The application of near-infrared magnetic circular dichroism spectroscopy to the study of heme proteins is discussed. Recent studies of yeast cytochromes c, c1 and b and of spinach cytochrome f leading to assignments of the axial ligation in these important electron transport proteins are summarised.

Vibrational Circular Dichroism (VCD) has proven to be useful for detecting conformational change and for characterizing secondary structures in polypeptides, proteins and nucleic acids. Recent progress in these areas is reviewed with an emphasis on the contribution of VCD studies to furthering the understanding of conformational aspects of these problems.

Vibrational optical activity has emerged as an important new technique for probing local solution conformation in chiral biological molecules. Vibrational circular dichroism, in particular, has provided information on conformations and intramolecular hydrogen bonding in sugars, amino acids and amino acid derivatives, small peptides, and catecholamine neurotransmitters and related molecules through application of the coupled oscillator and ring-current mechanisms.

A method is described which is capable of measuring circular dichroism, either natural or induced by an applied magnetic field, with time resolution of nanoseconds or better. Circular dichroism is determined from the change in polarization properties of an elliptically polarized probe beam. Such measurements can be made with high sensitivity, making rapid measurements feasible. Procedures for making rapid circular dichroism measurements and precautions which must be taken in obtaining and interpreting the signals are discussed and examples of the application of the techniques in inorganic photochemistry and biophysics are presented.

Many problems in cancer research deal with the functional change in a biomolecule due to an alteration in its structure or conformation. Such changes can come about from ligand binding, drug intercalation, radiation and/or chemical damage, or from changes in the molecule's environment. In many cases, such a small change in the structure or conformation of the molecule can have far reaching consequences. It is important, therefore, to develop research tools capable of studying small differences in conformations and/or small alterations in molecules of biological importance in aqueous media. We are proposing to apply wavelength modulation derivative spectroscopy, a high sensitivity method used in Condensed Matter Physics, to the study of small structural changes in biomolecules. Preliminary studies suggest that such infrared spectroscopy can be performed on biomolecules dissolved or dispersed in aqueous media, which overcomes a major obstacle - the need to work with nonaqueous solvents - in the application of infrared spectroscopy to the study of biomolecules.

Recent advances in FTIR spectroscopy provide the basis for investigating the role of individual chemical groups in the functional mechanisms of large biomolecules. Vibrations from these groups can be identified in FTIR difference spectra and, in some cases, in the total absorption spectra of biomolecules. Examples are given for two membrane proteins, rhodopsin and bacteriorhodopsin.

The study of pressure-induced protein:protein interactions, and in particular the pressure-induced denaturation and dissociation of oligomeric proteins, has gained considerable interest in recent years. It is now well recognized that the protein transformation known under the general name of denaturation is a physical process which affects the three dimensional protein structure,leading to chain unfolding and/or refolding. Denaturation can be induced by changes in temperature or pressure, and by the addition of denaturing agents. The rationale for using pressure to study protein denaturation is that pressure variations at constant temperature only affect the volume of the protein, unlike the more commonly used temperature denaturation, which affects both the volume and the kinetic properties of the protein.

Polymerizable, diacetylenic lecithins form cylindrical microstructures (tubules) in water at the liquid-crystalline to gel phase transition, or by isothermal crystallization from organic solvents. The mechanism and driving force for the formation of tubules is unknown. Our approach to understanding tubule formation has been to probe conformational order by vibrational and magnetic resonance spectroscopies during the polymorphic phase changes that precede tubule formation. FTIR spectral features and order parameters derived from Raman data both indicate that tubules in excess water show a surprising degree of conformational order in the aryl chains. In addition, low frequency Raman spectra indicate the presence of longitudinal acoustic modes (LAMS) which may be assigned to chain segments above and below the diacetylenic moieties. We are currently examining a number of deuterated analogs of these compounds with FTIR, Raman, and NMR spectroscopies in order to make more definitive assignments of the LAMS, and understand more clearly the order-disorder of particular regions of the chains.

