights into this unusual coordination geometry.     

Resonance Raman on the purple intermediates of the flavoenzyme D-amino acid oxidase

Resonance Raman (RR) spectra excited at 632.8 nm within a charge transfer absorption band were obtained for a catalytic intermediate, the purple complex of D-amino acid oxidase with D-proline or D-alanine as a substrate. The resonance enhanced Raman lines around 1605 and 1360 cm^?1 in either of the complexes were suggested to be derived from vibrational modes of reduced flavin molecule. Since the highest energy band at 1692 cm^?1 in the RR spectrum with D-alanine was shifted to 1675 cm^?1 upon [¹⁵N] substitution of alanine and ammonium, this Raman line in the spectrum with D-alanine or the line at 1658 cm^?1 with D-proline is assigned to the CN stretching mode of an imino acid corresponding to each amino acid. These results confirm the concept that the purple intermediate of D-amino acid oxidase consists of reduced flavin and an imino acid.     

Nuclear resonance vibrational spectroscopy--NRVS

The recent, synchrotron-based vibrational technique nuclear resonance vibrational spectroscopy (NRVS) is introduced. The method can be used for a number of M?ssbauer active isotopes including 57Fe, which has yielded most of the results to date. The NRVS experiment can be thought of as M?ssbauer spectroscopy with vibrational sidebands. Importantly, the NRVS experiment provides the complete set of bands corresponding to modes that involve motion of the iron atom. The method has a selectivity reminiscent of that of resonance Raman spectroscopy, but with the significant advantage that NRVS is not subject to the optical selection rules of Raman or infrared spectroscopy. Indeed, NRVS provides the ultimate limit in selectivity because only the vibrational dynamics of the probe nucleus contribute to the observed signal. All iron-ligand modes will be observed, including many that had not been previously observed. For hemes, these include in-plane iron vibrations that have not yet been reported by resonance Raman studies and the iron-imidazole stretch that has not been identified in six-coordinate porphyrins. Other modes that can be investigated include that of heme doming that is expected to be a low-frequency mode. The experimental setup at a beam line and sample requirements for iron-based derivatives are presented. Both powder and polarized single-crystal measurements can be made. The general features of data extraction and analysis are given. Data for heme and heme proteins are given. Examples of assignment of spectra for nitrosyl and carbonyl derivatives are given. These data demonstrate the importance of peripheral substituents on the vibrational spectrum of heme derivatives. Delocalization of modes appears to be common. Although this technique has only been available for a relatively short time, this early progress report indicates that NRVS has significant potential for probing the dynamics of Fe-containing molecules of biological interest.     

Resonance Raman spectroelectrochemistry of bacteriochlorophyll and bacteriochlorophyll cation radical.

The newly developed technique of resonance Raman spectroelectrochemistry (RRSE) is applied to the study of the radical ion species involved in the primary photochemistry of photosynthesis. By means of controlled potential coulometry combined with resonance Raman spectroscopic detection, the vibrational spectrum of the bacteriochlorophyll $\text{a}$ cation radical (BChl⁺·) has been obtained and compared with the corresponding spectrum of the parent molecule. The cation radical spectrum is significantly different from the neutral spectrum both in band frequencies and intensities. These results suggest that RR vibrational spectra may provide a new means of identifying and kinetically monitoring radical ion formation both in photosynthetic model systems and $\text{in vivo}$.     

Resonance Raman spectra of vitamin B12 and dicyanocobalamin

The Raman spectra of cyanocobalamin (vitamin B12) and dicyanocobinamide have been obtained from aqueous solutions at concentrations of approx. 1 10⁻⁴ M. The spectra were excited by laser radiation coincident in wavelength with the visible absorption, this resulting in selective enhancement of some vibrational modes through the rigorous resonance Raman effect. In spite of substantial chemical differences, cyanocobalamin and dicyanocobinamide give essentially identical spectra, indicating that only those modes associated with the common corrin ring system are resonance enhanced.     

Infrared and Raman spectroscopy of carbohydrates. Part VI: Normal coordinate analysis of V-amylose

A normal coordinate analysis of V-amylose has been performed for an isolated 6₁ helical chain. Negligible splitting from interactions of vibrations of successive residues is expected between A and E vibrational species due to the large size of the monomer unit. As a result, calculation of only the totally symmetric A modes represents an adequate approximation to the vibrational spectrum of helical polysaccharides. Using this method together with a valence force field we have obtained good agreement between the observed and calculated frequencies. In addition, the computed potential energy distribution and Cartesian displacement coordinates match previous experimental assignments, based on deuterium exchange. The analysis also supports the proposed mechanism for conversion of V-amylose to the more extended B-form. This conversion results in an observed frequency shift for the Raman line at 946 cm^?1 which is predicted by the calculations.     

