Wayne W. Wright

The hydration of amides in helices; a comprehensive picture from molecular dynamics, IR, and NMR

We examined the hydration of amides of α3D, 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 (∼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 13C‐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 310/α/π‐helical conformation.

Nitric oxide diffusion coefficients in solutions, proteins and membranes determined by phosphorescence

The reactivity of nitric oxide under a given condition is a complex function of its diffusivity and the concentration of reacting partners. Quenching by NO of luminescence from Ru and Pd chelates of mesoporphyrin IX, two molecules which exhibit phosphorescence at room temperature, was utilized to evaluate the gas concentration and apparent diffusion coefficients. The properties of Ru-mesoporphyrin, a dye not previously employed as a probe for O2 or NO, were determined and the assay was verified and used to quantify NO produced by decomposition of nitrosocysteine. The pseudo-second order quenching constants were obtained from Stern-Volmer plots measured under various conditions and used to calculate diffusion coefficients for nitric oxide in solutions, proteins and membranes. The diffusion coefficients were greater at 37 than at 25 degrees C and, at a given temperature, smaller in proteins and membranes than in water. The conclusion is that NO and O2 closely resemble each other in diffusivity but that NO is slightly less lipophilic, resulting in somewhat faster apparent diffusion in protein and slower diffusivity in lipid, relative to O2. Taking a mean diffusion coefficient for NO of 10(-7) cm2s-1, then within 10 s the mean path is 10(-3) cm, or less than the diameter of a single cell. However, at low NO and O2 concentrations, the halflife of NO will be considerably longer than 10 s, and consequently the path of NO diffusion much greater.

Protein in Sugar Films and in Glycerol/Water as Examined by Infrared Spectroscopy and by the Fluorescence and Phosphorescence of Tryptophan

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.

Mixed trehalose/sucrose glasses used for protein incorporation as studied by infrared and optical spectroscopy.

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.

Use of IR absorption of the carboxyl group of amino acids and their metabolites to determine pKs, to study proteins, and to monitor enzymatic activity

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.

Horseradish peroxidase monitored by infrared spectroscopy: effect of temperature, substrate and calcium

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.

Influence of static and dynamic disorder on the visible and infrared absorption spectra of carbonmonoxy horseradish peroxidase.

Spectroscopy of horseradish peroxidase with and without the substrate analog, benzohydroxamic acid, was monitored in a glycerol/water solvent as a function of temperature. It was determined from the water infrared (IR) absorption that the solvent has a glass transition at 170-180 K. In the absence of substrate, both the heme optical Q(0,0) absorption band and the IR absorption band of CO bound to heme broaden markedly upon heating from 10-300 K. The Q(0,0) band broadens smoothly in the whole temperature interval, whereas the IR bandwidth is constant in the glassy matrix and increases from 7 to 16 cm(-1) upon heating above the glass transition. Binding of substrate strongly diminishes temperature broadening of both the bands. The results are consistent with the view that the substrate strongly reduces the amplitude of motions of amino acids forming the heme pocket. The main contribution to the Q(0,0) bandwidth arises from the heme vibrations that are not affected by the phase transition. The CO band thermal broadening stems from the anharmonic coupling with motions of the heme environment, which, in the glassy state, are frozen in. Unusually strong temperature broadening of the CO band is interpreted to be caused by thermal population of a very flexible excited conformational substrate. Analysis of literature data on the thermal broadening of the A(0) band of Mb(CO) (Ansari et al., 1987. Biophys. Chem. 26:337-355) shows that such a state presents itself also in myoglobin.

Solvent dependent and independent motions of CO–horseradish peroxidase examined by infrared spectroscopy and molecular dynamics calculations

The role of the solvent matrix in affecting CO bound to ferrous horseradish peroxidase was examined by comparing band-widths of nu(CO) 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[bond]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.

Orientation distributions for cytochrome c on polar and nonpolar interfaces by total internal reflection fluorescence.

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 proteins zinc-substituted porphyrin was found to be 41 degrees and 50 degrees 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 9 degrees, 1 degrees, and 18 degrees, 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.

Phenylalanine fluorescence and phosphorescence used as a probe of conformation for cod parvalbumin

The fluorescence emission and triplet absorption properties of phenylalanine in cod fish parvalbumin type II, a protein that contains no Trp or Tyr, was examined in the time scale ranging from nanoseconds to microseconds at 25°C in aqueous buffer (pH 7.0). In the presence of Ca(II), the decay of fluorescence gave two lifetimes (5.9 and 53 ns) and the triplet lifetime was 425 μs. Upon removal of Ca, the fluorescence intensity decreased to values approaching that for free Phe, while the longest fluorescence decay component was 17 ns. At the same time, the decay of triplet showed complex nonexponential kinetics with decay rates faster than in the presence of Ca. Quenching and denaturation analyses suggest that the Phes are present in a hydrophobic environment in the Ca-bound protein but that the Ca-free protein is relatively unstructured. It is concluded that Phe luminescence in proteins is sensitive to conformation and that the long lifetime of Phe excited states needs to be considered when studying its photochemistry in proteins.