Refinement of 3D structure of bovine lens alpha A-crystallin.

In absence of 3D structures for alpha-crystallin subunits, alpha A and alpha B, we utilized a number of experimental and molecular modeling techniques to generate working 3D models of these polypeptides (Farnsworth et al., 1994. In Molecular Modeling: From Virtual Tools to Real Problems (Eds. Kumosinski, T.F. and Liebman, M.N.) ACS Symposium Series 576, Ch. 9:123-134, 1994, ACS Books, Washington DC). The refinement of the initial bovine alpha A model was achieved using a more accurate estimation of secondary structure by new/updated methods for analyzing the far UV-CD spectra and by neural network secondary structure predictions in combination with database searches. The spectroscopic study reveals that alpha-crystallin is not an all beta-sheet protein but contains approximately 17% alpha-helices, approximately 33% beta-structures and approximately 50% turns and coils. The refinement of the alpha A structure results in an elongate, asymmetric amphipathic molecule. The hydrophobic N-terminal domain imparts the driving force for subunit aggregation while the more flexible, polar C-terminal domain imparts aggregate solubility. In our quaternary structure of the aggregate, the monomer is the minimal cooperative subunit. In bovine alpha A, the highly negatively charged C-terminal domain has three small positive areas which may participate in dimer or tetramer formation of independently expressed C-terminal domains. The electrostatic potential of positive areas is modulated and become more negative with phosphorylation and ATP binding. The refined bovine alpha A model was used to construct alpha A models for the human, chick and dogfish shark. A high degree of conservation of the three dimensional structure and the electrostatic potential was observed. Our proposed open micellar quaternary structure correlates well with experimental data accumulated over the past several decades. The structure is also predictive of the more recent data.

Interaction of DNA with bovine lens alpha-crystallin: its functional implications.

Under normal conditions, lens aggregates of alpha-crystallin subunits, alpha A and alpha B, are found in the cytoplasm. However, during stress in nonlenticular tissues, alpha B translocates to the nucleus. A sequence study revealed that both subunits share a consensus sequence with other DNA binding proteins. These observations prompted us to investigate DNA binding with alpha-crystallin by UV-mediated photo-crosslinking. The data show that both single and double stranded DNA crosslink mainly with tetramers of alpha-crystallin subunits. The formation of tetramers appears to modify alpha-crystallin interactive properties and, therefore, its induction may have functional significance. These observations suggest that alpha-crystallin may have a nuclear function which includes DNA binding.

Effects of temperature and concentration on bovine lens alpha-crystallin secondary structure: a circular dichroism spectroscopic study.

Elucidation of the structure of alpha-crystallin, the major protein in all vertebrate lenses, is important for understanding its role in maintaining transparency and its function in other tissues under both normal and pathological conditions. Progress toward a unified consensus concerning the tertiary and quaternary structures of alpha-crystallin depends, in part, on an accurate estimation of its secondary structure. For the first time, three algorithms, SELCON, K2D and CONTIN were used to analyze far ultra-violet circular dichroism (UV-CD) spectra of bovine lens alpha-crystallin to estimate the secondary structure and to determine the effects of temperature and concentration. Under all experimental conditions tested, the analyses show that alpha-crystallin contains 14% alpha-helix, 35% beta-sheet and the remainder, random coil and turns. The results suggest that alpha-crystallin is best classified as a mixed protein. In addition, increased temperature and concentration of alpha-crystallin result in increased alpha-helices with a compensatory decrease in beta-sheets. Such structural alterations in alpha-crystallin may be functionally important during terminal differentiation of the lens fiber cells that is accompanied by increased protein concentrations and its role as a chaperone-like protein.

A comparison of structural relationships among alpha-crystallin, human Hsp27, gamma-crystallins and beta B2-crystallin.

