Structural homology of lens crystallins. III. Secondary structure estimation from circular dichroism and prediction from amino acid sequences.

Circular dichroism spectra (196-240 nm) of calf alpha-, beta H-, beta L- and gamma-crystallins were measured and analyzed over the entire wavelength range with five curve-fitting procedures for estimating protein secondary structure. For gamma-crystallin the estimates are in good agreement with the X-ray structure. For all four crystallins the estimates are very similar: 0-9% alpha-helix and 51-68% beta-sheet. This is in accordance with the three-dimensional homology of beta Bp- and gamma 2-crystallin polypeptide chains as postulated from their 30% sequence homology, and suggests that alpha A- and alpha B-crystallin chains may also have a corresponding structure. Secondary structure elements in the four amino acid sequences were predicted using two different comprehensive prediction methods. For gamma 2-crystallin the predictions of beta-sheet are in good agreement with the X-ray structure and with circular dichroism estimates. For beta Bp-crystallin only the C-terminal domain secondary structure predictions are considered satisfactory, which possibly relates to the proposed role of the N-terminal domain in subunit interactions. The combined predictions for alpha A- and alpha B-chains (3% helix, 49% sheet) are in excellent agreement with circular dichroism. Moreover, the good alignment of predicted beta-sheet segments in alpha-crystallin chains with known beta-sheet strands in gamma 2- (and presumably beta Bp-) crystallin strongly supports a similar 4-motif folding pattern in all four calf crystallin chains.

Physicochemical characterization of high-molecular-weight alpha-crystallin subpopulations from the calf lens nucleus.

Calf lens nuclear alpha-crystallin was separated into five molecular weight subpopulations by exclusion chromatography on Bio-Gel A-5m. These subpopulations were compared by amino acid analysis, ultraviolet absorption analysis, fluorescence, far- and near-ultraviolet circular dichroism, isoelectric focusing, SDS-polyacrylamide gel electrophoresis and sedimentation velocity analysis. Although only minor differences were detectable in most physicochemical properties, progressive changes were found in the near-ultraviolet circular dichroism spectra and in pellet hardness after centrifugation. Minute amounts of beta-crystallin polypeptides and a 43 kDa component were present in all five subpopulations. In addition, the highest molecular weight aggregates contain some gamma-crystallin polypeptides. A slow re-equilibration of separated subpopulations towards the initial distribution was observed by rechromatography.

Physicochemical characterization of lens proteins of the squid Nototodarus gouldi and comparison with vertebrate crystallins.

The main water-soluble proteins of squid lens (S-crystallins) have a molecular weight of 60 000, a sedimentation coefficient s020,w of 5.2 S, 20-30% alpha-helical secondary structure, and an unusually high methionine content (12%). The subunits of Mr 30 000 (major) and Mr 27 000 (minor) have related N-terminal amino acid sequences, but a very heterogeneous charge distribution with predominantly basic isoelectric points. Higher-Mr aggregates have similar secondary/tertiary structure and amino acid composition, but contain additional acidic subunits and Mr 35 000-40 000 subunits. S-crystallins resemble vertebrate beta-crystallins in their quaternary structure, and their N-terminal sequence shows analogy with the first 19 residues of calf beta/gamma-crystallin folding units. In the urea-soluble and urea-insoluble lens fractions polypeptides of Mr 58 000 and 80 000, respectively, predominate, which presumably correspond to the main cytoskeleton and membrane proteins. Water-soluble lens components of less than Mr 2000 were isolated which have ultraviolet absorption maxima at 327 and 370 nm.

Structure and stability of gamma-crystallins. II. Differences in microenvironments and spatial arrangements of cysteine residues.