Raman spectra and vibrational assignments for deuterated membrane lipids. 1,2-Dipalmitoyl phosphatidylcholine-d9 and -d62

Vibrational Raman spectra of polycrystalline 1,2-dipalmitoyl phosphatidylcholine-d9 (fully deuterated choline methyl groups) and 1,2-dipalmitoyl phosphatidylcholine-d62 (fully deuterated acyl chains) were recorded in the 3050- 2800, 2250-2050 and 1800-700 cm-1 regions. The fundamental vibrational modes were assigned primarily on the basis of isotopic frequency shift ratios, group frequency correlations and comparisons with specific model compounds. Since deuterium-substituted lipids provide well-isolated spectral probes, particularly in the carbon-deuterium stretching region, the dependence of the 2250-2050 cm-1 region on lipid phase was examined for the dipalmitoyl phosphatidylcholine-d62 species. The methylene CD2 deformation and twisting modes at 984 and 919 cm-1, respectively, also exhibit intense, isolated vibrational transitions which should prove useful for monitoring molecular order in mixed dueterated and undeuterated lipid systems. Except for the relatively weak choline methyl C-D and C-H stretching modes, the spectrum of 1,2-dipalmitoyl phosphatidylcholine-d9 is not distinguishable from that of the undeuterated system. For both the d9 and undeuterated species, the vibrational modes associated with the lipid head group region are sensitive to slight hydration.     

Vibrational spectroscopy of an algal Phot-LOV1 domain probes the molecular changes associated with blue-light reception

The LOV1 domain of the blue light Phot1-receptor (phototropin homolog) from Chlamydomonas reinhardtii has been studied by vibrational spectroscopy. The FMN modes of the dark state of LOV1 were identified by preresonance Raman spectroscopy and assigned to molecular vibrations. By comparing the blue-light-induced FTIR difference spectrum with the preresonance Raman spectrum, most of the differences are due to FMN modes. Thus, we exclude large backbone changes of the protein that might occur during the phototransformation of the dark state LOV1-447 into the putative signaling state LOV1-390. Still, the presence of smaller amide difference bands cannot be excluded but may be masked by overlapping FMN modes. The band at 2567 cm(-1) is assigned to the S-H stretching vibration of C57, the residue that forms the transient thio-adduct with the chromophore FMN. The occurrence of this band is evidence that C57 is protonated in the dark state of LOV1. This result challenges conclusions from the homologous LOV2 domain from oat that the thiolate of the corresponding cysteine is the reactive species.     

Raman spectroscopy of interferon-induced 2',5'-linked oligoadenylates

Raman spectra of model compounds and of 2',5'-oligoadenylates in D2O were utilized to assign the Raman bands of 2',5'-oligoadenylates. The Raman spectra of A2'pA2'pA, pA2'pA2'pA, and pppA2'pA2'pA contained features that were similar to those of adenosine, adenosine 5'-monophosphate (AMP), and adenosine 5'-triphosphate, respectively. When AMP and pA2'pA2'pA were titrated from pH 2 to 9, the normalized Raman intensity of their ionized (980 cm-1) and protonated (1080 cm-1) phosphate bands revealed similar pKa's for the 5'-monophosphates. The Raman spectrum of pA2'pA2'pA was altered slightly by elevations in temperature, but not in a manner supporting the postulate that 2-5A possesses intermolecular base stacking. Major differences in the Raman spectrum of 2',5'- and 3',5'-oligoadenylates were observed in the 600-1200-cm-1 portion of the spectrum that arises predominately from ribose and phosphate vibrational modes. Phosphodiester backbone modes in A3'pA3'pA and pA3'pA3'pA produced a broad band at 802 cm-1 with a shoulder at 820 cm-1, whereas all 2',5'-oligoadenylates contained a major phosphodiester band at 823 cm-1 with a shoulder at 802 cm-1. The backbone mode of pppA2'pA2'pA contained the sharpest band at 823 cm-1, suggesting that the phosphodiester backbone may be more restrained in the biologically active, 5'-triphosphorylated molecule. The Raman band assignments for 2',5'-oligoadenylates provide a foundation for using Raman spectroscopy to explore the mechanism of binding of 2',5'-oligoadenylates to proteins.     

Resonance Raman investigation of a soluble cytochrome c552 from alkaliphilic Bacillus firmus RAB

The environment of the heme site of a low-potential soluble cytochrome (c552) from alkaliphilic Bacillus firmus RAB has been characterized with resonance Raman scattering and compared to that of horse heart cytochrome c. The Raman data indicate that vibrational bands sensitive to the axial ligation of the heme, as well as modes sensitive to the heme peripheral environment in cytochrome c552, are distinct from those of horse heart cytochrome c. The spectra of cytochrome c552 display resonance Raman modes indicative of a methionine as the sixth ligand in the oxidized form, while the reduced form appears to contain a nitrogenous-based sixth ligand. In addition, Q-band excitation reveals differences among vibrational modes in cytochrome c552 that are sensitive to the amino acid environment surrounding the heme.     