The sensitivity of the indole chromophore and it excited state properties to solvent perturbations is assessed through the use of substituted indole derivatives in solution and complexed within a fis-cyclodextrin cavity. This system is presumed to be a prototype to study the effect of static perturbations of the indole chromophore on its' excited state relaxation mechanisms. A hypothesis is presented relating the electronic resonance structures of the indole molecule to its exciplex forming capabilities. Two locations on the indole ring are proposed to provide the most stable exciplex forming sites. One location, C-3, has long been recognized as a location for exciplex formation. However, a second site on the benzyl component of indole, C-5, may also provide a site for exciplex formation due to a different resonance structure. We postulate that this site, C-5, gains a negative charge through a resonance structure making it capable of forming an exciplex with polar molecules. This resonance structure involves the indole nitrogen donating it lone pair electrons to form a double bond with the six member ring of indole. Substitutions of the indole molecule that facilitate this resonance structure either through induction or resonance effects will produce an excited state indole molecule that will have the largest Stokes shift and will have enhanced susceptibility to solvent induced radiationless decay processes.

High resolution emission spectra, 1-photon and 2-photon excitation spectra, vibronic level decay times and saturation behavior have been measured for the polyenes diphenylbutadiene and diphenylhexatriene seeded in supersonic helium expansions. For isolated diphenylbutadiene hyper-Raman scattering enhanced by 2-photon resonances has also been observed. Take together with spectroscopic studies of the static vapors, these data provide a detailed picture of the nature of the most probable intramolecular excited state decay processes as a function of vibronic energy for these flexible molecules.

Non-resonant, electronic components of static and dynamic third-order molecular polarizabilities (y) for ethylene, linear polyenes, and benzene are calculated. The specific dynamic polarizabilities treated are those for the third-order nonlinear optical processes of third-harmonic generation and electric-field-induced second-harmonic generation. Also calculated are one- and two-photon spectroscopic properties of the 1ππ* states salient to the π-electronic component of y(yπ). The yπ's for the linear polyenes are defined principally by chain-axis-polarized virtual electronic transitions between three states: (1) the ground state, (2) the strongly one-photon allowed 1"1Bu"ππ* state, and (3) a strongly two-photon allowed n"1Ag" (n>2) ππ* state.

Much attention is paid in the recent years to the mobility properties of the components of cell surface membranes, especially specific receptors and antigens, since the description of the dynamic behavior of these macromolecules is necessary for the understanding of the mechanism of signal transduction through the biological membranes. The use of time-resolved optical methods for studying the rotational and lateral diffusional movements of membrane proteins was a subject for a number of reviews. This work provides a brief account of the use of time-resolved phosphorescence anisotropy decay measurements for studying the rotational dynamics of the integral membrane proteins on the surface of living cells.

Experimental and theoretical evidence is presented which suggests that two distinct forms of light-adapted bacteriorhodopsin may exist. We propose that these two forms have characteristic photocycles with significantly different primary quantum yields. INDO-PSDCI molecular orbital procedures and semiempirical molecular dynamics simulations predict that one ground state geometry of bR undergoes photochemistry with a primary quantum yield, Φ1, of ~ 0.27, and that a second ground state geometry, with a slightly displaced counterion, yields Φ1 ~ 0.74. This theoretical model is supported by the observation that literature measurements of Φ1 tend to fall into one of two categories- those that observe Φ1 ~ 0.33 or below, and those that observe Φ1 ~ 0.6 or above. The observation that all photostationary state measurements of the primary quantum yield give values near 0.3, and all direct measurements of the quantum yield result in values near 0.6, suggests that photochemical back reactions may select the bacteriorhodopsin conformation with the lower quantum yield. The two photocycles may have developed as a natural biological requirement that the bacterium have the capacity to adjust the efficiency of the photocycle in relation to the intensity of light and/or membrane electrochemical gradient

The picosecond fluorescence kinetics of tryptophan residues in bacteriorhodopsin (bR) and some perturbed analogues are measured to study the different tryptophan environments and their changes upon metal cation removal, partial delipidation, retinal removal, and M412 trapping. In bR, the emission shows four decay components designated C1R, C2R, C3R, and C4R in order of increasing lifetimes. The emission wavelength of C3R and C4R is near that found in aqueous solution, while that of C1R is the shortest. The removal of retinal triples the total emission intensity and reduces the number of components to two, suggesting that the observed variation of the lifetimes in bR results from the variation of the energy transfer efficiency between different tryptophans and retinal. We conclude that the C1R and C2R emission is from the closest tryptophans to the retinal. The quenching of the C3R emission by all metal cations, including those that cannot act as energy acceptors, e.g. Cali-, is attributed to protein conformation changes caused by metal cation binding which leads to a stronger energy transfer coupling between tryptophans and retinal. The additional quenching of the C2R emission in Eu3+ bound bR is proposed to result from direct energy transfer between tryptophans and Eu3+. No effect on tryptophan emission decay kinetics and intensity by adding metal cations to deionized delipidated bR indicates that the protein conformation is changed by delipidation. But the similar kinetics between bR and delipidated bR suggest that the delipidation provides a proper protein conformation to deprotonate the protonated Schiff base without metal cations. The formation of M412 at low temperature in glycerol leads to the quenching of only the faster emission decay component.

