Photochemically modified alpha-crystallin: a model system for aging in the primate lens.

The purpose of this study was to quantitatively study the changes that occur upon irradiation of 3-hydroxykynurenine (3-HK) in the presence of alpha-crystallin under conditions similar to those in the lens. The samples were prepared in 10 mM phosphate buffer at pH 7.4, bubbled with O2 or Ar and irradiated with 300-400 nm light. The amount of light absorbed by the samples (Iabs) was measured using azobenzene as an actinometer. Modifications to alpha-crystallin were monitored by ultraviolet-visible and fluorescence spectroscopy. Aerobic samples had increased absorption around 320 nm and above 400 nm while the 3-HK maximum at 368 nm decreased. The isolated modified protein showed that there was increased absorption throughout the spectrum. Changes in the anaerobic samples were similar to those of the aerobic but occurred more slowly. As irradiation time increased fluorescence emission of the isolated protein red shifted and quantum yields of fluorescence (phi f) were calculated at different irradiation time intervals by comparison to 3-HK. By comparing OD320/OD365 for the model system to values from primate lenses, Iabs can be correlated with age and transmission of the sample in the blue region of the spectrum and thus allows lenticular aging to be quantitated.     

Photooxidation of lens alpha-crystallin by hypericin (active ingredient in St. John's Wort)

Hypericin is the active ingredient in the over the counter antidepressant medication St. John's Wort. Hypericin produces singlet oxygen and other excited state intermediates that indicate it should be a very efficient phototoxic agent in the eye. Furthermore it absorbs in the UV and visible range, which means it can potentially damage both the lens and the retina. Lens alpha-crystallin, isolated from calf lenses, was irradiated in the presence of hypericin (5 x 10(-5) M, 10 mM ammonium bicarbonate, pH 7.0) and in the presence and absence of light (> 300 nm, 24 mW/cm2). Hypericin-induced photosensitized photopolymerization as assessed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. Further analysis of the oxidative changes occurring in alpha-crystallin using mass spectrometry showed specific oxidation of methionine, tryptophan and histidine residues, which increased with irradiation time. Hypericin did not damage the lens protein in the dark. Damage to alpha-crystallin could undermine the integrity of the lens directly by protein denaturation and indirectly by disturbing chaperone function. Therefore, in the presence of light, hypericin can induce changes in lens protein that could lead to the formation of cataracts. Appropriate precautions should be taken to protect the eye from intense sunlight while on this antidepressant medication.     

Lens alpha-crystallin and hypericin: a photophysical mechanism explains observed lens

Determining whether alpha-crystallin (the major lens protein) affects the photophysics of hypericin, a photosensitizing agent found in various plants, such as St. John's Wort, is important. Hypericin shows promise in cancer and human immunodeficiency virus therapy but may harm individuals taking St. John's Wort extracts (for mild to moderate depression). Hypericin causes hypericism, which is characterized by cellular damage in light-exposed areas. Ocular tissues are at risk for photosensitized damage; thus, we investigated the effects on hypericin photophysics by alpha-crystallin. We measured the transient absorption spectra and the 1270 nm luminescence of singlet (1Deltag) oxygen produced from hypericin in the presence of alpha-crystallin. alpha-Crystallin complexes hypericin, extending the lifetime of its triplet excited state; the Stern-Volmer slope is negative, but not linear, after a saturation curve. Damage to the lens protein by hypericin is known to occur via singlet oxygen, which oxidizes methionine, tryptophan and histidine residues. Binding to alpha-crystallin does not inhibit singlet oxygen formation by hypericin. alpha-Crystallin reacts with singlet oxygen with a rate constant of 1.3 x 10(8) M(-1) s(-1). Thus, we anticipate that hypericin will be an effective photosensitizer in the lens.     

Collision-induced conformational changes in glycine

We present quantum dynamical calculations on the conformational changes of glycine in collisions with the He, Ne, and Ar rare-gas atoms. For two conformer interconversion processes (III-->I and IV-->I), we find that the probability of interconversion is dependent on several factors, including the energy of the collision, the angle at which the colliding atom approaches the glycine molecule, and the strength of the glycine-atom interaction. Furthermore, we show that attractive interactions between the colliding atom and the glycine molecule catalyze conformer interconversion at low collision energies. In previous infrared spectroscopy studies of glycine trapped in rare-gas matrices and helium clusters, conformer III has been consistently observed, but conformer IV has yet to be conclusively detected. Because of the calculated thermodynamic stability of conformer IV, its elusiveness has been attributed to the IV-->I conformer interconversion process. However, our calculations present little indication that IV-->I interconversion occurs more readily than III-->I interconversion. Although we cannot determine whether conformer IV interconverts during experimental Ne- and Ar-matrix depositions, our evidence suggests that the conformer should be present in helium droplets. Anharmonic vibrational frequency calculations illustrate that previous efforts to detect conformer IV may have been hindered by the overlap of its IR-absorption bands with those of other conformers. We propose that the redshifted symmetric -CH(2) stretch of conformer IV provides a means for its conclusive experimental detection.     

