First-principles calculations of protein circular dichroism in the near ultraviolet

Electronic transitions in aromatic side chains are responsible for the characteristics of proteins in the near UV. We present the first systematic study of a large number of proteins focused on the accurate calculation of near-UV circular dichroism (CD) spectra. We report new parameter sets derived from ab initio calculations for benzene, phenol, and indole that describe the valence electronic transitions to the (1)L(b), (1)L(a), (1)B(b), and (1)B(a) states in the side chains of amino acids phenylalanine, tyrosine, and tryptophan. CD spectra were calculated, using the matrix method with the new side-chain parameters, for 30 proteins whose CD spectra and crystal structures have been made publicly available. The new parameter sets are fully self-consistent and yield near-UV spectra better than those obtained using previous parameter sets. The mean absolute errors for computed wild-type spectra in the near UV are reduced by a factor of approximately 2. A similiar reduction is found for the near-UV spectra (and difference spectra) of mutants involving aromatic amino acids. Empirical modifications to model vibronic coupling in the side-chain chromophore of phenylalanine offer no overall improvement. Protein CD calculations from first principles coupled with atomic-level modeling enhance the utility and interpretability of CD measurements in the near UV.     

Lethal cellular changes induced by near ultraviolet radiation.

There is clear evidence that significant quantities of lesions are induced in DNA by near-UV radiation and that these lesions, although susceptible to repair, may lead to cell death because of the simultaneous disruption of DNA repair systems by the same wavelengths. No particular DNA lesion can be linked to cell death in wild type strains. However, there are good grounds for speculating that a type of near-UV lesion exists which is rapidly "fixed" as a lethal event in cells as a result of the oxygen-dependent disruption of repair. There is a strong indication that the relative ability of various near-UV wavelengths to sensitize cells to heat, chemicals or other radiations is directly related to their efficiency in disrupting DNA repair systems in general. Some important specific questions remain. For example, it is important to ask why breaks formed at 365 nm and 405 nm, although apparently requiring a pol dependent pathway for their repair, do not produce the predicted lethal biological action in the strains tested. In general terms it is hoped to provide more comprehensive physico-chemical data in support of, or contradicting, the proposed model.     

ADP binding to TF1 and its subunits induces ultraviolet spectral changes

Adenine nucleotide binding sites on the coupling factor ATPase of thermophilic bacterium PS3 (TF1) were investigated by UV spectroscopy and by equilibrium dialysis. When ADP was mixed with TF1 in the presence and in the absence of Mg2+, an UV absorbance change was induced (t1/2 approximately 1 min) with a peak at about 278 nm and a trough at about 250 nm. Similar spectral changes were induced by ADP with the isolated beta subunits in the presence and in the absence of Mg2+, and with the isolated alpha subunits in the presence of Mg2+ although the magnitudes of the changes were different. From equilibrium dialysis measurement we identified two classes of nucleotide binding sites in TF1 in the presence of Mg2+, three high-affinity sites (Kd = 61 nM) and three low-affinity sites (Kd = 87 microM). In the absence of Mg2+, TF1 has one high-affinity site (Kd less than 10 nM) and five low-affinity sites (Kd = 100 microM). Moreover, we found a single Mg2+-dependent ADP binding site on the isolated alpha subunit and a single Mg2+-independent ADP binding site on the isolated beta subunit. From the above observations, we concluded that the three Mg2+-dependent high-affinity sites for ADP are located on the alpha subunit in TF1 and that the single high-affinity site is located on one of the beta subunits in TF1 in the absence of Mg2+.     

Structural and electronic characterization of heme moiety in oxygenated hemoproteins by using XANES spectroscopy.

Iron K-edge X-ray absorption near edge structure (XANES) spectra were measured for oxy-forms of cytochrome P-450cam (P-450cam), horseradish peroxidase (HRP) and myoglobin (Mb) by using Synchrotoron Radiation of Photon Factory (Tsukuba). A pronounced 1s-4p transition and some fine structures were well-resolved in the spectra obtained. Comparing the spectra, the features at the fine structures termed P, C and D, were similar among the three hemoproteins, suggesting a similar site-symmetry around the heme iron and the same Fe-O-O bond angle (about 115 degrees). On the other hand, absorption features at the edge region (7115-7135 eV) were slightly but significantly different from one another; the absorption intensity at 7115-7125 eV region increased in the order of Mb, HRP and P-450cam, while that at 7125-7135 eV decreased in the same order. A similar absorption feature was also obtained with their deoxy (ferrous high spin) forms. We assumed that the absorption at the lower energy region (7115-7125 eV) reflects the pi-character in the Fe-ligand bond, whereas that at the higher energy region (7125-7135 eV) does the sigma-character, on the basis of the previous and comprehensive studies of the XANES spectroscopy of the adsorbed molecules on the metal surface (McGovern et al. (1989) Handbook on Synchrotoron Radiation, Vol. 2, pp. 467-539). According to our assumption, our XANES results indicated that the pi-character of the Fe-ligand bond increases in the order of Mb, HRP and P-450cam, and that the pi-electron of the thiolate S- in P-450cam is donated to the Fe-O-O moiety, most probably to the antibonding pi* orbital of O2. Such an interpretation is consistent with the experimental findings or data accumulated so far by other methods, such as the resonance Raman spectroscopy.     