The flash-induced difference absorbance spectra of bacteriorhodopsin (BR) were measured at various times after an actinic flash using a diode array spectrophotometer (300-700 nm). Difference spectra for three late bacteriorhodopsin photocycle intermediates Mfast (Mf), mslow (Ms) and R are reported. The main distinguishing features of the 3 difference spectra at pH = 10.5 and 5 °C are as follows: Mf ΔAmax = 412 nm, a shoulder at 436 nm, no absorbance change at 350 nm, ΔAmin = 565 nm, ΔA412/ ΔA565 = 0.8-0.9. Ms: ΔAmax = 412 nm, a shoulder at 386 nm, ΔAmin = 570-575 nm, ΔA.412/ ΔA575 = 0.6. R: ΔAmax = 336 and 350 nm (double peak), minor peaks at 386 and 412 nms,ΔAmin = 585-590 nm; ΔA350/ ΔA585 = 0.2. The t1/2 of Mf, Ms and R and the relative weights of BR570 recovered with these rates are: 1 sec (50%), 3-5 sec (25%) and 35 sec (25%) respectively. These spectral features can also be seen at pH = 7, -16 °C, and at pH = 9-10.5, 20 °C. Based on some assumptions, the absorption maximum of R was calculated to be at ca. 550 nm. The extinction coefficient of R is approximately 70% that of light-adapted BR. We suggest that: 1) Mf decays into R and R decays to BR, and 2) there are two types of BR with independent photocycles; Ms and probably 0 are in the photocycle near neutral pH, and Mf and R in a photocycle predominant at higher pH.

Femtosecond time-resolved absorption and time-resolved resonance Raman spectroscopy are used to examine the molecular mechanism of the light-driven proton pump in bacteriorhodopsin. Transient absorption spectroscopy with 6-fs pulses is used to directly observe the excited-state, double bond torsional isomerization of the retinal chromophore. Time-resolved resonance Raman spectroscopy is used to determine the structure of the retinal prosthetic group in each of the subsequent photolytic intermediates. Based on these results, a new "C-T Model" for the molecular mechanism of the proton pump has been developed. The key feature of this model is an isomerization-driven conformational change of the protein from the T-form to the C-form which acts as a "reprotonation switch" and stores energy to drive later events in the photocycle.

Resonance Raman spectra are reported for the copper(II) complex of trans-octaethylisobacteriochlorin with excitation in the B and Qy absorption bands. Assignments are proposed for a number of the high-frequency (above 1400 cm-1) skeletal modes of the macrocycle. Normal coordinate calculations are also performed on the complex. These calculations indicate that the normal coordinates of isobacteriochlorins bear little resemblance to those of porphyrins or chlorins.

Typically, there are specific molecular interactions between a substrate and enzyme, which occur during enzymatic catalysis, that must be sufficient to not only reduce the transition state barrier appropriate for the reaction but also define a particular (usually small) set of chemical reactions. Both the origins and strengths of these interactions are fundamental issues in understanding how enzymes work. While more structural information is increasingly available from X-ray crystallographic studies, the extent of these interactions and the electronic character of the substrate and nearby protein groups within the active site generally must be simply surmised from the structural data and kinetic studies. Rarely are these molecular properties directly measured. We have approached this problem by determining the vibrational spectra of bound substrates using Raman spectroscopy. The observed vibrational frequencies are a measure of force constants between particular atoms, and these constants can be related in turn to bond orders and electronic distributions between these atoms.

Two techniques are described for obtaining the Raman spectra of transient intermediates formed upon the binding of ligands to proteins. In the first of these the far ultraviolet line at 239 nm is used to take the spectrum of a flowing mixture of protein and ligand. In the second technique, the Raman spectra are taken from the interior of an enzyme crystal as substrate diffuses in from the mother liquor and is converted to the product through a series of chemical intermediates. Methods for interpreting the data are discussed.