Pressure-tuning vibrational spectroscopy is a powerful tool for the study of structural and dynamic properties at the molecular level. The evaluation of changes in molecular structure associated with cell anomalies in biological tissues and cells became possible because of the development of pressure-tuning infrared spectroscopic techniques in our laboratory to obtain high quality infrared spectra of intact biological tissues and whole cells as a function of pressure. This development allowed us to investigate the molecular bases of a wide range of biological and biomedical problems. In this paper some causes of cell anomalies at the molecular level in human cancers are presented.

The secondary structure of the naturally occurring isoforms of myelin basic protein (MBP1-8) from human myelin was studied by Fourier transform infrared spectroscopy under a variety of experimental conditions. In aqueous solution each isoform was found to be unstructured. In the presence of negatively charged liquid bilayers MBP1-4 were shown to exhibit an amide I band maximum indicative of the adoption of (alpha) -helical secondary structures. A detailed analysis revealed that significant proportions of (beta) -sheet secondary structure were also present. MBP5 and MBP8, which have significantly less cationic charge than MBP1-4, exhibited an amide I maximum identical to that seen in solution, suggesting that no interaction with the bilayer occurred. Analysis of the lipid CH₂ and C equals O stretching vibrations also pointed towards significant interaction of MBP1-4 with the bilayer. The changes in intensity and frequency of these bands which typically accompany the phase transition in the pure bilayer were abolished by addition of the proteins. No such effect was seen for MBP5 and 8, the normal lipid phase transition being apparent. The implications of these results in the aetiology of multiple sclerosis is discussed.

A model for the dynamics of DNA dissolved in aqueous medium containing counterions is discussed. The dynamic behavior of B conformation poly(dA)-poly(dT) homopolymer DNA with sodium counterions is presented for two possible counterion-DNA interactions: totally site bound counterions and totally area bound counterions. We present an analysis of the eigenvectors of the modes below 100 cm^-1 and show that the location and density of hydrogen bond breathing modes and propeller twist modes depend on the counterion-DNA interaction model. The prediction of a plasmon mode around 25 cm^-1 for the site bound interaction model is discussed. We present an algorithm for calculating the dynamics of DNA with structural defects using this model.

The sensitizing hematoporphyrins and related species used in photosensitized tumor therapy (PTT) were revealed to be complex mixtures of monomers, dimers (mainly dihematoporphyrin ether or ester) and aggregates. Time resolved absorption spectroscopy was proved to be helpful for differentiation between these components. The spectral and temporal characteristics of the absorption changes ((Delta) A) of Hp, HpD and PS is aqueous and ethanol solutions were measured by picosecond pump-and-probe techniques. The (Delta) A spectrum of Hp in ethanol solution is formed by the bleaching of steady-state bands and the absorption from the excited S₁ state. The (Delta) A of Hp relaxes exponentially with time constant (tau) equals 10 divided by 17 ns (approximately equal to the fluorescence decay time of monomeric Hp). The (Delta) A of HpD in ethanol shows biexponential decay with time constants (tau) equals 10 divided by 17 and (tau) ₁ equals 1.5 divided by 4 ns. We suppose that lifetime 1.5 divided by 4 ns reflects the excitation energy relaxation in covalently linked oligomers and (tau) equals 10 divided by 16 ns is the lifetime of excited S₁ state of HpD monomers. It should be noted that the (tau) ₁ value depends on the existing different oligomers in HpD due to the HpD preparation conditions. The measured (Delta) A kinetics in aqueous Hp and HpD solution showed the different character. It can be fitted with the three exponents with time constants of (tau) ₂ equals 100 divided by 200 ps (short component), (tau) ₁ equals 1.5 divided by 4 ns (long component) and (tau) about 15 ns (monomeric component). The main changes in excitation relaxation in aqueous solutions is caused by the presence of equilibrium aggregates of porphyrins. We attribute the estimated lifetimes (tau) ₂ equals 100 divided by 200 ps and (tau) ₁ equals 1.5 divided by 4 ns in aqueous solutions to equilibrium aggregates and/or covalently linked dimers and oligomers of HpD, respectively.