The 3D structures of alpha-crystallin, a major eye lens protein, and related small heat shock proteins are unresolved. It has been assumed that alpha-crystallin is primarily a beta-sheet globular protein similar to alpha-crystallin (Siezen and Argos, Biochim. Biophys. Acta, 1983, 748, 56-67) containing sequence repeats in its two domains (Wistow, FEBS Lett. 1985, 181, 1-6). Positional flexibility of amino acid residues and far UV-circular dichroism spectroscopy were used to investigate structural relationships among these proteins. The utility of flexibility plots for predicting protein structure is demonstrated by the excellent correlation of these plots with the known 3D X-ray structures of beta/gamma-crystallins. Similar analyses of alpha-crystallin subunits, alpha A and alpha B, and human heat shock protein 27 show that the C-terminal domains and connecting segments of these proteins are very similar while the N-terminal domains have significant structural differences. Unlike beta/gamma-crystallins, both Hsp27 and alpha-crystallin subunits are asymmetrical with highly flexible C-terminal domains. Flexibility is considered essential for protein functional activity. Therefore, the C-terminal region may play an active role in alpha-crystallin and small heat shock protein function. Differences in flexibility profiles and estimated secondary structure distribution in alpha-crystallin by three recent/updated algorithms from far UV-CD spectra support our predicted 3D structure and the concept that alpha-crystallin and members of beta/gamma superfamily are structurally dissimilar.

Effect of thiobase incorporation into duplex DNA during the polymerization reaction.

6-Thioguanine, a thioderivative of the nucleic acid base guanine, is a metabolic inhibitor and possesses antitumor activity. In addition, it inhibits the synthesis of nucleotides and is incorporated readily into the nucleic acid during the polymerization reaction by substituting for guanine. The biological activity of 6-thioguanine has been related to the alterations in the dimensions and energetics of a duplex DNA as a result its incorporation in place of guanine (Bugg and Thewalt 1975). With the advancement in the computer hardware technology and emergence of sophisticated molecular modeling packages, it is now possible to simulate the biological activities of different drugs. An effort has been made to explain the biological activities of thoiderivatives of nucleic acid bases. For this, semiempirical MNDO quantum chemical calculations were performed on some non-complementary (thiosubstituted) base pairs to understand the effects of substitution of sulfur for oxygen in nucleic acid bases. The base pairs considered in this study are thioguanine-cytosine (TG-C), guanine-thiocytosine (G-TC) and thioguanine-thiocytosine (TG-TC). The results obtained suggest that the replacement of guanine by thioguanine and/or replacement of cytosine by thiocytosine weakens the stability of the double helical DNA by reducing the strength of the hydrogen bonds.

Electrostatic parameters of the theoretical quaternary structure of bovine alpha-crystallin.

By changing the ionic strength, the effects of charge modification on the electrostatic properties of our predicted "open' micellar quaternary structure composed of bovine alpha A subunits were determined. The electrostatic potential values (phi) at 6 arbitrary points surrounding the protein and at all atomic sites of the protein were calculated using the non-linear Poisson-Boltzmann equation. The effective charge (q) of our predicted aggregate ranged from 16 at 0.0022 M to 45 at 0.1472 M ionic strengths. The variation of potential (phi), as well as charge, is a hyperbolic function of ionic strength (R2, 0.901). Plotting the inverse charge (l/q) against inverse ionic strength (1/l) it is possible to calculate maximum charge (q(max)) (approximately 48) at saturation. This value is close to previously reported experimental (50 +/- 5), and our theoretical charge (45), values at physiological ionic strength (0.145 M). These data indicate that maximal repulsion among the alpha-crystallin aggregates occurs at or near physiological ionic strength. Also, half-maximal charge (q(max)/2) at 0.003-0.004 M indicates a transition state at very low ionic strength. The calculated volume available for the mobile solvent in our quaternary structure is approximately 70%. These data are in good agreement with experimental values for bovine alpha-crystallin in solution reported by Xia et al. (Biophys. J., 1994; 66: 861-872). This agreement provides support for our predicted models of alpha-crystallin and a level of confidence in the reliability of the theoretical calculations. Since an ionic gradient exists between the lens cortical and nuclear regions, the modification of charge on alpha-crystallin by varying ionic strength could contribute to the function of alpha-crystallin as a modulator of lens supermolecular order during fiber cell maturation.

alpha-Crystallin quaternary structure: molecular basis for its chaperone activity.

alpha-Crystallin, the major protein in all vertebrate lenses, functions as a chaperone. In the present analysis, an 'open' micellar structure composed of alpha A subunits is used to simulate chaperoning of partially heat denatured soluble gamma-crystallin. The interaction is both electrostatic and hydrophobic and satisfies experimental evidence for a 1:1 alpha/gamma molar ratio, a doubling of molecular mass and a minimal increase in the dimensions of the complex [J. Biol. Chem. (1994) 269, 13601-13608; Invest. Opthalmol. Vis. Sci. (1995) 36, 311-21]. These data are also in accord with Farahbaksh et al. [Biochemistry (1995) 34, 509-16]; i.e. the bound gamma-crystallin monomers are not in a central cavity, but are separated by alpha A subunits.

