Effect of the UV Modification of α-Crystallin on Its Ability to Suppress Nonspecific Aggregation

Recent studies have shown that structural modifications of α-crystallin during lens aging decrease it's effectiveness as a molecular chaperone. Some of these posttranslational modifications have been linked to UV radiation, and this study was undertaken to investigate the effect of UV irradiation on the ability of α-crystallin to suppress nonspecific aggregation. The effect of 3-hydroxykynurenine (3-HK) was also investigated as a model for its glucoside (3-HKG), a main lens chromophore that has been linked to photochemical changes in the human lens. Alpha- and γ-crystallin solutions (1 mg/mL, 1:0.125 wt/wt) were photolyzed (transmission above 295 nm) for various time intervals. Thermal denaturation of γ-crystallin with or without α-crystallin was carried out at 70°C and increases in light scattering were measured at 360 nm. We found that (1) irradiation of γ-crystallin increased its susceptibility to heat-induced scattering. The addition of α-crystallin protects it against thermal denaturation, although its ability to do so decreases the longer γ-crystallin is irradiated and (2) irradiation of α-crystallin decreases its ability to suppress nonspecific aggregation and the presence of 3-HK during irradiation decreases it further. Our results indicate that posttranslational modifications of α-crystallin due to UV irradiation affect the sites and mechanisms by which it interacts with γ-crystallin. The kinetics of γ-crystallin unfolding during thermal denaturation were also analyzed. We found that a simple two state model applies for nonirradiated γ-crystallin. This model does not hold when γ-crystallin is irradiated in the presence or absence of α-crystallin. In these cases, two step or multistep mechanisms are more likely.     

CONCENTRATION DEPENDENCE OF TRANSMISSION LOSSES IN UV-LASER IRRADIATED BOVINE α-, βH-,βL-AND γ-CRYSTALLIM SOLUTIONS

Experiments with calf lens protein fractions in aqeous buffer solutions at room temperature showed that β_H - and β_L - and γ-crystallin fractions became opaque following ultraviolet exposure at 308 nm, while the α-crystallin fraction remained transparent. Transmission loss, due to UV-irradiation, for all of the crystallin samples was studied in the concentration range of 0.1 mg/mL to 1.0 mg/mL, and for α- and γ-crystallin, in the range up to 5 mg/mL. With increased concnetrations of β_H-,β_L-and γ-crystallin, the rate of opacification increased. However, with α-crystallin, the loss of transmission was negligble for all of the concentrations and irradiation times studied. Opacification of the crystallins was accompanied by formation of higher molecular weight insoluble proteins as detected by SDS-PAGE.     

Chaperone Function of Rat Lens α-Crystallin

A solution of γ-crystallin became turbid upon beating at 65 °C for 30 minutes; however, addition of α-crystallin suppressed this thermal aggregation. It was found the effective chaperone function could be achieved with the molar ratio of α/γ greater than 1/20. In terms of crystallin subunit, five molecular α-crystallin subunits could afford chaperone for one molecular γ-crystallin. The gel filtration profile of the sample solution, containing α- and γ-crystallins and preincubation at 65 °C for 30 minutes, showed complex formation between α- and γ-crystallins, indicating α-crystallin was bound to thermally denatured γ-crystallin. A 1-anilinonaphthalene-8-sulfonic acid (ANS) fluorescence study showed that α-crystallin has more hydrophobic regions exposed after thermal incubation. In the presence of urea, both the α-crystallin chaperone activity and the ANS fluorescence intensity decreased. Accordingly, hydrophobic regions of α-crystallin play an indispensible role in its chaperone activity.     

