Fluorescence line narrowing is a sensitive tool to monitor subtle changes in protein conformation. We restudied the consequences of inhomogeneous broadening on the observed spectra. Vibronic absorption bands of systems with weak electron-phonon coupling can be described by a lorentzian function with width of the order of a few centimeters. For an ensemble of molecules with closely spaced electronic transitions, a single laser frequency will excite many molecules through the broad base (“tail”) of the lorentzian function. This, along with excitation of low-frequency modes, contributes to the unresolved background observed in line-narrowed spectra. Examples are shown for Zn cytochrome c, a fluorescent derivative of Fe cytochrome c. Spectra are compared for the protein in two solvents: glycerol/water or trehalose. Two types of cytochrome, from horse and yeast, are also compared.

The heme of horseradish peroxidase is buried in the protein, but a channel from the protein surface connects the aqueous solution to the heme site. Ferric horseradish peroxidase has an absorption band at 640 nm that is attributed to a charge-transfer (CT) transition between the a(2u) HOMO of T electrons of the porphyrin ring and the d(xy)/d(yz) orbital of the ferric ion. Because the water channel extends to the Fe, it seems likely that the CT band will be sensitive to the hydration of the protein. To study this premise, the protein was incorporated into trehalose/sucrose glasses and the hydration of the sugar glasses was varied. Absorption spectra of HRP in sugar glasses and in glycerol/water were taken in the range 10-300 K. The CT absorption band shows vibronic fine structure. The peak positions are the same in hydrated sugar and glycerol/water but the peak positions change in desiccated sugar glass. The data suggest that in hydrated, but not desiccated, sugar glass, water is retained in the heme pocket. Binding of the competitive inhibitor benzohydroxamic acid to the protein increases the CT absorption and resolution. The effect of benzohydroxamic acid on the Fe as calculated using a combination of density functional theory and molecular mechanics is to stabilize the spin state 3/2 with respect to 5/2. At low temperature the widths of the lines in the CT band are narrower for the protein in glycerol/water (glass transition at similar to150 K) than in trehalose/sucrose (glass formation at 65 degreesC). This indicates that the CT band is inhomogeneously broadened and sensitive to the solvent. The spectral narrowing of the CT absorption occurs as the temperature decreases over the temperature range studied. Water, as indicated by the OH stretch, also shifts in this range. The findings are discussed in terms of how buried water and nearby charges can modulate the activity of the heme.

We examined the hydration of amides of alpha(3)D, a simple, designed three-helix bundle protein. Molecular dynamics calculations show that the amide carbonyls on the surface of the protein tilt away from the helical axis to interact with solvent water, resulting in a lengthening of the hydrogen bonds on this face of the helix. Water molecules are bonded to these carbonyl groups with partial occupancy ( approximately 50%-70%), and their interaction geometries show a large variation in their hydrogen bond lengths and angles on the nsec time scale. This heterogeneity is reflected in the carbonyl stretching vibration (amide I' band) of a group of surface Ala residues. The surface-exposed amides are broad, and shift to lower frequency (reflecting strengthening of the hydrogen bonds) as the temperature is decreased. By contrast, the amide I' bands of the buried (13)C-labeled Leu residues are significantly sharper and their frequencies are consistent with the formation of strong hydrogen bonds, independent of temperature. The rates of hydrogen-deuterium exchange and the proton NMR chemical shifts of the helical amide groups also depend on environment. The partial occupancy of the hydration sites on the surface of helices suggests that the interaction is relatively weak, on the order of thermal energy at room temperature. One unexpected feature that emerged from the dynamics calculations was that a Thr side chain subtly disrupted the helical geometry 4-7 residues N-terminal in sequence, which was reflected in the proton chemical shifts and the rates of amide proton exchange for several amides that engage in a mixed 3(10)/alpha/pi-helical conformation.

The role of the solvent matrix in affecting CO bound to ferrous horseradish peroxidase was examined by comparing band-widths of νCO for the protein in aqueous solutions and in trehalose/sucrose glasses. We have previously observed that the optical absorption band and the CO stretching mode respond to the glass transition of glycerol/water in ways that depend upon the presence of substrate (Biochemistry 40 (2001) 3483). It is now demonstrated that the CO group band-width for the protein with bound inhibitor benzhydroxamic acid is relatively insensitive to temperature or the glass transition of the solvent. In contrast, in the absence of inhibitor, the band-width varies with the temperature that the glass is formed. The results show that solvent dependent and independent motions can be distinguished, and that the presence of substrate changes the protein such that the Fe–CO site is occluded from the solvent conditions. Molecular dynamic calculations, based upon X-ray structures, showed that the presence of benzhydroxamic acid decreases the distance between His42 and Arg38 and this leads for closer distances to the O of the CO from these residues. These results are invoked to account for the observed line width changes of the CO band.