Ellipsometric techniques for time-resolved measurements of visible and near-UV CD and MCD have been introduced in the last few years and used to follow the nanosecond dynamics of protein systems containing suitable chromophores. Recent technical advances are described that permit the extension of fast time-resolved CD measurements into the far-UV (190 - 290 nm) spectral region. This development opens the possibility of using CD spectroscopy to monitor secondary structure changes in real time, at physiological temperatures, during processes such as protein folding. Fast CD techniques also permit the measurement of CD and MCD of photoselected protein chromophores before rotational reorientation restores isotropy. Theoretical expression for the CD and MCD of photoselectively oriented absorbers suggest that additional molecular information might be revealed by such measurements.

Vibrational circular dichroism (VCD) spectra provide information on the solution conformations of chiral molecules. The CH-, OH-, NH-, and C equals O-stretching VCD features are particularly useful conformational probes. Molecules of pharmaceutical interest studied by VCD include biomimetic iron carriers, unsaturated peptides, the anticancer drug taxol and a series of type I antiarrhythmic drugs.

Protein secondary structure has been analyzed using a Fourier transform infrared spectroscopic method in the amide III region. Although extensive work has been done on protein secondary structure using the amide I region (1700 - 1600 cm^-1), the amide III region has not been utilized in the past for its potential in protein structural analysis. One of the major reasons for non-use of the amide III vibrations is perhaps the very weak signal in the amide III frequency region (1200 - 1350 cm^-1). However, benefits of using the amide III region are substantial. For example, water vibrations do not interfere with the protein spectrum unlike in the amide I region. In the amide III region, the protein spectrum is better resolved into individual bands than in the amide I region. This feature allows for a greater ease in peak definement of the protein spectra. In the amide III region, the bands for the individual secondary structures ((alpha) -helix, (beta) -sheet and random coils) do not overlap as much as they do in the amide I region. This lack of overlapping allows for easier and a more reliable means of peak assignment, and secondary structure band positions are easier to determine. Amide III region of protein IR spectra appears to be a valuable tool in estimating the amount of secondary structure present in proteins.

The protein response to the photodissociation, escape and subsequent rebinding of carbon monoxide in myoglobin is studied using time-resolved infrared (TRIR) spectroscopy. All phases of these reactions are investigated, from ultrafast phenomena (picoseconds) to relatively slow processes (milliseconds). Conformational changes in myoglobin (Mb) are detected by time-resolved infrared absorption changes in the amide I band. On the hundreds of nanoseconds to milliseconds timescale, a 'real-time' apparatus is used. This apparatus is based on a tunable diode laser operating in the region of 1650 cm^-1. The time course of changes in the amide I band are shown to follow the recombination of CO with photolyzed Mb. On the basis of the rise times of the amide I and Fe-CO signals, it is concluded that protein motion is complete within 100 ns. A time-resolved difference spectrum in the amide I region is generated from single wavelength transients taken throughout the amide I envelope. A static difference spectrum is also generated by subtracting FTIR spectra of carbonmonoxy and deoxy myoglobin. The two difference spectra are compared and are interpreted in terms of the three-dimensional structures of deoxy and carbonmonoxy Mb. Preliminary picosecond TRIR data are also given for the ultrafast response of the protein immediately following photodissociation of CO.

Recent infrared spectroscopic studies of the ultrafast responses of optically triggered changes in proteins are discussed. Examples from research on bacteriorhodospin and reaction centers illustrate the potential of time resolved infrared spectroscopy in the field of protein dynamics.

The photochemistry of vision involves the ultrafast cis-trans photoisomerization and efficient excited-state deactivation of a polyene aldehyde chromophore. We have been studying the photophysics of smaller model systems that also exhibit some of these properties. Resonance Raman and preliminary picosecond time-resolved absorption experiments on butadiene and cis- and trans-1,3,5-hexatriene in vapor and solution phases have been analyzed to characterize the structure and dynamics of both the optically allowed B state and the lowest singlet excited A state. Solvent polarizability is found to have a large effect on the B-state torsional potentials, particularly in trans-hexatriene. Related studies including picosecond two-color pump-probe Raman measurements on the ultrafast photoreactions of cis-stilbene are also described. These experiments probe the initial vibrational energy distribution and subsequent relaxation of the initially formed trans-stilbene, as well as the partitioning between the cis-trans isomerized and ring-closed products. Significant solvent polarity effects are observed on both the excited-state photochemistry and the ground-state vibrational relaxation.