Oxidation-Induced Structural Alterations and Its Effect on Chaperone Function of Rat Lens α-Crystallin

An ascorbate-FeCl₃-EDTA-H₂O₂ system was used to oxidize rat lens α-crystallins. Under this oxidative insult, the chaperone activity of α-crystallin toward γ-crystallin was shown to decrease significantly, which is quite different from the result reported by Wang and Spector. (Invest. Ophthalmol. Vis. Sci. 1995 , 36, 311-321.) Fluorescence spectroscopy and circular dichroism were employed to characterize the structural changes of oxidized α-crystallin. It was found that fluorescence intensity of l-anilinonaphthalene-8-sul-phonate (ANS) bound to oxidized α-crystallin increased comparing to that bound to normal α-crystallin, suggesting oxidation causes the exposure of more hydrophobic regions. Further, α-crystallin's fluorescence intensity in response to tryptophan residues showed a pseudo first order decline. Amino acid analysis of normal versus oxidized α-crystallin confirmed actual decline in tryptophan levels, showing about 80% of tryptophan being modified after 10-hour oxidation. Circular dichroism showed both changes in the secondary and tertiary structures of oxidized α-crystallin, characterized by a large loss of aromatic-type amino acid interactions and a large loss of β-sheet structure. In conclusion, modified tryptophan, secondary and tertiary structural changes of α-crystallin correlate best with the reduction of chaperone function, the curves all showing a linear slope for 10 hours, then plateauing. These results indicate that the decrease of α-crystallin chaperone activity is attributed to the structural changes.     

Identification of Photooxidation Sites in Bovine α-Crystallin

Abstract— Because UV irradiation of proteins can produce reactive oxygen species and exposure to UV light has been implicated in cataractogenesis, the sites of photooxidation of bovine α-crystallin, a major lens protein with molecular chaperone activity, were identified using tandem mass spectrometry (MS/MS). Bovine α-crystallin was irradiated with UV light (293 nm) for 1, 4 and 8 h, digested with trypsin and analyzed by matrix-assisted laser de-sorption ionization, time-of-flight mass spectrometry (MALDI) to identify the oxidized sequences. Tryptic peptides were purified by reverse-phase HPLC and oxidized peptides were sequenced by MS/MS to determine the sites of oxidation. Tryptophan fluorescence decreased exponentially with increasing time of UV exposure and peptides containing residues 1-11 of α-crystallin and 1-11, 12-22 and 57-69 of α-crystallin were determined to be oxidized by shifts of 16 D or multiples of 16 Da above the mass of the unmodified peptide. The MALDI analysis revealed single oxidation of all four sequences, which increased with increasing time of UV exposure and possible double oxidation of α 12-22. The specific sites of photooxidation indicate that the N-terminal regions of α-and β-crystallin are exposed to an aqueous environment and are in the vicinity of tryptophan residues from neighboring subunits.     

The Effect of Arg on the Structure Perturbation and Chaperone Activity of α-Crystallin in the Presence of the Crowding Agent,Dextran

α-Crystallin is a protein that is expressed at high levels in all vertebrate eye lenses. It has a molecular weight of 20 kDa and is composed of two subunits: αA and αB. α-Crystallin is a member of the small heat shock protein (sHsps) family that has been shown to prevent protein aggregation. Small molecules are organic compounds that have low molecular weight (<800 Da). Arginin (Arg) is a small molecule and has been shown to prevent protein aggregation through interaction with partially folded intermediates. In this study, the effect of Arg on the chaperone activity of α-crystallin in the presence of dextran, as a crowding agent, against ordered and disordered aggregation of different target proteins (α-lactalbumin, ovotransferrin, and catalase) has been investigated. The experiments were done using visible absorption spectroscopy, ThT-binding assay, fluorescence spectroscopy, and CD spectroscopy. The results showed that in amorphous aggregation and amyloid fibril formation, both in the presence and absence of dextran, Arg had a positive effect on the chaperone action of α-crystallin. However, in the presence of dextran, the effect of Arg on the chaperone ability of α-crystallin was less than in its absence. Thus, our result suggests that crowding interior media decreases the positive effect of Arg on the chaperone ability of α-crystallin. This is a very important issue, since we are trying to find a mechanism to protect living cells against the toxic effect of protein aggregation.     