The gamma-crystallin fractions II, III and IV from calf eye lens were treated with the thiol-specific fluorescent probe 2-(4'-maleimidylanilino)naphthalene-6-sulfonate (MIANS), in order to determine the reactivity of the seven (gamma-II) or six (gamma-III, gamma-IV) cysteine residues. Two classes of reactive cysteines were distinguished by variations in fluorescence intensity with increasing molar excess of the probe, and approximately three cysteines were nonreactive in each gamma-crystallin. From the position of the emission maximum, it is apparent that MIANS-labeled cysteines of gamma-IV are in the least hydrophobic environment. Fluorescence energy transfer was observed from tryptophan to MIANS-labeled cysteines in both gamma-II and gamma-III crystallins, with efficiencies of 86% and 89%, respectively, but not in gamma-IV crystallin. We suggest that the spatial arrangements and microenvironments of cysteine residues of gamma-crystallins are sufficiently different from each other to account for the variations in fluorescence characteristics of the MIANS-labeled proteins and the lack of energy transfer in gamma-IV crystallins.

Structural homology of lens crystallins. II. Homology expressed by correlation coefficients and hydropathy profiles.

Bovine lens alpha A- and alpha B-crystallin polypeptides show extensive sequence homology with each other, but apparently none with beta Bp- and gamma 2-crystallin. Despite only 30% sequence homology, the latter two proteins are assumed to have a strong correspondence in tertiary structure, consisting of four structurally similar folding units of antiparallel beta-sheet. We have tested for internal structural repeats in all crystallins, and structural homology between crystallins, by comparing various physical properties of the amino acid residues, such as bulkiness and propensity to form beta-sheet and beta-turn structure. Two procedures used a combination of five physical parameters to calculate correlation coefficients. The 4-fold structural repeat in gamma 2-crystallin and the internal duplication in beta Bp-crystallin were readily detectable, as was also the strong structural homology between corresponding folding units in beta Bp- and gamma 2-crystallin. However, for alpha-crystallin polypeptides, no conclusive support was obtained for either a four-unit or a six-unit folding, the two models previously considered by us. The third procedure compared smoothened hydropathy plots, representing hydrophilic and hydrophobic regions along the polypeptide sequences. Hydropathy profiles were found to show strong correspondence, particularly between alpha B-crystallin and beta Bp-crystallin. These observations support a similar 4-fold folding pattern for all bovine crystallins. A possible role in subunit interactions of the N-terminal folding unit, which has hydrophobic surface characteristics in both alpha- and beta-crystallin polypeptides, is proposed.

Three classes of sulfhydryl group in bovine alpha-crystallin according to reactivity to various reagents.

alpha-Crystallin from calf eye lens is found to have 30 sulfhydryl groups per 800 000 daltons by modification with 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) and 4,4'-dithiopyridine in 6 M urea. These -SH groups can be divided into three different classes in native alpha-crystallin from their reactivity with DTNB, 4,4'-dithiopyridine, iodoacetamide and ethylenimine. Results obtained with these reagents point to the presence of 7 +/- 1 thiol groups (Class I) which are likely to be surface exposed, with a concomitant 40--45% quenching of tryptophan fluorescence in DTNB-modified alpha-crystallin. Another 10 +/- 1 thiol groups (Class II) must be in a hydrophobic environment since they react only with the hydrophobic reagents, causing a further decrease in fluorescence intensity. Class III, 13 +/- 2 thiol groups, is inaccesible to any of these reagents. Introduction of up to 15 negatively charged thionitrobenzoate groups or seven positively charged aminoethyl groups in the alpha-crystallin molecule at pH 7.5--8.0 did not change the state of aggregation as judged from the sedimentation coefficients.

Structure and stability of gamma-crystallins. I. Spectroscopic evaluation of secondary and tertiary structure in solution.