Chromophore interactions in flavocytochrome c552: a resonance Raman investigation

Resonance Raman spectra with both Soret and visible excitation have been obtained for Chromatium flavocytochrome c552 and its isolated diheme subunit under varying conditions of pH and inhibitor binding. The spectra are generally consistent with previously established classification schemes for porphyrin ring vibrations. The presence of covalently bound flavin in the protein was apparent in the fluorescent background it produced and in flavin-mediated photoeffects observed in heme Raman spectra obtained at high laser power. No flavin modes were present in the Raman spectra, nor was any evidence of direct heme-flavin interaction found by using this technique; however, a systematic perturbation of heme B1g vibrational frequencies was found in the oxidized holoprotein. The heme vibrational frequencies of c552 are compared to those of the diheme peptide and of other c-type cytochromes. They are consistent with an interpretation that involves pH-dependent changes in axial ligation and treats the hemes and flavin as isolated chromphores communicating via protein-mediated interactions.     

Secondary and tertiary structure of the A-state of cytochrome c from resonance Raman spectroscopy.

Ferricytochrome c can be converted to the partially folded A-state at pH 2.2 in the presence of 1.5 M NaCl. The structure of the A-state has been studied in comparison with the native and unfolded states, using resonance Raman spectroscopy with visible and ultraviolet excitation wavelengths. Spectra obtained with 200 nm excitation show a decrease in amide II intensity consistent with loss of structure for the 50s and 70s helices. The 230-nm spectra contain information on vibrational modes of the single Trp 59 side chain and the four tyrosine side chains (Tyr 48, 67, 74, and 97). The Trp 59 modes indicate that the side chain remains in a hydrophobic environment but loses its tertiary hydrogen bond and is rotationally disordered. The tyrosine modes Y8b and Y9a show disruption of tertiary hydrogen bonding for the Tyr 48, 67, and 74 side chains. The high-wavenumber region of the 406.7-nm resonance Raman spectrum reveals a mixed spin heme iron atom, which arises from axial coordination to His 18 and a water molecule. The low-frequency spectral region reports on heme distortions and indicates a reduced degree of interaction between the heme and the polypeptide chain. A structural model for the A-state is proposed in which a folded protein subdomain, consisting of the heme and the N-terminal, C-terminal, and 60s helices, is stabilized through nonbonding interactions between helices and with the heme.     

Dependence of the Raman signature of genomic B-DNA on nucleotide base sequence.

The vibrational spectra of four genomic and two synthetic DNAs, encompassing a wide range in base composition [poly(dA-dT). poly(dA-dT), 0% G + C; Clostridium perfringens DNA, 27% G + C; calf thymus DNA, 42% G + C; Escherichia coli DNA, 50% G + C; Micrococcus luteus DNA, 72% G + C; poly(dG-dC).poly(dG-dC), 100% G + C] (dA: deoxyadenosine; dG: deoxyguanosine; dC: deoxycytidine; dT: thymidine), have been analyzed using Raman difference methods of high sensitivity. The results show that the Raman signature of B DNA depends in detail upon both genomic base composition and sequence. Raman bands assigned to vibrational modes of the deoxyribose-phosphate backbone are among the most sensitive to base sequence, indicating that within the B family of conformations major differences occur in the backbone geometry of AT- and GC-rich domains. Raman bands assigned to in-plane vibrations of the purine and pyrimidine bases-particularly of A and T-exhibit large deviations from the patterns expected for random base distributions, establishing that Raman hypochromic effects in genomic DNA are also highly sequence dependent. The present study provides a basis for future use of Raman spectroscopy to analyze sequence-specific DNA-ligand interactions. The demonstration of sequence dependency in the Raman spectrum of genomic B DNA also implies the capability to distinguish genomic DNAs by means of their characteristic Raman signatures.     

Resonance Raman investigations of chloroperoxidase, horseradish peroxidase, and cytochrome c using Soret band laser excitation.

Resonance Raman spectra of the heme protein chloroperoxidase in its native and reduced forms and complexed with various small ions are obtained by using laser excitation in the Soret region (350-450 nm). Additionally, Raman spectra of horseradish peroxidase, cytochrome P-450cam, and cytochrome c, taken with Soret excitation, are presented and discussed. The data support previous findings that indicate a strong analogy between the active site environments of chloroperoxidase and cytochrome P-450cam. The Raman spectra of native chloroperoxidase are found to be sensitive to temperature and imply that a high leads to low spin transition of the heme iron atom takes place as the temperature is lowered. Unusual peak positions are also found for native and reduced chloroperoxidase and indicate a weakening of porphyrin ring bond strengths due to the presence of a strongly electron-donating axial ligand. Enormous selective enhancements of vibrational modes at 1360 and 674 cm-1 are also observed in some low-spin ferrous forms of the enzyme. These vibrational frequencies are assigned to primary normal modes of expansion of the prophyrin macrocycle upon electronic excitation.