Advances in computer technology: impact on the practice of medicine.

Advances in computer technology provide a wide range of applications which are revolutionizing the practice of medicine. The development of new software for the office creates a web of communication among physicians, staff members, health care facilities and associated agencies. This provides the physician with the prospect of a paperless office. At the other end of the spectrum, the development of 3D work stations and software based on computational chemistry permits visualization of protein molecules involved in disease. Computer assisted molecular modeling has been used to construct working 3D models of lens alpha-crystallin. The 3D structure of alpha-crystallin is basic to our understanding of the molecular mechanisms involved in lens fiber cell maturation, stabilization of the inner nuclear region, the maintenance of lens transparency and cataractogenesis. The major component of the high molecular weight aggregates that occur during cataractogenesis is alpha-crystallin subunits. Subunits of alpha-crystallin occur in other tissues of the body. In the central nervous system accumulation of these subunits in the form of dense inclusion bodies occurs in pathological conditions such as Alzheimer's disease, Huntington's disease, multiple sclerosis and toxoplasmosis (Iwaki, Wisniewski et al., 1992), as well as neoplasms of astrocyte origin (Iwaki, Iwaki, et al., 1991). Also cardiac ischemia is associated with an increased alpha B synthesis (Chiesi, Longoni et al., 1990). On a more global level, the molecular structure of alpha-crystallin may provide information pertaining to the function of small heat shock proteins, hsp, in maintaining cell stability under the stress of disease.

Interaction of ATP and lens alpha crystallin characterized by equilibrium binding studies and intrinsic tryptophan fluorescence spectroscopy.

alpha-Crystallin, the most prevalent protein in vertebrate lenses, is a high molecular weight aggregate composed of alpha A and alpha B subunits. Evidence is presented that ATP, a major phosphorus metabolite of the lens binds to alpha-crystallin extracted from calf lenses. The following parameters were obtained from equilibrium binding studies conducted at 37 degrees C: binding sites per 400 kDa aggregate = 10 and Ka = 8.1 x 10(3) M-1; and an essentially identical Ka of 7.84 x 10(3) M-1 and 22 binding sites were determined for a 850 kDa aggregate. The cooperativity parameter, alpha H, approximates unity which denotes that the binding of ligand is at independent sites. Binding was not significant at 22 degrees C and was absent at 4 degrees C. The specificity of the binding site for ATP was established by intrinsic tryptophan fluorescence spectroscopy. In the presence of increasing concentrations of ATP (0.05-0.3 mM), tryptophan fluorescence decreases in a concentration dependent manner to a minimum of 0.2 mM above which there is a non-linear response. Quenching of fluorescence was not evident with P(i), AMP or ADP. GTP elicited a minimal quenching of fluorescence only at the highest concentration (0.30 mM). Modulation of both supramolecular organization and lens metabolism is predicted as a consequence of ATP/alpha-crystallin binding.

Effect of catalytically active recruited crystallins on lens metabolism.

The impact on duck lens metabolism of recruited epsilon-crystallin/LDH-B4 and tau-crystallin/enolase was investigated by NMR spectroscopy. A comparison of the duck lens metabolite profile with that of the calf in which recruited crystallins are absent revealed significant increases in ATP, alpha-glycerophosphate (alpha-GP) and pyridine dinucleotide concentrations. The alterations in the concentrations of ATP and alpha-GP appear to be related to both the high concentration of NAD and the elevated reduced to oxidized NADH/NAD+ ratio.

31P NMR studies of the ATP/alpha-crystallin complex: functional implications.

Evidence is presented for the binding of ATP to alpha-crystallin in the lens by 31P NMR spectroscopic measurements. The chemical shift data as well as the T1 and T2 values indicate that P beta and P gamma of ATP are of prime importance in binding. In addition, it is demonstrated that the association of alpha-crystallin with purified fiber cell membranes is significantly enhanced by the addition of ATP. These results suggest that ATP modulates the functional behavior of alpha-crystallin.

Procion yellow fluorescent microscopy: a new method of studying arterial and venous pathology.