Crosslinking and Oxidation of 1, 2-Polybutadiene by UV Irradiation

Photoinduced crosslinking and oxidation of 1, 2-poly-butadiene film were carried out with UV irradiation in vacuo and in air. A small amount of the structures of trans-vinylene, diene, and triene were observed in the infrared spectrum of the nonirradiated film, and bands attributable to conjugated double bond (230, 280 nm) were also observed in ultraviolet spectrum. In the film irradiated in vacuo, intensities of the bands assigned to trans-vinylene, methylene, and methyl groups increased with the increase in irradiation time, but the intensity of the band of pendent vinyl group decreased. With irradiation in air, the formation of carbonyl and hydroxyl groups and ether linkages was observed. Formation of H₂ with the irradiation in vacuo and of H₂ O with the irradiation in air was observed. The gel fraction increased with an increase in irradiation time in the absence and presence of oxygen. The gel fraction and the formation of carbonyl groups were markedly accelerated by irradiation with UV rays of wavelength near 280 nm and increased with decreasing concentration of pendent vinyl groups. Postreaction of the irradiated film took place in the dark. The gel fraction and the intensity of the carbonyl group increased by radical reaction with α, α'-azobisisobutyronitrile. On the basis of these results, mechanisms of photoinduced crosslinking and oxidation of 1, 2-polybutadiene were discussed.     

Living cationic polymerization of a coumarin‐substituted vinyl ether and reversible photoinduced crosslinking of the resulting homopolymers and amphiphilic block copolymers

Living cationic polymerization of 4‐methyl‐7‐(2‐vinyloxyethoxy)coumarin (CMVE) was achieved using SnCl₄ in the presence of nBu₄NBr as an added salt at 0 °C. The number‐average molecular weight of the resulting polymers increased in direct proportion to the monomer conversion while retaining relatively low polydispersity. Structural analysis revealed that the resulting polymers carried pendant coumarinyl moieties. These coumarinyl moieties were crosslinked by irradiation with UV light at λ_max = 366 nm, and the crosslinked sites were then cleaved by irradiation with UV light at λ_max = 254 nm. The crosslinking behaviors of the polymers were studied by UV and FTIR spectroscopic measurement. PolyCMVE was soluble in dichloromethane but was found to be insoluble upon UV light irradiation. We also synthesized amphiphilic block polymers bearing coumarinyl moieties by living cationic copolymerization with an amphiphilic vinyl ether. The resulting block polymers were soluble in an aqueous medium and also formed micelle‐like aggregates. Upon UV irradiation of aqueous solutions above the critical micelle concentration, an efficient crosslinking reaction occurred. Photoinduced structural changes of these polymer aggregates in the solution state were further investigated. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

UV-B-induced secondary conformational changes in lens alpha-crystallin.

The changes in turbidity and protein secondary structure of alpha-crystallin after a 72 h UV-B (302 nm) irradiation in aqueous solution have been determined by UV spectrophotometry and Fourier transform infrared (FT-IR) microspectroscopy with reflection mode. The relative transmission of alpha-crystallin aqueous solution gradually decreases with the exposure time, indicating that the transparent alpha-crystallin aqueous solution becomes opaque with prolonged UV-B irradiation. The turbidity induced by UV-B shows first-order kinetics due to the photo-induced aggregation. The modification of the secondary structure of the alpha-crystallin molecule in aqueous solution caused by this aggregation might enhance the alpha-helix and beta-turn structures from 8.14 to 14.92% and from 24.46 to 35.54%, respectively; reduce the beta-sheet structure from 60.20% to 43.77%; and leave the random coil structure almost unaltered. The secondary conformation of alpha-crystallin changes gradually but evidently with its increase of turbidity during UV-B exposure.     

TRYPTOPHAN PROTECTION OF PHAGE FROM ULTRAVIOLET IRRADIATION

Abstract— Bacteriophage SP02c12 and its isolated DNA were irradiated at 254 nm in tryptophan solutions. At a concentration of 10 m M , the amino acid exerted a protective effect on intact virus. The magnitude of this effect depended upon the length of time elapsed between mixing and irradiation. No protection was observed for solutions irradiated immediately after mixing. Tryptophan did not have a significant effect on UV sensitivity of the isolated viral DNA. No covalent crosslinking of tryptophan to DNA was observed.     