Sugars are known to stabilize proteins. This study addresses questions of the nature of sugar and proteins incorporated in solid sugar films. Infrared (IR) and Raman spectroscopy was used to examine trehalose and sucrose films and glycerol/water solvent. Proteins and indole-containing compounds that are imbedded in the sugar films were studied by IR and optical (absorption, fluorescence, and phosphorescence) spectroscopy. Water is able to move in the sugar films in the temperature range of 20-300 K as suggested by IR absorption bands of HOH bending and OH stretching modes that shift continuously with temperature. In glycerol/water these bands reflect the glass transition at approximately 160 K. The fluorescence of N-acetyl-L-tryptophanamide and tryptophan of melittin, Ca-free parvalbumin, and staphylococcal nuclease in dry trehalose/sucrose films remains broad and red-shifted over a temperature excursion of 20-300 K. In contrast, the fluorescence of these compounds in glycerol/water solvent shift to the blue as temperature decreases. The fluorescence of the buried tryptophan in Ca-bound parvalbumin in either sugar film or glycerol/water remains blue-shifted and has vibronic resolution over the entire temperature range. The red shift for fluorescence of indole groups exposed to solvent in the sugars is consistent with the motion of water molecules around the excited-state molecule that occurs even at low temperature, although the possibility of static complex formation between the excited-state molecule and water or other factors is discussed. The phosphorescence yield for protein and model indole compounds is sensitive to the matrix glass transition. Phosphorescence emission spectra are resolved and shift little in different solvents or temperature, as predicted by the small dipole moment of the excited triplet state molecule. The conclusion is that the sugar film maintains the environment present at the glass formation temperature for surface Trp and amide groups over a wide temperature excursion. In glycerol/water these groups reflect local changes in the environment as temperature changes.

The spectroscopy of horseradish peroxidase with and without the substrate analogue benzohydroxamic acid (BHA) was monitored in different solvents as a function of the temperature in the interval from 10 to 300 K. Thermal broadening of the Q(0,0) optical absorption band arises mainly from interaction of the electronic pi --> pi transition with the heme vibrations. In contrast, the width of the IR absorption band of CO bound to heme is controlled by the coupling of the CO transition moment to the electric field of the protein matrix. The IR bandwidth of the substrate free enzyme in the glycerol/H2O solvent hardly changes in the glassy matrix and strongly increases upon heating above the glass transition. Heating of the same enzyme in the trehalose/H2O glass considerably broadens the band. The binding of the substrate strongly diminishes the temperature broadening of the CO band. This result is consistent with the view that the BHA strongly reduces the amplitude of vibrations of the heme pocket environment. Unusually strong thermal broadening of the CO band above the glass transition is interpreted to be caused by thermal population of a very flexible excited conformational substate. The thermal broadening of the same band in the trehalose glass is caused by an increase of the protein vibrational amplitude in each of the conformational substates, their population being independent of the temperature in the glassy matrix.

Porphyrin electronic transitions in heme proteins provide a useful tool for probing the protein environment, since the surrounding protein affects the porphyrin pi-electron cloud. Perturbations can arise from structural distortions of the porphyrin ring, from the internal electric field generated by charged and polar groups, or from axial ligation to the heme iron. In this work, cytochrome c in aqueous solution or in glasses of trehalose or glycerol/water was examined as a function of temperature to evaluate the effect of fluctuations on the heme. The amide I band of cytochrome c in trehalose remains constant over a wide temperature excursion, indicating that interactions between the protein and the matrix do not change with temperature. The width of the Q(0,0) optical transition measured at low temperature (i.e., < 100 K) reflects the temperature at which the glass was formed, while the temperature profiles of the widths for the protein in different solvents and glasses are similar at high temperature. The results were interpreted in terms of contributions from solvent-coupled and solvent-uncoupled motions. Molecular dynamics simulations of cytochrome c in explicit solvent were performed to investigate the structural distortions in the protein, and semiempirical quantum mechanics (Zindo/ S) was used to calculate the resultant changes in the spectroscopic transitions. A correlation between the calculated transition energies and the structural distortions in both the heme and the surrounding protein environment was observed and was invoked to characterize the origins of the temperature-dependent broadening of the electronic transitions seen in the visible spectra.