The three major bovine gamma-crystallin fractions (gamma-II, gamma-III and gamma-IV) are known to have closely related (80-90%) amino acid sequences and three-dimensional folding of the polypeptide backbone. Their chiroptical and emission properties, as measured by circular dichroism (CD) and fluorescence, are now shown to differ distinctly. The far-ultraviolet CD spectra indicate that all three gamma-crystallins have predominantly beta-sheet conformation (45-60%) with only subtle differences in secondary structure. The fluorescence emission maxima of gamma-II, gamma-III and gamma-IV, due to the four tryptophan residues, appear at 324, 329 and 334 nm, respectively, suggesting that tryptophan residues are buried in environments of decreasing hydrophobicity. Corresponding differences in quantum yield may be due to fluorescence quenching by neighboring sulfur-containing residues. Titratable tyrosines are maximal for gamma-III, as manifested from difference absorption spectra at alkaline pH. The near-ultraviolet CD spectra differ in position, magnitude and sign of tryptophan and tyrosine transitions. In addition, a characteristic CD maximum at 235 nm, presumably due to tyrosine-tyrosine exciton interactions, differs in magnitude for each gamma-crystallin. This study shows that the environment and interactions of the aromatic residues of the individual gamma-crystallin fractions are quite different. These variations in tertiary structure may be significant, in terms of stability of gamma-crystallins towards aggregation and denaturation, for understanding lens transparency and cataract formation in general.

Rat lens gamma-crystallins. Characterization of the six gene products and their spatial and temporal distribution resulting from differential synthesis.

We have isolated, purified and characterized six individual gamma-crystallin polypeptides present in the rat lens. Comparison of their amino acid compositions with the known structure of the six gamma-crystallin genes permits a one-to-one correspondence to be made between each protein synthesized and the encoding gene. This demonstrates that each of the six genes is actually expressed in vivo. Two classes of three gamma-crystallins each, which we have designated classes gamma ABC and gamma DEF, are known to exist, on the basis of internal sequence homology. We have measured the temperature-dependent phase-separation characteristics of solutions of the six purified gamma-crystallins, and find that the three members of the gamma DEF class (gamma 2-2, gamma 3-1 and gamma 4-1) are all cryo-proteins with relatively high phase-separation temperatures, whereas the three gamma ABC crystallins (gamma 1-1, gamma 1-2 and gamma 2-1) do not show phase separation above -7 degrees C. We have measured the spatial distribution in rat lens of each of the alpha-, beta- and gamma-crystallins as a function of age from 1 to 420 days, using size-exclusion and ion-exchange high-pressure liquid chromatography (HPLC). Our findings in the cortical layer permit us to establish the differential synthesis of each of the crystallins during lens development. Particular attention has been devoted to the spatial and temporal distribution of the six individual gamma-crystallins. Up to birth, synthesis of the three components of the gamma DEF class predominates, and in particular that of gamma 2-2. In subsequent development the three components of the gamma ABC class assume a greater proportion of monomeric crystallins synthesized, while beta s-crystallin synthesis predominates in late development. Our analysis of different layers within single lenses provides novel information on spatial gradients of the water-soluble and water-insoluble protein fractions as a function of age. We consider the consequences of these findings for lens transparency and opacity in both rat and mouse lens. We show that the high concentrations of gamma DEF-crystallins appear to be responsible for the opacity known to occur in young rat lenses. We conclude from these observations that close control of the differential synthesis of gamma-crystallins plays an important role in maintaining lens transparency during development.

Subtilases: the superfamily of subtilisin-like serine proteases.

Subtilases are members of the clan (or superfamily) of subtilisin-like serine proteases. Over 200 subtilases are presently known, more than 170 of which with their complete amino acid sequence. In this update of our previous overview (Siezen RJ, de Vos WM, Leunissen JAM, Dijkstra BW, 1991, Protein Eng 4:719-731), details of more than 100 new subtilases discovered in the past five years are summarized, and amino acid sequences of their catalytic domains are compared in a multiple sequence alignment. Based on sequence homology, a subdivision into six families is proposed. Highly conserved residues of the catalytic domain are identified, as are large or unusual deletions and insertions. Predictions have been updated for Ca(2+)-binding sites, disulfide bonds, and substrate specificity, based on both sequence alignment and three-dimensional homology modeling.

Cloning of usp45, a gene encoding a secreted protein from Lactococcus lactis subsp. lactis MG1363.