Radiation synthesis of seroalbumin nanoparticles

Synthesis of small polymeric particles can be achieved by ionizing radiation technology via intramolecular crosslinking by gamma rays onto soluble polymer molecules in random coil conformation. Differently soluble globular proteins are naturally densely packed structures. Fragmentation and aggregation processes have been reported for irradiated globular proteins solutions with ionizing radiations.In this work we describe protein-based nanoparticles prepared by gamma irradiation of a soluble and globular protein, such as seroalbumin, as the basic building blocks keeping its original conformational shape. Protein nanoparticles in the range 20–40 nm were detected after gamma irradiation of the aqueous protein solution in the presence of polar organic solvents. Nanoparticles were characterized by DLS, fluorescence, and UV and CD spectroscopy, showing that the protein molecules keep their general three-dimensional structure into the created nanoparticle.     

THE EFFECTS OF NEAR-UV RADIATION ON HUMAN LENS β-CRYSTALLINS: PROTEIN STRUCTURAL CHANGES and THE PRODUCTION OF O2_ and H2O2

beta-Crystallins (beta 1-, beta 2- and beta 3-crystallin) comprise nearly half the protein of the human lens. The effect of near-UV radiation, which is one of the possible risk factors in cataract formation, on the beta-crystallins is investigated in this study. Protein intersubunit crosslinking, change in charge of the protein subunits to more acidic species and changes in protein tertiary structure (conformation) by 300 nm irradiation are reported. The fluorescence yield of protein tryptophan residues decreases by 300 nm irradiation. There is an increase in nontryptophan fluorescence (lambda cx 340 nm, lambda cm 400-600 nm), and in protein absorption at 340 nm, due to the formation of tryptophan photooxidation products. Both tryptophan and its oxidation products can be photoexcited by 300 nm irradiation and the latter are known to be good photosensitizers. The results provide evidence for the generation of H2O2 in the irradiated human beta-crystallin solutions by the Type I photosensitizing action of the chromophores absorbing at 300 nm. The H2O2 is generated via the intermediate production of O2 anion; the latter spontaneously dismutates to H2O2, presumably via O2- protein interactions. The amount of H2O2 generated per absorbed photon is compared for various solutions of beta 1-, beta 2- and beta 3-crystallins from human lenses of different age.     

Morphological Investigation of Poly[methyl(H)silane-co-diphenylsilane] Irradiated by XeCl Excimer Laser

The morphological properties of poly[methyl(H)silane-co-diphenylsilane] copolymer (PSH) under XeCl (308 nm) laser irradiation were investigated. For this purpose, PSH films were exposed to XeCl excimer laser, 308 nm, at various UV doses (122 and 366 mJ/cm²), with 11 and 33 mJ/pulse and 1 Hz pulse repetition rate. The morphology of the PSH film surface was investigated by the atomic force microscopy (AFM) technique. AFM identifies that the films of the copolymer form a wormlike morphology before irradiation and conical defects are created on the polymer surface and grow with the laser irradiation.     

In Vitro Renaturation of Alkaline Family G/11 Xylanase via a Folding Intermediate: α-Crystallin Facilitates Refolding in an ATP-Independent Manner

In this study, alkaliphilic family G/11 xylanase from alkali-tolerant filamentous fungi Penicillium citrinum MTCC 6489 was used as a model system to gain insight into the molecular aspects of unfolding/refolding of alkaliphilic glycosyl hydrolase protein family. The intrinsic protein fluorescence suggested a putative intermediate state of protein in presence of 2 M guanidium hydrochloride (GdmCl) with an emission maximum of 353 nm. Here we studied the refolding of GdmCl-denatured alkaline xylanase in the presence and the absence of a multimeric chaperone protein α-crystallin to elucidate the molecular mechanism of intramolecular interactions of the alkaliphilic xylanase protein that dictates its extremophilic character. Our results, based on intrinsic tryptophan fluorescence and hydrophobic fluorophore 8-anilino-1- naphthalene sulfonate-binding studies, suggest that α-crystallin formed a complex with a putative molten globule-like intermediate in the refolding pathway of xylanase in an ATP-independent manner. A 2 M GdmCl is sufficient to denature alkaline xylanase completely. The hydrodynamic radius (R_H) of a native alkaline xylanase is 4.0, which becomes 5.0 in the presence of 2 M GdmCl whereas in presence of the higher concentration of GdmCl R_H value was shifted to 100, indicating the aggregation of denatured xylanase. The α-crystallin·xylanase complex exhibited the recovery of functional activity with the extent of ~43%. Addition of ATP to the complex did not show any significant effect on activity recovery of the denatured protein.     