Ethanol or/and captopril-induced precipitation and secondary conformational changes of human serum albumin

We determined the secondary structure of solid-state native human serum albumin (HSA) and its precipitates induced by ethanol, captopril, or a captopril/ethanol mixture. A transmission Fourier transform infrared (FT-IR) microspectroscopy equipped with a thermal analyzer was used. The secondary structural composition of solid-state native HSA was 54% alpha-helices (1655 cm(-1)), 22% beta-turns (1679 cm(-1)), and 23% beta-sheets (1633 cm(-1)). After ethanol treatment, a new peak was observed at 1690 cm(-1), and the peak at 1633 cm(-1) was more apparent in the HSA precipitates. The corresponding compositions consisted of 59% alpha-helices, 17% beta-turns, and 24% beta-sheets. After treatment with captopril with or without ethanol, the percentage of alpha-helices and beta-turns decreased in both HSA precipitates, but the percentage of beta-sheets increased. The temperature-dependent structural transformation from alpha-helices/random coils to beta-sheets for the solid-state HSA samples occurred at markedly different onset temperatures. The onset temperature for native HSA was 85 degrees C, and that for HSA precipitates obtained from ethanol, captopril, or captopril/ethanol was 100, 48 or 57 degrees C, respectively. The thermal-induced structural transformation from alpha-helices/random coils to beta-sheets implies a partial unfolding structure in these HSA samples.     

Characterization of secondary and tertiary conformational changes of beta-lactoglobulin adsorbed on silica nanoparticle surfaces

Nanoparticles possess unique properties as a result of their large surface area per unit volume and therefore can be functionalized by the immobilization of enzymes for a variety of biosensing applications. Changes in the tertiary conformation of beta-lactoglobulin adsorbed on 90 nm silica nanoparticles with time were inferred using tryptophan fluorescence and Fourier transform infrared spectroscopy (FTIR) for different surface concentrations, temperature, pH, ionic strength, and 2,2,2-trifluoroethanol (TFE) and dithiothreitol (DTT) concentrations. Rapid initial unfolding followed by a much slower rate at longer times was observed, with the extent of unfolding being higher at lower surface concentrations, higher ionic strengths, higher temperature, higher TFE and DTT concentrations, and pI. The effect of temperature on the unfolding of adsorbed protein on the nanoparticle surface was similar to that in the bulk even though the extent of unfolding was higher for adsorbed protein molecules. The results of the extent of change in tertiary conformation using FTIR as indicated by the change in the ratio of amide II'/amide I were consistent with those obtained by tryptophan fluorescence whereas the rates of conformational changes given by FTIR were found to be much faster. Circular dichroism (CD) spectra showed that altering the surface concentration by itself did not change the secondary structure of beta-lactoglobulin on the surface. TFE was found to increase the alpha helix content at the expense of the fraction of the beta sheet, whereas the beta sheet was converted to an unordered conformation in the presence of DTT.     

Mast cells in UV-B-induced immunosuppression

Degranulating dermal mast cells in UV-B-irradiated skin have been implicated for many years in the mechanisms of irradiation erythema. There is now considerable evidence that dermal mast cells are important to the processes by which both UV-B radiation and cis-urocanic acid (cis-UCA) suppress immune responses to sensitizing antigens applied to non-irradiated/non-cis-UCA-exposed sites. Mast-cell-depleted mice are resistant to the immunosuppressive effects of UV-B radiation and cis-UCA for 'systemic' immunomodulation. However, these mice gain responsiveness if the dorsal skin is reconstituted six weeks prior to irradiation or cis-UCA administration at that site with cultured bone-marrow-derived mast cells from +/+ mice. The molecular triggers for initiating mast-cell degranulation are being actively sought. Evidence suggests that histamine, and not tumour necrosis factor alpha, is the major mast-cell product that signals altered immune responses to sensitizing antigens applied to non-irradiated, non-cis-UCA-exposed sites. Histamine may have multiple roles, but experiments with indomethacin administered to mice have shown that one process involves induction of prostanoid production.     