We have cloned usp45, a gene encoding an extracellular secretory protein of Lactococcus lactis subsp. lactis strain MG1363. Unidentified secreted 45-kDa protein (Usp45) is secreted by every mesophilic L. lactis strain we tested so far and it is chromosomally encoded. The nucleotide sequence of the usp45 gene revealed an open reading frame of 1383 bp encoding a protein of 461 amino acids (aa), composed of a 27-aa signal peptide and a mature protein initiated at Asp28. The gene contains a consensus promoter sequence and a weak ribosome-binding site; the latter is rather uncommon for Gram-positive bacteria. Expression studies in Escherichia coli showed efficient synthesis and secretion of the protein. Usp45 has an unusual aa composition and distribution, and it is predicted to be structurally homologous with P54 of Enterococcus faecium. Up to now, no biological activity could be postulated for this secreted protein.

Biosynthesis and secretion of a precursor of nisin Z by Lactococcus lactis, directed by the leader peptide of the homologous lantibiotic subtilin from Bacillus subtilis.

The DNA sequence encoding the leader peptide of the lantibiotic subtilin from Bacillus subtilis was fused to the sequence encoding pronisin Z, and this hybrid gene was expressed in a Lactococcus lactis strain that produces nisin A. This strain simultaneously secreted nisin A and a protein of approximately 6 kDa. Amino acid sequencing of the purified 6 kDa protein and structural analysis of its main tryptic fragment by two-dimensional 1H-NMR showed that it consists of the unmodified leader peptide of subtilin, without the N-terminal methionine residue, linked to a fully matured nisin Z part. The hybrid protein and its main tryptic fragment [ITPQ]-nisin Z, showed at least 200-fold lower antimicrobial activities than nisin Z against three different indicator strains.

Characterization of the Lactococcus lactis pepN gene encoding an aminopeptidase homologous to mammalian aminopeptidase N.

The nucleotide sequence of the pepN gene from Lactococcus lactis encoding a zinc-metallo aminopeptidase has been determined. The open reading frame of 2,538 base pairs encodes a protein with a calculated M(r) of 95,368, which agrees with the apparent M(r) of 95,000 of the gene product which was identified by polyclonal antibodies raised against the purified aminopeptidase. The amino acid sequence of the aminopeptidase of L. lactis was found to be similar to the corresponding enzymes of human, rat and mouse, with almost 30% of the residues identical. Also, a highly conserved area was identified which has similarity with the active site of thermolysin. A zinc-binding site, as well as the catalytic site for PepN, is predicted to lie within this conserved stretch. Putative promoter regions upstream of PepN were confirmed by primer extension analysis.

The structure of the lantibiotic lacticin 481 produced by Lactococcus lactis: location of the thioether bridges.

The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis ssp. lactis. This polypeptide contains 27 amino acids, including the unusual residues dehydrobutyrine and the thioether-bridging lanthionine and 3-methyllanthionine. Lacticin 481 belongs to a structurally distinct group of lantibiotics, which also include streptococcin A-FF22, salivaricin A and variacin. Here we report the first complete structure of this type of lantibiotic. The exact location of the thioether bridges in lacticin 481 was determined by a combination of peptide chemistry, mass spectrometry and NMR spectroscopy, showing connections between residues 9 and 14, 11 and 25, and 18 and 26.

Interactions of lens proteins. Ultrafiltration is unsuitable to detect self- or mixed-association.

Crystallins from calf lens were subjected to ultrafiltration through an Amicon XM-300 membrane to determine whether specific interactions between identical proteins (self-association) or different proteins (mixed-association) could be detected and quantified. Single crystallins at different concentrations, simple mixtures and total lens extracts were studied separately. alpha-Crystallin (Mr 800 000) is nearly fully retained (greater than 95%) by XM-300. Retention of beta-crystallins (Mr 50 000-200 000) is found to be much higher than expected from their molecular weights. Ultrafiltration of gamma-crystallin (Mr 20 000) solutions of 1.0-22.6 g/l shows that retention increases as a function of protein concentration. In solutions of single crystallins, self-association effects could not be separated from concentration polarization effects at the membrane surface. In mixtures of crystallins, mixed-association could not be separated from self-association, concentration polarization and excluded volume effects on self-association.

