A dynamic quaternary structure of bovine alpha-crystallin as indicated from intermolecular exchange of subunits

The structural bovine eye lens protein alpha-crystallin was dissociated in 7 M urea and its four subunits, A1, A2, B1, and B2, were separated by means of ion-exchange chromatography. Homopolymeric reaggregates of these subunits were prepared by removal of the denaturant via dialysis. It was found that subunits were exchanged upon incubation of mixtures of two homopolymers under native conditions. New hybrid species were formed within 24 h as demonstrated by isoelectric focusing. Moreover, native alpha-crystallin molecules also exchanged subunits when incubated with homopolymeric aggregates of B2 subunits. Subunit exchange between native alpha-crystallin molecules is postulated, and a "dynamic quaternary structure" is presented that allows the polydisperse protein to adapt to changes in cytoplasmic conditions upon aging of the lens tissue.     

The effect of various acylating agents on the catalytic properties and structure of aspartate aminotransferase]

Changes of quaternary structure and conformation of molecule concomitant with inactivation were observed in the course of aspartate transaminase acylation by maleic, citraconic, dimethylmaleic and succinic anhydrides. It was established that acylation of 10-12 xi-amino groups of lysine did not induce the dissociation of transaminase into subunits. Further acylation of amino groups (2 groups if dimethylmaleic anhydrade was used as acylating agent) induced dissociation of transaminase dimer into subunits. These data were obtained by sedimentation analysis. The dissociation was accompanied with a sharp decrease of correlation time (from 18 nsec to 9 nsec) of the paramagnetic label covalently bound to the protein. The obtained results allow us to distinguish three types of xi-aminogroups of aspartate transaminase: exposed (about 12 residues), "contact" (2 residues) located in the vicinity to complementary surfaces of subunits and buried (about 6 residues). The stepwise inactivation occurred during the acylation as a result of conformational changes or appearance of sterical hindrances in the cataytic site of the enzyme. The thiol groups were not modified in transaminase molecule under experimental conditions used. Aspartate transaminase treated with citraconic or dimethylmaleic anhydride may be deacylated under mild conditions. After reacylation the quaternary structure was reconstituted and catalytic activity was almost fully restored.     

On the structure of alpha-crystallin: construction of hybrid molecules and homopolymers

The alpha A2 and alpha B2 subunits of bovine alpha-crystallin were purified by chromatofocussing in urea and assembled into homopolymers. Light-scattering measurements indicated their molecular masses were 360 and 420 kDa. The alpha A2 and alpha B2 polypeptides were also used to construct a series of hybrid molecules with alpha A/alpha B ratios ranging from 7:1 to 1:7. Sedimentation velocity analyses, isoelectric focussing under non-deaggregating conditions, circular dichroism spectroscopy and immunochemical analysis indicated that all of the subunits had copolymerized to alpha-crystallin-like aggregates with complete regeneration of the native structure. The polymers could be distinguished on the basis of their differing affinities for the antiserum. This was directly related to the proportion of alpha A2 subunits in each polymer. It was concluded that the alpha A2 and alpha B2 subunits are structurally equivalent and occupy equivalent site in the alpha-crystallin aggregates. It was also concluded that a micellar-like quaternary structure was consistent with most previous observations on the protein.     

Modification of alpha-crystallin subunit chains and formation of alpha-neoprotein molecules: role of SH groups