Dichroism of transient absorbance changes in the red spectral region using oriented chloroplasts. I. Field indicating absorbance changes

The light-induced transient absorbance changes which are affected by valinomycin have been studied using magnetically oriented spinach chloroplasts and a polarized measuring beam. The ΔA spectra for the two polarizations parallel and perpendicular to the plane of the photosynthetic membranes have been recorded in the spectral range 630–750 nm. Large polarization effects are found in all the bands of the ΔA spectrum, shifts in the position of the extrema are observed and the two spectra cross each other at various wavelengths. A comparison of these spectral features with available data on the dichroism of the Stark effect on monomolecular films of chlorophyll a and b indicates similarities favoring the already well documented hypothesis of the electrochromic nature of these absorbance changes in vivo.The data on this electrochromic effect can be correlated with the linear dichroism of oriented chloroplasts and the ΔA_∥?ΔA_⊥ spectrum in the 645–655 nm region gives further evidence of the orientation out of the membrane plane of the red transition moment of chlorophyll b.     

Heme-linked ionization and ligand binding produce identical changes of proximal heme stereochemistry in reduced horseradish peroxidase. Evidence for existence of two protein conformations

The visible and near infrared magnetic circular dichroism spectra of chemically reduced horseradish peroxidase at neutral and alkaline pH values and 5-coordinate protoheme-(2-methylimidazole) at pH 9.1 were compared at 4.2 K with those of photolysis products of their carbon monoxide complexes. From the results obtained we concluded that: (i) there are two protein conformations of HRP which determine the geometry of the Fe-N(His) bond; (ii) the transition from one conformation (heme stereochemistry) to another can be induced by either heme-linked ionization or ligand binding; (iii) a trigger mechanism for switching between two conformations has to exist.     

Structural and spectral response of green fluorescent protein variants to changes in pH

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become a useful tool in molecular and cell biology. Recently, it has been found that the fluorescence spectra of most mutants of GFP respond rapidly and reversibly to pH variations, making them useful as probes of intracellular pH. To explore the structural basis for the titration behavior of the popular GFP S65T variant, we determined high-resolution crystal structures at pH 8.0 and 4.6. The structures revealed changes in the hydrogen bond pattern with the chromophore, suggesting that the pH sensitivity derives from protonation of the chromophore phenolate. Mutations were designed in yellow fluorescent protein (S65G/V68L/S72A/T203Y) to change the solvent accessibility (H148G) and to modify polar groups (H148Q, E222Q) near the chromophore. pH titrations of these variants indicate that the chromophore pKa can be modulated over a broad range from 6 to 8, allowing for pH determination from pH 5 to pH 9. Finally, mutagenesis was used to raise the pKa from 6.0 (S65T) to 7.8 (S65T/H148D). Unlike other variants, S65T/H148D exhibits two pH-dependent excitation peaks for green fluorescence with a clean isosbestic point. This raises the interesting possibility of using fluorescence at this isosbestic point as an internal reference. Practical real time in vivo applications in cell and developmental biology are proposed.     

Enzymic nature of the protein moiety of protochlorophyllide holochrome

The enzymic nature of the protein moiety of protochlorophyll(ide) holochrome was studied by following the fate of the [(14)C]protochlorophyll(ide) formed when dark-grown barley (Hordeum vulgare) or bean (Phaseolus vulgaris) leaves are incubated in the dark with 3 mm 4-delta-[(14)C]aminolevulinic acid. It was found that: [List: see text]Since turnover of protochlorophyll(ide) was not observed, these results show that there is a free exchange between the old "endogenous" and the new delta-aminolevulinic-acid-induced protochlorophyll(ide) molecules on the active site of the holochrome protein. These results are consistent with the hypothesis that the holochrome protein acts as an enzyme.     

Ultrastructural changes in the erythrocytes irradiated with ultraviolet light

The ultrastructure of erythrocytes of donor blood and blood of patients suffering from kidney insufficiency was studied with the help of scanning and transmission electron microscopes before and after it was treated with ultraviolet light. It was concluded that after being treated with ultraviolet light, the ultrastructure of donor blood erythrocytes undergoes significant positive changes which are being characterized by normalization of the form of cells, decrease of associative ties between them, the cytoplasm content becomes more homogenous. There was a noticeable increase of a number of discocytes and a decreased number of associative ties between the cells after the patients' own blood was treated with ultraviolet light. The violation of the wholesomeness of erythrocytes membrane was not discovered. The indications are that the observed therapeutical effect after treating blood with ultraviolet light, is explained, to a certain extent, by the ultrastructural modification of erythrocytes membrane.