Interactions of lens proteins. Self-association and mixed-association studies of bovine alpha-crystallin and gamma-crystallin.

Concentrated solutions of calf alpha-crystallin (up to 45 g/l) and gamma-crystallin (up to 67 g/l) were subjected to frontal exclusion chromatography at pH 7.3, ionic strength 0.17 and 20 degrees C. The experimental concentration dependence of the weight-average partition coefficient was compared with theoretical expressions, which include considerations of thermodynamic non-ideality effects, for the concentration dependence of a single solute and of a solute undergoing reversible self-association. Two types of association pattern were examined, discrete dimerization and indefinite self-association. The partition chromatography results are consistent with an indefinite self-association of gamma-crystallin, governed by an isodesmic association constant of 6.7 X 10(-3) l/g. alpha-Crystallin appears to self-associate either very weakly, with a maximal association constant of 0.9 X 10(-3) l/g, or not at all; the distinction depends on the assessment of the non-ideality coefficients. The consequences of excluded volume effects on these self-association equilibria at high total protein concentration are discussed. Mixtures of alpha-crystallin and gamma-crystallin were analyzed by frontal exclusion chromatography (up to 14 g/l) and sedimentation velocity (up to 115 g/l): no interaction was observed.

The indefinite self-association of lysozyme: consideration of composition-dependent activity coefficients.

An improved iterative method for computing association constants from sedimentation equilibrium results obtained with self-interacting protein systems is presented which accounts for the composition-dependence of the activity coefficients of all oligomeric species. The method is based on the calculation of viral coefficients from covolume and charge considerations, the statistical mechanical basis of which is discussed in relation to the DLVO theory. The method is applied to results obtained with lysozyme in diethylbarbiturate buffer of pH 8.0 and ionic strength 0.15 at 15 degrees C. It is shown that these results, encompassing a range of total solute concentration up to 19.7 g/liter are consistent with self-association patterns comprising either a monomer--dimer--trimer system or an isodesmic indefinite self-association of the monomer, the latter being favored. A firmer distinction between these possibilities is sought on the basis of the dependence of the weight-average partition coefficient, determined by frontal gel chromatography, on total solute concentration (up to 56.6/liter). This analysis accounts for the composition-dependence of the ratio of the activity coefficients of partitioning monomer in mobile and stationary phases. It is concluded that all results are consistent with an indefinite self-association of lysozyme governed by a single association constant of 4.61 x 10(2) liter/mole.

Chromatographic evidence of the self-association of oxyhemoglobin in concentrated solutions: its biological implications.

Expressions that take into account the effects of thermodynamic non-ideality, described in terms of a high-order virial expansion, are derived for the concentration-dependence of the weight-average partition coefficient in exclusion chromatography of a single solute and of a solute undergoing reversible self-association. Comparison of the concentration-dependences predicted by those expressions with results obtained for bovine and human oxyhemoglobins on CPG-10-120 porous glass beads in 0.156 I phosphate-chloride buffer, pH 7.3, shows that neither oxyhemoglobin conforms with the concept of it being a single alpha 2 beta 2 entity with Stokes radius of 3.13 nm, the experimental value. Previously published osmotic pressure and sedimentation equilibrium results are also shown to be inconsistent with this concept. On the other hand, both sets of exclusion chromatography results are consistent with the joint operation of thermodynamic non-ideality and reversible association of the alpha 2 beta 2 species. From the magnitude of the equilibrium constant, derived for either of two possible modes of association, it is calculated that only half of the oxyhemoglobin would be in the alpha 2 beta 2 states under conditions of oxygen saturation and a concentration of 320 g/liter, that pertaining in the red blood cell. The consequences of this association phenomenon are discussed in relation to the oxygen binding curves obtained by others in the presence and absence of 2,3-diphosphoglycerate (DPG). An explanation is provided of the observed dependence on hemoglobin concentration of oxygen-binding in the presence of DPG, and of the absence of such an effect in DPG-free solutions. It is concluded that the control of oxygen binding to hemoglobin in the physiological situation involves the joint operation of self-association and allosteric effects.