Cooperative conformational changes in globular proteins

In globular proteins, the complicated steric arrangement of the polypeptide chain is determined by several interdependent cooperative interactions. These macromolecules are capable of reacting to changes in the environmental conditions such as temperature and pressure or the concentration of a wide range of compounds by changing their conformation and hence their biological and chemical properties. They are thus suitable for regulation processes or information storage in solution with a wide range of time constants. Environmental effects of this kind can be followed and explained in part at the molecular level by investigation of the time-dependent reversible unfolding of a number of proteins that can be described to a good approximation by a strongly cooperative “all-or-none” transition between two states.     

Chirality and conformational changes in 4,5-diaryltriphenylenes

Line-shape analysis of temperature dependent NMR spectra of several substituted 4,5-diphenyl-triphenylenes has been performed to determine the free energy of activation for rotation (ΔG_rot^*) of the phenyl groups. The rotational barrier (ΔG_rot^*) depends on the presence and position of substituents on the phenyl groups; it is the largest in compounds with ortho-substituents. The independent determined free energy of activation of racemization (ΔG_rac^*) is about equal to ΔG_rot^* in 4-phenyl-5-(3,5-dimethylphenyl)triphenylene, but in 4,5-bis-(3,5- dimethylphenyl) triphenylene ΔG_rac^* is much larger than ΔG_rot^*. It is concluded that the racemization does not occur via a process in which the phenyl groups remain parallel but via a molecular movement in which the phenyl groups turn around each other like cog wheels.     

Vibrational spectroscopic studies on the disulfide formation and secondary conformational changes of captopril-HSA mixture after UV-B irradiation

The effects of pH and ultraviolet-B (UV-B) irradiation on the secondary structure of human serum albumin (HSA) in the absence or presence of captopril were investigated by an attenuated total reflection (ATR)/Fourier transform infrared (FTIR) spectroscopy. The UV-B exposure affecting the stability of captopril before and after captopril-HSA interaction was also examined by using confocal Raman microspectroscopy. The results indicate that the transparent pale-yellow solution for captopril-HSA mixture in all pH buffer solutions, except pH 5.0 approximately 7.0, changed into a viscous form then a gel form with UV-B exposure time. The secondary structural transformation of HSA in the captopril-HSA mixture with or without UV-B irradiation was found to shift the maxima amide I peak in IR spectra from 1652 cm(-1) assigned to alpha-helix structure to 1622 cm(-1) because of a beta-sheet structure, which was more evident in pH 3.0, 8.0 or 9.0 buffer solutions. The Raman shift from 1653 cm(-1) (alpha-helix) to 1670 cm(-1) (beta-sheet) also confirmed this result. Captopril dissolved in distilled water with or without UV-B irradiation was determined to form a captopril disulfide observed from the Raman spectra of 512 cm(-1), which was exacerbated by UV-B irradiation. There was little disulfide formation in the captopril-HSA mixture even with long-term UV-B exposure, but captopril might interact with HSA to change the protein secondary structure of HSA whether there was UV-B irradiation or not. The pH of the buffer solution and captopril-HSA interaction may play more important roles in transforming the secondary structure of HSA from alpha-helix to beta-sheet in the corresponding captopril-HSA mixture than UV-B exposure. The present study also implies that HSA has the capability to protect the instability of captopril in the course of UV-B irradiation. In addition, a partial unfolding of HSA induced by pH or captopril-HSA interaction under UV-B exposure is proposed.     

An improved method combining two electrophoretic procedures: application to the separation of lens alpha-crystallin isoforms

Polypeptides having different net electric charges and very similar molecular weights, visualized as one single band in sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE), can be readily analyzed by an improved method combining two electrophoretic procedures. The methodology consists of the identification and isolation of selected protein bands from SDS-PAGE, their equilibration in an isoelectric focusing (IEF) sample buffer, and their casting and separation in an IEF flat-bed gel. This method requires no extra equipment, is highly reproducible, is suitable for quantitative and comparative studies, and is especially useful in the case of small samples. As a particular example, we analyze here the subunit composition of alpha-crystallins of young and embryonic quail lenses.     

Solvent-induced conformational changes of polypeptides probed by electrospray-ionization mass spectrometry.