Photosensitized Effects of Rose Bengal on Structure and Function of Lens Protein "Alpha-Crystallin"

The conformational changes of the bovine lens protein "α-crystallin" have been investigated in the presence of the photosensitizer Rose Bengal (RB), in the dark as well as after visible light irradiation. Absorption and fluorescence emission spectra of RB [5 × 10⁻⁶  m ] and Fourier transform-IR spectra of α-crystallin [5 mg mL⁻¹] were significantly altered upon RB α-crystallin complex formation. RB was found to bind to α-crystallin in a molecular pocket characterized by a low polarity, with Trp most likely involved in this interaction. The binding constant ( K _b) has been estimated to be of the order of 2.5 (mg/mL)⁻¹. The intrinsic fluorescence of α-crystallin was quenched through both dynamic and static mechanisms. Light-induced photosensitized effects showed structural modifications in α-crystallin, including tertiary and secondary structure (an increase in unordered structure) alterations. Notwithstanding those photoinduced structural variations detected in α-crystallin when complexed with RB, the protein still retains its ability to play the role of chaperone for β-crystallin.     

Crosslinking of cotton cellulose with succinic acid in the presence of titanium dioxide nano-catalyst under UV irradiation

The nanometer titanium dioxide (TiO₂), succinic acid (SUA), and the mixture of nano- TiO₂ and SUA were dispersed or dissolved in pure water, then irradiated with UV at the wavelength of 254 nm under stirring for different time periods respectively. The pH and conductivity values and UV-vis spectra of those solutions were recorded immediately. The powders obtained from the filtrated and vacuum dried from those solutions were examined with Fourier Transform Infrared Spectroscopy and Wide Angle X-Ray Diffraction meter. It shows that the crystalline structure and IR and UV-vis spectra of SUA adsorbed on nano-TiO₂ particles are changed. The photo-reduced succinic acid in the presence of nanometer titanium dioxide under UV irradiation can create aldehyde group on SUA and free radical of SUA to improve the catalytic effects of crosslinking reaction between SUA and cotton cellulose.     

PROTEIN-RNA CROSSLINKS IN RIBOSOMES: ULTRAVIOLET ACTION SPECTRA. COMPARISON WITH INACTIVATION ACTION SPECTRUM

Abstract— RNA-protein crosslinking by UV of different wavelengths was studied in 70S E. coli ribosomes by three techniques: sucrose gradient centrifugation in the presence of sodium dodecyl sulfate (SDS), RNA solubilization in LiCI-urea concentrated solutions and RNA adsorption on nitrocellulose filters in the presence of SDS.
The centrifugational technique shows that the crosslinking reaction occurs in two steps, the first one corresponding to the fixation of a few protein molecules on 16 or 23 s RNAs and the second one corresponding to extensive RNA-protein crosslinking so that most protein molecules are no longer released by SDS from 30S and 50S subunits.
The initial rates for the first step of crosslinking were evaluated by the solubilization and adsorption techniques at 7 (or 6) wavelengths of irradiation between 223 and 290 mm. The action spectrum for RNA solubilization in LiCl-urea is perturbed at 223 nm by the breakage of protein chains. The action spectrum for retention on nitrocellulose filters seems to be exempt of this defect. It corresponds at high wavelengths to a nucleic chromophore and at low wavelengths to a proteic one. This means that RNA-protein crosslinking may occur through RNA and protein excitation. The similarity between the action spectrum for RNA retention on nitrocellulose filters and the action spectrum for inactivation of ribosomal synthesis activity suggests that RNA-protein crosslinking may be responsible for inactivation of ribosomes by UV.     

