Specific UV photodissociation of tyrosyl-containing peptides in multistage mass spectrometry.

UV photodissociation (UVPD) at 262 nm has been carried out on protonated tyrosyl-containing peptides formed by trypsin digestion of apo-transferrin. Under UVPD, the main event is the fragmentation of the C(alpha)-C(beta) bond of the tyrosyl residues leading to a radical ion 107 Da below the precursor ion. The dissociation rate of this specific cleavage appears to be strongly dependent on the peptide sequence and is more prominent on the singly protonated species than on the doubly protonated state. The fragmentation spectra resulting from collisional activation of the protonated even-electron native peptides and of the odd-electron radical species prepared by UVPD are dominated by y-type backbone cleavages. A comparison of their respective y-ion pattern shows complementarities since the combination of both increases the sequence coverage of the peptide sequence. The specific detection of the neutral loss of 107 Da from peptides witnesses the content of at least one tyrosyl residue and, though preliminary, is proposed as a potential new filtering strategy during protein database searching.

Experimental determination of the radiation dose limit for cryocooled protein crystals.

Radiation damage to cryocooled protein crystals during x-ray structure determination has become an inherent part of macromolecular diffraction data collection at third-generation synchrotrons. Generally, radiation damage is an undesirable component of the experiment and can result in erroneous structural detail in the final model. The characterization of radiation damage thus has become an important area for structural biologists. The calculated dose limit of 2 x 10(7) Gy for the diffracting power of cryocooled protein crystals to drop by half has been experimentally evaluated at a third-generation synchrotron source. Successive data sets were collected from four holoferritin and three apoferritin crystals. The absorbed dose for each crystal was calculated by using the program raddose after measurement of the incident photon flux and determination of the elemental crystal composition by micro-particle-induced x-ray emission. Degradation in diffraction quality and specific structural changes induced by synchrotron radiation then could be compared directly with absorbed dose for different dose/dose rate regimes: a 10% lifetime decrease for a 10-fold dose rate increase was observed. Remarkable agreement both between different crystals of the same type and between apoferritin and holoferritin was observed for the dose required to reduce the diffracted intensity by half (D(1/2)). From these measurements, a dose limit of D(1/2) = 4.3 (+/-0.3) x10(7) Gy was obtained. However, by considering other data quality indicators, an intensity reduction to I(ln2) = ln2 x I(0), corresponding to an absorbed dose of 3.0 x 10(7) Gy, is recommended as an appropriate dose limit for typical macromolecular crystallography experiments.

Structural characterization of the apo form of a calcium binding protein from Entamoeba histolytica by hydrogen exchange and its folding to the holo state.

One of the calcium binding proteins from Entamoeba histolytica (EhCaBP) is a 134 amino acid residue long (M(r) approximately 14.9 kDa) double domain EF-hand protein containing four Ca(2+) binding sites. CD and NMR studies reveal that the Ca(2+)-free form (apo-EhCaBP) exists in a partially collapsed form compared to the Ca(2+)-bound (holo) form, which has an ordered structure (PDB ID ). Deuterium exchange studies on the partially structured apo-EhCaBP reveal that the C-terminal domain is better structured than the N-terminal domain. The protein can be reversibly folded and unfolded upon addition of Ca(2+) and EGTA, respectively. Titration shows a slow initial folding of the apo form with increasing Ca(2+) concentration, followed by a highly cooperative folding to its final state at a certain threshold of Ca(2+). Ca(2+) and the EGTA titration taken together show that site II in the N-terminal domain has the highest affinity for Ca(2+) contrary to earlier studies. Further, this study has thrown light on the relative Ca(2+) binding affinity and specificity of each site in the intact protein. A structural model for the partially collapsed form of apo-EhCaBP and its equilibrium folding to its completely folded holo state has been suggested. Large conformational changes seen in transforming from the apo to holo form of EhCaBP suggest that this protein should be functioning as a sensor protein and might have a significant role in host-parasite recognition.

Spectral analysis of pigment photobleaching in photosynthetic antenna complex LHCIIb.