Evaporation of water from a 1/1 mixture of trehalose and sucrose gives rise to optically clear glasses that are transparent in the UV and visible ranges and do not crystallize when they are prepared at ambient temperatures. Two proteins, liver alcohol dehydrogenase and parvalbumin, and the tryptophan derivative N-acetyl-tryptophanamide were incorporated into the glasses. Infrared spectroscopy of the amide I band reveals that the proteins retain secondary structure in the glass over a temperature range of 20-300K. The amide II band of the protein and the HOH bending band of residual water in the glass shift with temperature changes, consistent with increased H-bonding strength as temperature is lowered. Phosphorescence of tryptophan can be seen from the proteins at room temperature, which shows the immobilization of the protein by the glass and the curbing of oxygen diffusion. It is suggested that using mixed sugars to form glasses is a way to immobilize proteins over a wide temperature range without distortions from solvent crystals.

The formation of chemisorbed monolayers of yeast cytochrome c on both uncharged polar and nonpolar soft surfaces of organic self-assembled monolayers (SAM) on solid inorganic substrates was followed in situ by polarized total internal reflection fluorescence. Two types of nonpolar surfaces and one type of uncharged polar surface were used. The first type of nonpolar surface contained only thiol endgroups, while the other was composed of a mixture of thiol and methyl endgroups. The uncharged polar surface was provided by the mixture of thiol and hydroxyl endgroups. The thiol endgroups were used to form a covalent disulfide bond with the unique surface-exposed cysteine residue 102 of the protein. The mean tilt angle of the protein's zinc-substituted porphyrin was found to be 41degrees and 50degrees for the adsorption onto the nonpolar and uncharged polar surfaces, respectively. The distribution widths for the pure thiol and the thiol/methyl and thiol/hydroxyl mixtures were 9degrees, 1degrees, and 18degrees, respectively. The high degree of the orientational order and good stability achieved for the protein monolayer on the mixed thiol/methyl endgroup SAM makes this system very attractive for studies of both intramolecular and intermolecular electron transfer processes.

Infrared and optical spectra of carbonmonoxy horseradish peroxidase were monitored as a function of pH and substrate binding. The analyses of experimental results together with semiempirical calculations show that the CO-porphyrin complex is sensitive to environmental changes. The electronic Q(0,0) band of the porphyrin and the CO stretching mode respond to external perturbations with different symmetry dependencies. In this way, the complex is nonisotropic, and the combined spectral analyses constitute a valuable tool for the investigation of structure. In the absence of substrate and at pH 6.0, the low-spin heme optical Q(0,0) absorption band is a single peak that narrows as the temperature decreases. Under these conditions, the CO vibrational stretch frequency is at 1903 cm(-1). Addition of the substrates benzohydroxamic acid or naphthohydroxamic acid produces a split of approximately 320 cm(-1) in the Q(0,0) absorption band that is clearly evident at < 100 K and shifts the CO absorption to 1916 cm(-1). Increasing the pH to 9.3 also causes a split in the Q(0,0) optical band and elicits a shift in nu(CO) to a higher frequency (1936 cm(-1)). The splitting of the Q(0,0) band and the shifts in the IR spectra are both consistent with changes in the local electric field produced by the proximity of the electronegative carbonyl of the substrate near the heme or the protonation and/or deprotonation of the distal histidine, although other effects are also considered. The larger effect on the Q(0,0) band with substrate at low pH and the shift of nu(CO) at high pH can be rationalized by the directionality of the field and the orientation dependence of dipolar interactions.

Low-temperature UV-vis absorption and Stark-effect hole-burning spectra of Zn substituted cytochrome c are studied experimentally and theoretically using quantum mechanical and Poisson-Boltzmann electrostatics models. Both the Q and Soret bands show resolved splitting at temperatures below similar to 180 K. The trend observed in the splittings when comparing cytochromes from different species is found to be the same as that observed for the Q(0,0) band of ferrous cytochrome c. The relative magnitudes of the Q and Soret splittings are found to be consistent with predictions based on Goutennan's four orbital model. For horse heart and yeast cytochrome c, which show the greatest difference in the UV-visible band splittings, Stark effect measurements on persistent spectral holes in the Q(0,0) band indicate that the protein-induced polarization is distinctly different for these two species. Incorporation of the protein electrostatic field as virtual point charges into quantum mechanical calculations utilizing the INDO/s semiempirical Hamiltonian is used to demonstrate that the effects of the protein on the heme electronic structure can be considerably different for the two proteins, consistent with the experimental observations.