Electrospray-ionization (ESI) mass spectrometry is used to monitor higher order structural changes of polypeptides induced by alteration of the pH or organic solvent composition in the protein solution environment. A bimodal charge-state distribution is observed in the ESI mass spectrum of ubiquitin (relative molecular mass 8565) in solutions containing small amounts (less than 20%) of organic solvents. The distribution of peaks at high m/z (low-charge state) is found to represent the protein in its native, globular state; the higher-charge-state distribution is characteristic for a more extended conformation. Addition of methanol denaturant in excess of 40% v/v is needed to eliminate the low-charge-state distribution completely. Lesser amounts of acetonitrile, acetone, or isopropanol (approximately 20%) are required to denature the ubiquitin protein. Other proteins showing conformational effects in their ESI mass spectra are also illustrated. While the ESI spectra are related to solution phase structure, ESI-tandem mass spectrometry of multiply charged molecular ions of different conformation is suggested as a probe of gas-phase protein three-dimensional structure.     

Time-dependent conformational changes of polyelectrolyte complexes in solution

Changes in the conformation of the polyelectrolytes when contacted with oppositely charged polyelectrolytes or when subjected to shifts in solution conditions (pH), have been studied in this work along with reversibility of the changes using fluorescence spectroscopy. While changes due to both of the above are marked, they are measurably different from each other. Thus, the extent of coiling of the complexes formed between the anionic polyelectrolyte, maleicacid anhydride–propene and the cationic polyelectrolytes was much higher than that achieved by the change in pH alone. Also, while the changes due to pH shifts were fast and reversible, that due to complexation between oppositely charged ones involved first a rapid uncoiling followed by slow recoiling to a new structure. Interestingly, shifting the coiled conformation to an even more coiled one resulted in a new reversible state, but shifting to a stretched state by complexation led to a somewhat irreversible structure. Also maximum interaction obtained between the anionic and one cationic polyelectrolyte was markedly higher than that between the former and another cationic polymer. These observations using fluorescence spectroscopy was consistent with that obtained by the potentiometric titration. The study clearly shows the importance of the manner in which the polyelectrolytes are equilibrated to desired solution conditions. These results are interpreted here in terms of deprotonation/protonation of the polyelectroytes upon pH change and complexation with oppositely charged ones resulting in screening of charges as well as stiffening.     

Analytical assays based on detecting conformational changes of single molecules.

One common strategy for the detection of biomolecules is labeling either the target itself or an antibody that binds to it. Herein, a different approach, based on detecting the conformational change of a probe molecule induced by binding of the target is discussed. That is, what is being detected is not the presence of the target or the probe, but the conformational change of the probe. Recently, a single-molecule sensor has been developed that exploits this mechanism to detect hybridization of a single DNA oligomer to a DNA probe, as well as specific binding of a single protein to a DNA probe. Biomolecular recognition often involves large conformational changes of the molecules involved, and therefore this strategy may be applicable to other assays.     

UV-B-induced generation of free radicals in blood platelets

UV-B irradiation of blood-platelet concentrates is used in transfusion practice to prevent the development of post-transfusion alloimmunization and inactivate viruses and bacteria in the concentrates. UV-B radiation may affect the blood-platelet metabolism and function; therefore we have investigated the effect of UV-B irradiation on free radical production in blood platelets. Our results show that exposure of pig blood platelets to UV-B radiation (0.36 and 1.08 J/cm2) induces the generation of free radicals measured by the chemiluminescence method (respectively 28 and 148.6% above the control). The superoxide radical level after UV-B irradiation measured by the cytochrome c reduction method shows only a slight increase (p > 0.05). Free radical generation induced by UV-B radiation is dependent partly on blood-platelet activation and enzymatic pathways, since we have shown that wortmannin, an inhibitor of phosphatidylinositide 3-kinase, reduces the level of radicals formed in blood platelets after UV-B irradiation. This indicates that free radicals generated in blood platelets after stimulation by UV-B radiation are involved in platelet activation and metabolism of platelet polyphosphoinositides.     