The kinetic constraints that are imposed on cytochrome oxidase in its dual function as the terminal oxidant in the respiratory process and as a redox-linked proton pump provide a unique opportunity to investigate the molecular details of biological O₂ activation. By using flow/flash techniques, it is possible to visualize individual steps in the O₂-binding and reduction process, and results from a number of spectroscopic investigations on the oxidation of reduced cytochrome oxidase by O₂ are now available. In this article, we use these results to synthesize a reaction mechanism for O₂ activation in the enzyme and to simulate time-concentration profiles for a number of intermediates that have been observed experimentally. Kinetic manifestations of the consequences of coupling exergonic electron transfer to endergonic proton translocation emerge from this analysis. An important conclusion is that, in achieving efficiency in this redox-coupled proton translocation mechanism, rate limitation in dioxygen activation in cytochrome oxidase is transferred to protonation reactions that occur late in the reduction reaction. As a consequence, potentially toxic intermediate oxidation states of dioxygen accumulate to substantial concentration during the reduction reaction, which allows us to detect and characterize these species.

We summarize here our recent advancements in application of Raman spectroscopy to studies of protein-ligand interactions. Two main experimental strategies are shown to yield a wealth of information regarding the specific binding 'handles' which contribute to protein-ligand affinity and selectivity. Non-resonance Raman difference spectroscopy, in which the vibrational spectrum of the bound ligand is obtained by subtraction of the apo-protein spectrum from that of the complex is applied to chymotrypsin and aspartate aminotransferase. In both cases, vibrational information derived from previous resonance Raman measurements is shown to be misleading due to photochemically-induced changes in substrate conformation. Isotope editing extends the application of Raman difference spectroscopy to systems where measurements of the apo-protein spectrum is not a viable option because the bound ligand induces too many protein conformational changes that show up in the difference spectrum or in cases where the apoprotein is unstable. A difference spectrum is formed between the two protein-ligand complexes, one of which is specifically labeled with a stable isotope. Vibrational modes which are associated with the isotropic tag show as spectral shifts in the difference spectrum, while all other bands cancel. The power of this method is shown by studies the binding of phosphate of GDP to EF-Tu, the elongation factor from E. Coli. and of glucose-phosphate to phosphoglucomutase, PGM, where ¹⁸O labeling of phosphate reveals binding-induced changes in the vibrational modes of the phosphate moiety.

Transient picosecond Raman spectroscopy is capable of differentiating vibrational relaxation from conformational changes by comparing the Stokes and anti-Stokes dynamics. We report pump-probe picosecond Raman experiments on oxy- and deoxyhemoglobin (oxyHb and deoxyHb, respectively) using 8 ps 532 nm pump pulses and 8 ps 355 nm probe pulses. Heme- to-protein vibrational cooling has been directly observed in deoxyHb for the first time, and the deconvolved cooling time constant is measured to be 2 - 5 ps. By applying our mode-specific Stokes and anti-Stokes technique to oxyHb, we find that any geminate recombination of photodeligated O₂ must occur in either less than two picoseconds or longer than a nanosecond.