Immunoadsorbents with bound antibodies restricted to determinants dependent on alpha-crystallin's quaternary structure permitted the fractionation of the population of 125I-labeled alpha-crystallin molecules, treated by iodoacetic acid, into molecules in which the native structure was still preserved and molecules with a completely different quaternary structure than the native protein. Parallel experiments with [14C]iodoacetic acid yielded information on the percentage of blocked SH groups in each of the above two fractions. The presence of molecules formed by A with B-chain association was established by sequential binding first to an immunoadsorbent with antibodies restricted to determinants located on alpha-crystallin's A-subunit chains as ligand and second, after desorption, to an immunoadsorbent with antibodies to B chains as ligand. With the aid of these techniques, it was established that (i) The modified alpha-crystallin molecules with quaternary determinants of the native protein contained a maximum of 23% blocked SH groups, indicating that the carboxymethylation involved only the fast-reacting surface SH groups. (ii) The modified alpha-crystallin molecules without the native protein's quaternary structure were built by a different association between A and B subunits than in alpha-crystallin, indicating formation of alpha-neoprotein molecules. (iii) Monomeric A chains with all SH groups carboxymethylated, and monomeric B chains in a ratio of 1A:5B, 2A:1B, and 5A:1B in urea solution, associate on dialysis, forming alpha-neoprotein molecules.     

Effects of citraconylation on enzymatic modification of human proinsulin using trypsin and carboxypeptidase B

Insulin is a polypeptide hormone which is produced by the β‐cell of pancreas and controls the blood glucose level in the human body. Enzymatic modification of human proinsulin using trypsin and carboxypeptidase B generally causes high accumulation of insulin derivatives, leading to more complicated purification processes. A simple method including citraconylation and decitraconylation in the enzymatic modification process was developed for the reduction of a major derivative, des‐threonine human insulin. Addition of 3.0 g citraconic anhydride per g protein into the reaction solution led to the citraconylation of lysine residues in human proinsulin and reduction of relative des‐threonine insulin content from 13.5 to 1.0%. After the enzymatic hydrolysis of the citraconylated proinsulin, 100% of lysine residues can be decitraconylated and restored by adjusting pH to 2–3 at 25 °C. Combination of hydrogen peroxide addition and citraconylation of proinsulin expressed in recombinant Escherichia coli remarkably improved the conversion yield of insulin from 52.7 to 77.7%. Consequently, citraconylation of lysine residues blocked the unexpected cleavage of human proinsulin by trypsin, minimized the formation of des‐threonine insulin and hence increased the production yield of active insulin. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Identification of "buried" lysine residues in two variants of chloramphenicol acetyltransferase specified by R-factors.

Two variants of chloramphenicol acetyltransferase which are specified by genes on plasmids found in Gram-negative bacteria were subjected to amidination with methyl acetimidate to determine the relative reactivity of surface lysine residues and to search for unreactive or "buried" amino groups which might contribute to stabilization of the native tetramers. Representative examples of the type-I and type-III variants of chloramphenicol acetyltransferase were found to have one lysine residue each in the native state which appears to be inaccessible to methyl acetimidate. The uniquely unreactive residue of the type-I protein is lysine-136, whereas the lysine that is "buried" in the type-III enzyme is provisonally assigned to residue 38 of the prototype sequence. It is suggested that the lysine residue in each case participates in the formation of an ion pair at the intersubunit interface and that the two amino groups in question occupy functionally equivalent positions in the quaternary structures of their respective enzyme variants. Lysine-136 of type-I enzyme is also uniquely unavailable for modification by citraconic anhydride, a reagent used to disrupt the quaternary structure of the native enzyme. Contrary to expectation, exhaustive citraconylation fails to dissociate the tetramer, but does destroy catalytic activity. Removal of citraconyl groups from modified chloramphenicol acetyltransferase is accompanied by a full region of catalytic activity. Analysis of the rate of hydrolysis of citraconyl groups from the modified tetramer by amidination of unblocked amino groups with methyl [14C]acetamidate reveals difference in lability for several of the ten modified lysine residues. Although the unique stability of the quaternary structure of chloramphenicol acetyltransferase may be due to strong hydrophobic interactions, it is argued that lysine-136 may contribute to stability via the formation of an ion pair at the subunit interface.     