Perspective: pre-chemistry conformational changes in DNA polymerase mechanisms

In recent papers, there has been a lively exchange concerning theories for enzyme catalysis, especially the role of protein dynamics/pre-chemistry conformational changes in the catalytic cycle of enzymes. Of particular interest is the notion that substrate-induced conformational changes that assemble the polymerase active site prior to chemistry are required for DNA synthesis and impact fidelity (i.e., substrate specificity). High-resolution crystal structures of DNA polymerase β representing intermediates of substrate complexes prior to the chemical step are available. These structures indicate that conformational adjustments in both the protein and substrates must occur to achieve the requisite geometry of the reactive participants for catalysis. We discuss computational and kinetic methods to examine possible conformational change pathways that lead from the observed crystal structure intermediates to the final structures poised for chemistry. The results, as well as kinetic data from site-directed mutagenesis studies, are consistent with models requiring pre-chemistry conformational adjustments in order to achieve high fidelity DNA synthesis. Thus, substrate-induced conformational changes that assemble the polymerase active site prior to chemistry contribute to DNA synthesis even when they do not represent actual rate-determining steps for chemistry.     

pH-Dependent conformational changes in bisporphyrincalix[4]arene

pH-Dependent conformational changes in bisporphyrincalixarene in which the calix[4]arene core fixes the tetrapyrrole chromophores in the cyclophane orientation relative to each other were studied by spectrophotometric titration and ¹H NMR spectroscopy. It was shown that the distance between the reaction centers of the porphyrin fragments of the macrocycle can be controlled by varying the solution acidity. The ranges of reagent concentrations where functionally significant reversible conformational changes of the dimeric porphyrin occur were determined.     

Flow-induced conformational changes in gelatin structure and colloidal stabilization

Flow can change the rate at which solutes adsorb on surfaces by changing mass transfer to the surface, but moreover, flow can induce changes in the conformation of macromolecules in solution by providing sufficient stresses to perturb the segmental distribution function. However, there are few studies where the effect of flow on macromolecules has been shown to alter the structure of macromolecules adsorbed on surfaces. We have studied how the local energy dissipation alters the adsorption of gelatin onto polystyrene nanoparticles ( r = 85 nm). The change in the nature of the adsorbed layer is manifest in the change in the ability of the nanoparticles to resist aggregation. Circular dichroism spectroscopy was used to assess conformational changes in gelatin, and dynamic light scattering was used to assess the colloid stability. Experiments were conducted in a vortex jet mixer where energy density and mixing times have been quantified; mixing of the gelatin and unstable nanoparticles occurs on the order of milliseconds. The adsorption of the gelatin provides steric stabilization to the nanoparticles. We found that the stability of the gelatin-adsorbed nanoparticles increased with increasing mixing velocities: when the mixing velocities were changed from 0.9 to 550 m/s, the radius of the nanoclusters (aggregates) formed 12 h after the mixing decreased from 2620 to 600 nm. Increasing temperature also gave rise to similar trends in the stability behavior with increasing temperature, leading to increasing colloid stability. Linear flow birefringence studies also suggested that the velocity fields in the mixer are sufficiently strong to produce conformational changes in the gelatin. These results suggest that the energy dissipation produced by mixing can activate conformational changes in gelatin to alter its adsorption on the surfaces of nanoparticles. Understanding how such conformational changes in gelatin can be driven by local fluid mechanics and how these changes are related to the adsorption behavior of gelatin is very important both industrially and scientifically.     

Effects of oligopeptide's conformational changes on its adsorption

We report the effects of peptide adsorption to cross-linked polymers (adsorbents) by its conformational changes. Two adsorbents, APhe and ALeu, were prepared and expected to show high affinity to the oligopeptide VW-8 (NH(2)-Val-Val-Arg-Gly-Cys-Thr-Trp-Trp-COOH) according to our previous studies. These absorbents bared the residues of phenylalanine and leucine, respectively, and carried both hydrophobic and electrical groups. The adsorbent AAsp, which carried only the electrostatic groups, was also prepared as a reference. Both APhe and ALeu were found to exhibit higher VW-8 capacity than AAsp, in which APhe showed the highest VW-8 capacity (13.6 mg/g). The VW-8 adsorption to ALeu and APhe was analyzed using a variety of techniques, including the surface plasmon resonance (SPR) technology, nuclear magnetic resonance (NMR) spectra and isothermal titration calorimetry (ITC). The comprehensive experimental data together indicated that APhe could induce a conformational change of VW-8 from a random-coil to a β-strand structure due to its ability to provide the strong ring stacking and electrostatic interactions, which is believed to be responsible for its highest adsorption affinity (K(a)=2.59×10(7) M(-1)). In contrast, the hydrophobic interactions provided by ALeu were not strong enough to induce a VW-8 conformational change to a regular structure, and therefore it exhibited a relatively lower affinity to VW-8 (K(a)=6.23×10(5) M(-1)). The results presented in this work showed that peptide adsorption can be influenced by its conformational changes induced by suitable adsorbents via strong non-covalent interactions.