Light-induced photooxidation of chlorophyll (Chl) a, b and xanthophylls was investigated in LHCIIb, the antenna pigment-protein complex of photosystem II. Absorption difference spectra at normal and low temperatures show initially (at less than 25% Chl a decay) a selective bleaching of a red-shifted Chl b with absorption bands at 487 and 655 nm, Chl b (460/650 nm) and Chl a (433/670 nm), which changes to a less selective photooxidation pattern at deeper bleaching stages. Difference absorption spectra and HPLC analyses indicate different photooxidation rates of pigments in the order neoxanthin>Chl a>lutein approximately Chl b. Despite significant pigment loss as monitored with absorption spectra, CD spectra indicate an essentially complete persistence of the protein secondary structure. Fluorescence excitation spectra suggest the conversion of a small fraction of Chl a into pheophytin a which acts as a fluorescence quencher, possibly through temporary charge separation process. The strong features in the electroabsorption (Stark effect) spectra due to chlorophyll b at 655 nm and a xanthophyll at 510 nm, and the spectral changes mentioned above are assigned to Chl molecules located at several binding sites in LHCIIb protein and are discussed in the context of spatial configuration and interactions of pigment molecules.

Algal rhodopsins: phototaxis receptors found at last.

The discovery of two distinct Chlamydomonas sensory receptors responsible for phototaxis reveals additional diversity among the microbial rhodopsins. Sequence and architecture comparisons among this growing family highlight key components for light-responsive functions.

Light-induced hydrolysis and rebinding of nonisomerizable bacteriorhodopsin pigment.

Bacteriorhodopsin (bR) is characterized by a retinal-protein protonated Schiff base covalent bond, which is stable for light absorption. We have revealed a light-induced protonated Schiff base hydrolysis reaction in a 13-cis locked bR pigment (bR5.13; lambda(max) = 550 nm) in which isomerization around the critical C13==C14 double bond is prevented by a rigid ring structure. The photohydrolysis reaction takes place without isomerization around any of the double bonds along the polyene chain and is indicative of protein conformational alterations probably due to light-induced polarization of the retinal chromophore. Two photointermediates are formed during the hydrolysis reaction, H450 (lambda(max) = 450 nm) and H430 (lambda(max) = 430 nm), which are characterized by a 13-cis configuration as analyzed by high-performance liquid chromatography. Upon blue light irradiation after the hydrolysis reaction, these intermediates rebind to the apomembrane to reform bR5.13. Irradiation of the H450 intermediate forms the original pigment, whereas irradiation of H430 at neutral pH results in a red shifted species (P580), which thermally decays back to bR5.13. Electron paramagnetic resonance (EPR) spectroscopy indicates that the cytoplasmic side of bR5.13 resembles the conformation of the N photointermediate of native bR. Furthermore, using osmotically active solutes, we have observed that the hydrolysis rate is dependent on water activity on the cytoplasmic side. Finally, we suggest that the hydrolysis reaction proceeds via the reversed pathway of the binding process and allows trapping a new intermediate, which is not accumulated in the binding process.

A deletion in the PHYD gene of the Arabidopsis Wassilewskija ecotype defines a role for phytochrome D in red/far-red light sensing.

The PHYD gene of the Wassilewskija (Ws) ecotype of Arabidopsis contains a 14-bp deletion (the phyD-1 mutation) beginning at amino acid 29 of the reading frame, resulting in translation termination at a nonsense codon 138 nucleotides downstream of the deletion end point. Immunoblot analyses showed that Ws lacks phyD but contains normal levels of phyA, phyB, and phyC. By backcrossing into the Ws and Landsberg erecta genetic backgrounds, we constructed sibling pairs of PHYD+ and phyD-1 lines and of phyB- PHYD+ and phyB- phyD- lines. Hypocotyl lengths after growth under white or red light increased sequentially in strains that were B+D+, B+D-, B-D+, and B-D-. In the Ws genetic background, an increase in petiole length, a reduction in cotyledon area and in anthocyanin accumulation in seedling stems, a diminished effect of an end-of-day pulse of far-red light on hypocotyl elongation, and a decrease in the number of rosette leaves at the onset of flowering were also seen sequentially in these lines. Thus, phyD, which is approximately 80% identical in amino acid sequence to phyB, acts in conjunction with phyB in regulating many shade avoidance responses. The existence of the apparently naturally occurring phyD-1 mutation indicates that phyD is not essential in some natural environments.