Infrared spectral frequencies of amide vibrational modes are sensitive to secondary structure. In this work, evidence is presented that accessibility to water additionally affects spectral positions. The dimeric alpha -helical coiled-coil GCN4-P1' was C-13 labeled in the amide carbonyl groups of buried Leu or exposed Ala. At 20 degreesC, the amide I' peak for C-13 Ala amide is at 1585 cm(-1), whereas the position for C-13 Leu is at 1606 cm(-1). These shifts permit the distinction of solvent-exposed and buried amide groups. Lowering temperature increases H-bond strength, producing a shift to lower frequency. In the temperature range from 10 to 273 K in aqueous glycerol, the amide transitions assigned to solvent-exposed regions of the helices undergo the strongest temperature-dependent shifts, similar to that of the peptide bond model compound, N-methylacetamide, in the same aqueous solvent. In addition, spectral shifts of the amide bands for N-methylacetamide and the solvent-exposed component of the proteins follow the glass transition temperature of the cryosolvent. In contrast, the amide transitions assigned to alpha -helical segments that are expected to have little interaction with water undergo the weakest shifts. The amide I' band of the alpha -helical protein parvalbumin also shows subpeaks that shift differently with temperature, and on the basis of their temperature dependence and frequency can be assigned to solvent exposed or buried regions. The spectral shifts are discussed in terms of changes in hydrogen bond strengths, including contributions from volume expansion of the sample, and variations in the average hydrogen bond angle, induced by population of low-frequency librational modes involving the solvent and protein. The results on the isotopically labeled peptides conclusively show that alpha -helical regions that are or are not solvent exposed can be distinguished both by the position of the amide I' peak and by the temperature-dependent shifts.

Horseradish peroxidase was examined as a function of Ca and substrate binding using infrared spectroscopy in the temperature range of 10–300 K. The Ca complex could be identified by the carboxylate stretches. The amide peak positions indicate that the protein remains stable from room temperature to 10 K. Shifts in these peaks are consistent with increased hydrogen bonding as temperature decreases, but the protein conformation is maintained at cryogenic temperatures. The substrate, benzohydroxamic acid, produced no detectable change in the infrared spectrum, consistent with X-ray crystallography results. With removal of Ca, the protein maintained its overall helicity.

The effect of Ca2+ binding to parvalbumin was monitored by probes of conformation including absorption, fluorescence, circular dichroism (CD), infrared (IR) spectroscopy and differential scanning calorimetry. These experimental studies were compared with molecular dynamics computations on the structures of the Ca-bound and Ca-free forms of cod parvalbumin. The UV CD spectra show that removal of calcium results in a decrease in the alpha-helical content of the protein. The IR amide I' and III' regions are very much affected by Ca removal and are indicative of significant perturbation of secondary structure. The fluorescence of tryptophan, the IR markers, and UV ellipticity all show changes with temperature, pointing to a lowering of protein stability upon Ca removal. These results are consistent with the structures obtained for both the Ca-bound and Ca-free proteins after 200 ps of solvated molecular dynamics simulations which show a decrease in the secondary structure upon Ca removal.

The absorption spectra of 20 amino acids (Gly, Ala, Val, Leu, Ile, Ser, Thr, Asp, Asn, Glu, Gln, Lys, His, Arg, Phe, Trp, Cys, Met, Pro, and hexafluorovaline) and some of their metabolites (α-ketoglutarate, oxalacetate, pyruvate, succinate, citrate, and acetate) were determined in the infrared (IR) region from 1300 to 1700 cm−1 under conditions that are appropriate for biological studies (i.e., in phosphate-buffered D2O solution). The strongest transition in this region is , with an extinction coefficient ∼1 mM−1 cm−1, and an emphasis was made to demonstrate use of this transition for enzymatic assays and to study proteins. To these ends, these relevant features were demonstrated. The value for is a function of the residue pK: the higher the frequency, the lower the pK of the carboxylic acid. The high extinction of permits detection of carboxyl groups in parvalbumin, a protein that is rich in Asp and Glu. The IR profiles for the amino acids and their metabolite products are sufficiently characteristic so that IR can be used to monitor enzymatic reactions involving amino acids. We show that transaminase reactions, which interconvert amino and keto acids, can be monitored by IR. © 1997 John Wiley & Sons, Inc. Biospectroscopy 3: 457–467, 1997

The carboxylate groups of organic acids give strong absorption in the infrared between approximately 1550 and 1650 cm-1. For acetate and chloroacetate derivatives, the infrared (IR) frequency of the carboxylate antisymmetric stretching mode (v(a)OCO) is related to the square root of the pK of the acid, with a shift of approximately 20 cm-1 to higher frequency for a pK drop in the range 5-3. It follows that v(a)OCO may respond to conditions on the protein surface. In this paper, the IR amide I' and carboxylate absorptions of cytochrome c from horse, yeast, and tuna are compared with model compounds such as Val-Glu and microperoxidase-11, the 11 amino acid fragment of horse cytochrome c containing the covalently bound heme. For microperoxidase-11, the contribution from all four carboxylates can be accounted for and the 1567 cm-1 absorption is assigned to the heme propionates. For the proteins, the carboxylate absorption band is inhomogeneous, i.e., there is a distribution of frequencies. Both the amide I' and carboxylate bands are sensitive to protein conformation as shown by their different pH, salt, and redox dependence.