Exclusion chromatography of concentrated hemoglobin solutions. Comparison of the self-association behavior of the oxy and deoxy forms of the alpha 2 beta 2 species.

Theory pertaining to the interpretation of partition chromatography results obtained with self-associating protein systems studied at high total concentrations is extended to permit consideration of situations in which both monomeric and dimeric states partition. This development, which includes considerations of thermodynamic nonideality effects, permits a quantitative correlation of human oxyhemoglobin results reported previously and obtained in this work employing a different stationary matrix of controlled-pore glass beads. The two sets of results, obtained at pH 7.3 and 20 degree C, indicate that the alpha 2 beta 2 species of oxyhemoglobin self-associates. Two types of association pattern, discrete dimerization and an indefinite self-association, are examined. This is done for a realistic range of values for the radius, r. of the effective hard sphere appropriate to the calculation of the covolume of the alpha 2 beta 2 species in the assessment of the thermodynamic nonideality contribution. Assessed values of the isodesmic association constant range from 66 +/- 23 M-1 (r - 2.84 nm) to 154 +/- 26 M-1 (r = 3.13 nm). This mode of indefinite association is marginally favored over dimerization when the larger value of r is considered, the two patterns becoming virtually indistinguishable for the lower value of r. Partition chromatography results are also presented for human deoxyhemoglobin up to a total concentration of 225 g/l, and are analyzed in a similar fashion to show that the indefinite self-association pattern is favored, governed by an isodesmic constant in the range 91 +/- 9 M-1 (r = 2.84 nm) to 223 +/- 84 M-1 (r = 3.13 nm). Comparison of the constants assessed for the oxy and deoxy systems permits discussion of the concept that oxygen binds preferentially to the alpha 2 beta 2 species of deoxyhemoglobin in comparison with its polymers.

Models for the analytical ultracentrifuge behavior of Helix pomatia alpha-hemocyanin. I. Experimental testing of previous models.

The 'microheterogeneity model' (R.J. Siezen and R. van Driel, Biochim. Biophys. Acta 295 (1973) 131) and the 'incompetent whole molecule model' (G. Kegeles, Arch. Biochem. Biophys. 180 (1977) 530) for the dissociation of Helix pomatia alpha-hemocyanin whole molecules to half molecules were tested experimentally, using ultracentrifugation and stopped-flow dilution analysis. Results of differential sedimentation experiments followed by stopped flow analysis of separated fractions of 60 S and 100 S molecules were not entirely as predicted by the incompetent model, the agreement depending on the pH and ionic strength of analysis. A considerable amount of stopped-flow dilution response could be attributed to material sedimenting between 60 and 100 S. This material appears to be the main equilibrating fraction, and its amount is considerably larger than that predicted by the microheterogeneity model. Increased hydrostatic pressure was found to enhance this fraction, whereas fixation or low ionic strength reduced or eliminated this fraction. Nonequilibrium components of 30, 50 and 80 S were detected and partially purified by differential sedimentation.

Squid lens cytoskeleton and membrane proteins.

Cytoskeletal and membrane proteins were isolated from respectively the urea-soluble and urea-insoluble fractions of the squid lens. The main cytoskeletal polypeptide has a molecular weight (63000) and an amino acid composition similar to those of vertebrate intermediate filament proteins, including mammalian lens vimentins. Intermediate filaments, and bundles thereof, were regenerated from the squid lens urea-soluble fraction upon removal of urea. The main membrane polypeptide of 140000 Mr has an amino acid composition entirely different from that of the main intrinsic membrane protein of 26000 Mr which is found in all vertebrates. Although non-EDTA-extractable, the 140000 Mr squid membrane polypeptide is best classified as extrinsic, since it has a high polarity (mainly acidic residues) and can be degraded completely upon trypsin treatment of squid lens membranes.