A new means of studying vessel wall pathology is presented. This technique utilizes Procion yellow, a vital fluorescent dye, to stain and delineate the connective elements, matrix, and elastic tissue within the arterial wall. In addition, penetration of the dye into cells provides evidence of membrane pathology. A canine model was used, and clamped segments of femoral artery were stained and examined. The appreciation of this approach for the examination of the vessel wall shows alterations in tissue elements that have not been reported previously. This technique makes possible a unique means of studying connective tissue elements as well as membrane integrity.

Regional variations in electrolytes related to resistivity during bovine lens maturation.

Little is known regarding the behavior of ions in protein-rich cytoplasm characteristic of lens fiber cells. Resistivity is dependent upon the electrolyte concentration available to conduct an applied current and the mobility of these electrolytes. In the present study, the relative importance of these factors in the increasing cortico-nuclear resistivity gradient reported for both calf and bovine lens homogenates was analysed. Relative ion mobility for regions of the lens was determined by the calculation of the ratio of resistivity of lens homogenates to resistivity of aqueous solutions of freely mobile KCl at the same molarity. The increasing resistivity ratios in the calf cortex, transition zone and nucleus suggest an increasingly impaired ion mobility from the outer to the inner lens regions.

Investigation of lens glycolytic enzymes: species distribution and interaction with supramolecular order.

Distribution of several glycolytic enzymes in the lenses of different vertebrate species and their organization in the calf lenses were studied. Though no general pattern of enzyme activities in different species is discernible, high activities of TPI followed, in decreasing order, by GAPDH, enolase, PK, LDH and aldolase appear to be more common. Our observation on the unusually high activities of aldolase in the pig, enolase in the sheep and LDH in the duck lens are interesting in view of the already known dual function of LDH as an enzyme and a structural protein (epsilon-crystallin) in duck. Controlled treatment with detergents Brij-58 and Triton X-100 caused distinctly differential purturbations in the lens cells. In spite of fiber membrane disruption and partial actin dissolution by Brij-58, no significant increase in the release of glycolytic enzymes compared to control was observed. This suggests that none of the enzymes existed as a completely soluble and freely diffusible fraction. But treatment with a strong detergent (Triton X-100) caused the release of higher amounts of enzymes suggesting either a direct or indirect interaction with the cytomatrix components. Aldolase appears to be maximally bound in the cytosol followed by TPI, GAPDH, LDH and PK in decreasing order. Although thin lens slices were incubated with the detergents for a total period of 40 min and the loss of fiber architecture and organization confirmed by light microscopy, in the Triton X-100 treated tissues less than 25% of the total activity of any enzyme except TPI appeared in the bathing medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-dependent changes in resistivity and electrolytes related to lens development and growth in the rat.

Alterations in resistivity measurements of lens homogenates, lens percent water and cation concentrations of sodium and potassium show a complex pattern in the Sprague-Dawley rat during lens development and maturation. During the neonatal period, the data provide evidence for three distinct periods: days 5-12, a pre-critical maturation period (pre-CMP), a steep decline in cation concentration and a minimal change in percent water were accompanied by an expected sharp rise in resistivity; days 12-16, critical maturation period (CMP), a further decrease in ion concentration and water was concomitant with the unexpected observation of no significant change in resistivity; and days 16-30, post-CMP, no significant changes were observed for cation concentrations, percent water, or resistivity. From 30 to 100 days, an adult nuclear maturation period (NMP), a decrease in cation concentration and percent water was reflected in a rise in resistivity. A comparison of 100 and 500 day lenses revealed that the concentrations of Na and K, and percent water are essentially unchanged. The K/Na ratio, which had decreased from an initial value of 5.9 at 5 days, stabilized at approximately 4 by day 100. A comparison of the resistivity measured in both lens homogenates and KCl solutions at identical molar strengths revealed that, for the ages studied, ion concentration and ion mobility play important roles in determining this parameter. The underlying cause for age related variations in lens susceptibility to cataractogenic insult is most likely related to complex changes in composition and resistivity which undoubtedly reflect adjustments in molecular organization. The proposition that the lens is a 'free flowing' syncytium of cells appears unwarranted.

L-alpha-glycerophosphate binding to bovine gamma-crystallin: a potential link between metabolism and supramolecular order.