Phototransformation of pea phytochrome A induces an increase in alpha-helical folding of the apoprotein: comparison with a monocot phytochrome A and CD analysis by different methods.

The photoreversible conformational change associated with the Pr-->Pfr transformation of a dicot phytochrome A (Pisum sativum, pea) has been probed by circular dichroism (CD) studies. Three different CD analysis methods have been used to determine the secondary structure of pea phytochrome A in both Pr and Pfr forms. We have shown that the secondary structure of dicot pea phytochrome A is very similar to the structure of monocot oat phytochrome A which was determined earlier [Sommer & Song (1990) Biochemistry 29, 1943-1948]. As with oat phytochrome A, an increase in the alpha-helical folding of the apoprotein takes place when photochrome in the Pr form is phototransformed to the Pfr form. This conformational change might well be a general characteristic of all phytochrome A's.

[The cytotoxicity of oxidized lipoproteins]. / Etude de la cytotoxicité des lipoprotéines oxydées.

The peroxidation of low-density lipoproteins (LDL) and their subsequent cytotoxicity is believed to be involved in the atherogenesis. The aim of this work was to determine the possible involvement of lipid peroxides in the cytotoxicity of lipoproteins. We report a new experimental model system constituted by lipoproteins treated by short-UV radiations which result in a major lipid peroxidation without functional alteration of apoproteins. UV radiations induced the following lipid modifications: the content of polyunsaturated fatty acids decreased considerably in all lipid classes; the level of natural antioxidants, i.e. vitamin E and carotene, dropped dramatically; conjugated dienes and thiobarbituric acid reactive substances (TBARS) were notably increased; apoproteins from LDL exhibited little structural modification but no functional alteration. The cytotoxicity was studied in lymphoblastoid cell lines established from lymphocytes derived from normal subjects or from patients with familial hypercholesterolemia. High density lipoproteins (HDL) were shown to inhibit the cytotoxicity induced by low doses of peroxidized LDL; the protective effect of HDL was complete up to the ratio ApoB/ApoA close to 1. In addition, vitamin E and catechin, two well known antioxidants, blocked the cytotoxicity of peroxidized LDL. These results corroborate the possibility of the synergic effect of HDL and antioxidant molecules for the protection of cells against oxidized LDL that are incorporated through the LDL-receptor pathway.

[Energizing of F1-ATPase after irradiation with visible light]. / Energizatsiia F1-ATFazy pri obluchenii vidimym svetom.

It was shown that ATP synthesis by F1-ATPase sensitized by visible light was combined with oxidation of SH groups and decrease of initial flavin fluorescence in the F1-ATPase preparation. It was suggested that it was an endogenous flavin group that regulated redox transitions between SH-S-S groups which was essential for the catalysis in vivo.

The riboflavin-sensitized photooxidation of horseradish apoperoxidase.

Native horseradish peroxidase, as well as its reduced and carboxymethylated form, and the apoenzyme, showed resistance to photodynamic action. Sensitivity to this action was detected only in reduced and carboxymethylated apoenzyme, when the photooxidation of its histidine residues was observed. When analyzing the bulk hydrophobic character (Hf) and the accessibility coefficients (Br) in those amino acid residues which can be subjected to photooxidation in horseradish peroxidase, it was found that all of them are situated in hydrophobic zones with low accessibility coefficients. This could justify the high resistance of this enzyme to photodynamic action. The only exception is tryptophan-117, which has low values of Hf and Br, and therefore its resistance to photodynamic action can only be explained in terms of its location and environment. Tryptophan-117 would be situated in a zone of antiparallel beta-structure, according to Chou and Fasman's predictive method for protein conformation.