Purified Na+, -adenosine triphosphatase (ATPase) in membrane vesicles has been covalently labelled with the triplet probe eosin S'-isothiocynate. The rotational mobility of the protein has been investigated by measurement of time-resolved depolarization of the emitted phosphorescence, induced by a laser pulse. The probe was specifically attached to a lysine residue of the protein located at the putative ATP binding site. The anisotropy of the emission was recorded over the temperature range 3°-42 °c. The total anisotropy was found to be temperature­ dependent with an initial value of about 0.2 at 3 °c and about 0.13 at 42 °C. The overall decrease of total anisotropy with temperature reversed in the temperature range around 13 °C where a peak value of total anisotropy was observed. The anisotropy decay curve was found to fit a double exponential decay process composed of a rapidly rotating component with a rotational correlation time of 20µs-10µs and a slower rotating component with a rotational correlation time of 400µs-100µs, over the temperature range of 3 °C-42 °C. These motions areindividually assigned to the motions of the monomer and oligomer states of the protein molecules, rotating about its axis normal to the plane of the membrane. It was also found that the associated weighting functions were also temperature-dependent; the weighting function of the slowly rotating species increases with increase in temperature, while the weighting function of the rapidly rotating species exhibits the opposite trend. It was estimated that about 80% of the total anisotropy signal was contributed by the fast rotation, and the remainder from slow rotation at 3°C and this reduced to 60% at 42°C.

Nonthennal effects of millimeter wave (MMW) radiation upon the vibrational spectra parameters of o.-Gly, 13-Ala, DL­ Trp, L-Tyr, L-His aminoacids and AMP nucleotide are presented. MMW induced intensity changes up to 35% in IR absorption spectra of Trp, Tyr and AMP films were discovered. Irradiation effect was observed in a number of bands corresponding to different chemical bonds and forms of vibrations Resonance character of MMW effect was regarded as its distinctive feature. In IR reflection spectra of o.-Gly, 13-Ala, L-His single crystals the band structure changes were found together with the intensity redistribution. The MMW action had a resonance nature which appeared to be more pronounced for NH;and coo· deformational vibrations. The amplitude of changes approached 55% of initial intensity. It was shown that specimens' temperature did not vary (accurate to 0,5° C) in the process of irradiation. Heating up to 320 K resulted only in well known temperature effects upon IR bands, that differs essentially from the non-thermal MMW action observed.

We report here our observation of Raman spectrum of the Granulosis Virus of cabbage butterfly Preris rapae (PrGV) and investigate the protein structure of the inclusion body of PrGV. The intense Amide III line at 1248 cm^-1 and Amide I line at 1668 cm^-1 in the Raman spectrum of PrGV contains a predominantly random-Coil or (beta) -turn secondary structure. The intensities ratio of the tyrosyl lines 850 cm^-1 and 830 cm^-1 was found to be 1.25, it indicates the tyrosyl residues are exposed to a solvent. According to s-s stretching vibration frequency 509 cm^-1, the protein molecules of PrGV have gauche-gauche-gauche linkage. The strong peak at 1367 cm^-1 is typical of Tryptophans buried in a hydrophobic environment.

The spectrum of photosensitized singlet oxygen luminescence has been investigated in air- saturated CCl₄, hexafluorobenzene and freon 113 at the wavelengths shorter fundamental at (lambda) < 1200 nm. Novel bands have been detected. The 1073 nm band was observed in all solvents with all photosensitizers used. The data suggest that it accompanies the ¹(Delta) _g (v equals 1) yields ³(Sigma) _g (v equals 0) transition in thermally activated singlet oxygen molecules. The 1160 nm band was detected in CCl₄. It probably corresponded to simultaneous vibronic transitions involving singlet oxygen and Cl-atoms in solvent molecules. The spectra and intensity of luminescence in the visible region strongly depended on the chemical structure and fluorescence properties of the photosensitizers. When the main fluorescence maximum of a photosensitizer was shorter than 630 nm, the 635, 703 and 780 nm, luminescence bands corresponding to the singlet-oxygen dimols were observed. When main fluorescence maxima of photosensitizers or products of their photodestruction were at (lambda) < 700 nm, the luminescence spectra corresponded to fluorescence of photosensitizers and products of their photodestruction. The data suggest that this luminescence is emitted by singlet oxygen dimols activated in collisions with photosensitizer molecules and by molecules of the photosensitizers and products of their destruction excited as a result of energy migration from singlet oxygen dimols. Some photosensitizers as metal-free tetra-4-tret-butyl-phthalocyanine and bis(tri-n-hexylsiloxy)silicon-2,3-naphthalocyanine are extremely strong amplifiers of the dimol emission. This shows that phthalocyanines and naphthalocyanines might be used for luminescence detection of monomols and dimols of singlet oxygen in systems of biomedical importance.