The highly selective nature of protein-ligand interactions provides a sensitive mechanism for the modulation of cellular activity by proteins. In the eye lens the supramolecular order of the lens crystallins, which is expected to be susceptible to protein electrostatic charge, in part defines transparency. The binding of charged ligands to proteins is one way of achieving an alteration in protein electrostatic charge. Evidence is presented that L-alpha-glycerophosphate, a major phosphorus metabolite of eye lens metabolism, binds to the globular protein, gamma-crystallin with moderately high affinity and in a positive cooperative manner. The following binding parameters were obtained from equilibrium measurements: minimum number of binding sites, n = 2; Kassoc = 6.2 +/- 0.5 x 10(3) M-1; cooperativity parameter, alpha H = 1.9 +/- 0.1. Interactive computer graphics display techniques were used to locate putative ligand binding sites, and in turn, to identify the possible molecular interactions responsible for the binding of ligand to protein at one of the sites. One putative binding site was located in the cleft between the two domains of gamma II-crystallin. Arginyl residues 79 and 147 are involved in ligand binding as are the peptide carbonyl oxygens of residues Tyrosyl-50 and Aspartyl-156. Five hydrogen bonds between the ligand and the protein structure are predicted for the binding of L-alpha-glycerophosphate, whereas only 3 occur for the binding of the "unnatural" D-enantiomorph. Modulation of both lens protein supramolecular organization and lens metabolism is predicted to be a consequence of L-alpha-glycerophosphate binding to gamma-crystallin in the lens.

Impact of cellular and molecular organization on lens metabolism.

The relationship between the metabolic gradient within the ocular lens and its cellular and molecular organization is discussed. The lens shares a number of organizational similarities with other stratified ectodermal tissues. All of these tissues were avascular and therefore, dependent upon the simple diffusion of nutrients from the surrounding medium. They contain non-metabolizing cell layers which are continually produced by an irreversible maturation process. This process is not random aging but a well orchestrated sequence of events dependent upon the physico-chemical properties of specialized proteins typical of each tissue. In the lens, these proteins are the crystallins. The signal for this maturation is decreased metabolism in conjunction with cell dehydration. Metabolism of these maturing cells is decreased by limited nutrient penetration because of barriers to nutrient diffusion and the high rate of utilization of nutrients by the more metabolically active cells near the basement membrane. Additionally, the regulation of anaerobic metabolism is dependent upon the maintenance and dissolution of an organized array of enzymes and the effects of dehydration, i.e. excluded volume effects and modified macromolecular organization. In the lens, the absence of metabolism during the maturation of the inner fiber cell layers is as important as the presence of active metabolism where differentiation and active protein synthesis occur. Therefore, any stress that disrupts tissue organization will have a detrimental effect on lens integrity.

Morphological studies of an ion-dependent perinuclear cataract model.

The perinuclear region of the rabbit lens is susceptible to alterations in the ionic composition of incubation medium. Rabbit lenses and a comparable cell type, red blood cells, were stressed during ex vivo incubations in isotonic modified Earle's medium with 131 mM NaCl replaced by either 232 mM sucrose or 131 mM choline chloride at pH 7.2 (normal) or 9.2. Our parallel NMR study revealed that these experimental media maintain normal intracellular pH and phosphorus metabolite levels. The present study demonstrates that lens transparency, normal fiber cell ultrastructure and volume were maintained in either sodium chloride or choline chloride containing media at normal or elevated pH. Similarly, normal morphology, mean cell volume (MCV) and mean cell hemoglobin concentration (MCHC), 86.8 +/- 0.03 micron 3 and 33.2 +/- 1.0 g dl-1, respectively, were maintained in red blood cells in either sodium chloride or choline chloride containing media. In sodium chloride deficient media at both normal and elevated pH the lens developed a nuclear cataract based on slit-lamp examination; however, SEM examination showed that fiber cell morphological abnormalities were confined to a narrow band, 50 micron wide, in the perinuclear region of the transition zone. Damage consisted of ruptured cell membranes and an absence of identifiable interdigitations with the combination of sodium chloride deficiency and elevated pH. The major abnormality produced during incubation in sodium chloride deficient medium at normal pH was the presence of numerous smooth-surfaced cellular protrusions along the vertices of the perinuclear fiber cells. In addition, the sodium chloride deficient medium, pH 9.2, produced a volume loss both in the lens and RBC (4.5 +/- 1.5% and 5.6 +/- 1.1%, respectively). The sodium chloride deficient medium, pH 7.4, produced no volume loss in the lens or red blood cells (MCV 86.0 +/- 0.05 micron 3). Further studies indicated that the cataract induced by sodium chloride deficiency (pH 9.2) is irreversible. The mechanism for perinuclear opacification due to ion deficiency remains to be elucidated.