A polymeric photobase generator containing oxime–urethane groups: Crosslinking reaction and application to negative photoresist

A polymeric photobase generator containing oxime–urethane groups was prepared by copolymerization of methyl methacrylate and methacryloxyethyl benzophenoneoxime urethane, and its photo and thermal crosslinking reaction after irradiation was examined from the measurement of UV and IR absorption spectral changes, insoluble fraction, and molecular weight changes. The photo‐crosslinking reaction of the copolymer film was more efficient when irradiations were carried out with 310 nm UV light in the presence of benzophenone than with 254 nm UV light without the addition of benzophenone. The crosslinking reaction increased after postexposure baking (PEB), and this thermal crosslinking reaction mechanism was studied from the identification of the photolysis products of a model compound, benzophenoneoxime phenylurethane, by a high‐performance liquid chromatography. The results indicate that the thermal crosslinking reaction of the copolymer after PEB is due to the formation of urea‐type chemical bonds. Resist properties of the copolymer were examined from the measurement of normalized thickness and micropattern development. A negative tone image with a resolution of 2 μm was obtained with this copolymer, having a sensitivity (D) of 1200 mJ/cm² and contrast (γ_n) of 1.31, when irradiation was carried out with 310 nm UV light in the presence of benzophenone following chloroform development. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 975–984, 2004     

FAR UV IRRADIATION OF DNA IN THE PRESENCE OF PROTEINS, AMINO ACIDS OR PEPTIDES

Abstract— The DNA of bacteriophage SP02c12 was subjected to 254 nm irradiation in solutions containing lysozyme or histone. In these solutions, the protein-DNA mass ratios and the ionic strengths of the solvents were varied to change the amount of protein associated with the DNA. Lysozyme-DNA binding constants were measured under the same conditions. The sensitivity of phage DNA to biological inactivation by UV increased as the amount of lysozyme bound per DNA strand increased. Although binding constants could not be measured for the DNA-histone interaction, this protein had a protective effect which was greater under conditions which cause enhanced binding. No crosslinking of either protein could be detected even at doses ten-fold greater than those giving a surviving fraction of 0.01.
Irradiation was also performed in the presence of various amino acids and short peptides. These were chosen to include amino acids which: (1) are positively charged, (2) absorb UV of this wavelength or (3) form UV-induced crosslinks to DNA. None of the amino acids tested affected sensitivity of the DNA to biological inactivation. Peptides containing a UV-absorbing amino acid and a positively charged amino acid enhanced sensitivity. For each of these peptides, a mixture of the constituent amino acids had the same effect as the peptide itself. Under the conditions used, no evidence for formation of DNA-amino acid crosslinks was found. The results indicate that proteins and peptides can sensitize DNA to UV inactivation by mechanisms other than covalent crosslink formation. Such mechanisms could include energy or electron transfer or alterations in the conformation of the DNA.     

Indirect Photo-Induced Phosphorylation Via a C-Ester Caged Troika Acid

Abstract

Use of a photoremovable “caging” group allows the generation of reactive molecules under mild conditions. Photo-induced phosphorylations typically have involved attachment of the photosensitive group at phosphorus.[1] We now have investigated indirect photolytic activation of an unmodified phosphonic acid group using broad band UV (Hg lamp), 308 nm XeCl excimer laser or 355 nm YAC laser irradiation of the o-nitrobenzyl C-ester of “troika acid” [(E)-1²]. In alcohols or neutral buffer, irradiation of (E)-2 gave phosphorylation of the solvent plus phosphorocyanidate, the expected 2-isomer product.²All thrce UV sources gave -1:2 E:Z product distribution in MeOH. In the (E)-1 methyl C-ester, the oxime functionality absorbed strongly near 205 nm (E_max 5200), weakly at 308 nm and negligibly above 355 nm, and no photoisomerization was seen using the 355 nm source. Thus, oxime isomerization in (E)-2 at least using 355 nm irradiation. requires the o-nitrobenzyl group, and possibly involves an energy- or charge-wansfer effect. Phosphorylation of EtOH/t-BuOH mixtures by photolysis of (E)-2 showed little alkyl selectivity. consistent with photoinduced formation of an intermediate. plausibly (E)-1, which undergoes spontaneous dissociative fragmentation via a monomeric metaphosphate-like species.