Interaction between the constituent A and B lens alpha-crystallin subunits leading to formation of alpha-neoprotein molecules

A and B constituent subunits associated in lens alpha-crystallin were found to interact with added B chains forming alpha-neoprotein molecules with lower A to B chain ratios than 2 A to 1 B in alpha-crystallin. Addition of 1% excess of B chains to the one in alpha-crystallin, which resulted in a ratio of 1.98 A to 1 B in the mixture, caused a change of quaternary structure in 30% of alpha-crystallin molecules within 18 h. At a ratio of 1.86 A to 1 B, all alpha-crystallin molecules were affected at this time. A maximum number of 495 B chains was found to form an association with 1 A chain, initially bound in alpha-crystallin. Such a high number may indicate that the reaction involves monomeric A chains binding aggregated macromolecules of B chains. It is in such form that B chains occur as macromolecules with an average molecular weight of 0.7 X 10(6) in aqueous solution. The alpha-neoprotein molecules selected for studies in this report had A to B chain ratios of 1.75:1, 1:1, and 0.2:1. Each behaved in immunodiffusion tests like single molecular entities. Antigenic determinants located on A as well as on B chains associated with each other in alpha-crystallin were found to be identical with determinants on the chains associated in the above alpha-neoprotein molecules. Determinants dependent on the quaternary structure of alpha-neoprotein and of alpha-crystallin molecules were completely different. Some of the quaternary determinants of various alpha-neoproteins were type specific and did not occur in molecules with different A to B chain ratios. Other quaternary determinants occurred in all alpha-neoproteins. An excess of A chains did not revert alpha-neoproteins to alpha-crystallin. However, alpha-neoprotein molecules did interact with added B chains forming neomolecules with lower A to B chain ratios.     

The reaction of aldolase with 2-methylmaleic anhydride

1. The reaction of rabbit muscle aldolase with 2-methylmaleic anhydride is described. All the protein amino groups can be reversibly blocked. 2. As the reaction proceeds, the enzyme activity decreases until, at about 50% citraconylation of amino groups, the enzyme is completely inhibited. At this stage, little or no dissociation of the enzyme tetramer is observed and 75% of the activity is recoverable on unblocking the amino groups. 3. At 80% blocking, the enzyme is completely dissociated but little enzymic activity is recoverable after unblocking. Inability to recover activity after citraconylation and unblocking correlates with the onset of dissociation of the citraconyl-aldolase seen on ultracentrifugation. 4. The only irreversible modification of the enzyme primary structure detectable after the citraconylation and unblocking reactions is the partial loss of thiol groups. It is probable that this is responsible for the inability to reform active enzyme from the citraconylated subunit. 5. Other reversible side reactions of maleic anhydride and citraconic anhydride that may occur with proteins are discussed.     

Circular dichroism of chemically modified human plasma alpha1-antitrypsin. Interaction with porcine elastase.

Chemical modifications of human plasma alpha1-antitrypsin with reagents which modify lysyl residues (citraconic anhydride, acetic anhydride, formaldehyde and 2,4,6-trinitrobenzenesulfonic acid) and arginyl residued (1,2-cyclohexanedione) were examined with regard to their effect upon the elastase inhibitory capacity of the glycoprotein. 2,4,6-Trinitrobenzenesulfonic acid was employed to quantitate the remaining free amino groups (epsilon-NH2 groups of lysine) and the extent of modifications. Amino acid analysis was utilized in the same capacity for the guanidino groups of arginyl residues. The elastase inhibitory capacity of alpha1-antitrypsin was destroyed following trinitrophenylation, citraconylation and acetylation. Circular dichroism of the native and modified derivatives revealed major changes in conformation following trinitrophenylation and citraconylation while CD profiles of acetylated and reductively methylated derivatives differed from that of the native profile considerably less. Reductively methylated alpha1-antitrypsin retained its elastatse inhibitory capacity. The reaction of 1,2-cyclohexanedione with alpha1-antitrypsin did not effect in a loss in inhibitory capacity. Gel filtration studies of native and modified alpha1-antitrypsin on Sephadex G-100 demonstrated an increased molecular weight presumably through molecular aggregation, in the citraconylated and trinitrophenylated derivatives, but not in the cases of the other derivatives. Based upon these studies and previous investigations of our laboratory, it was concluded that (1) alpha1-antitrypsin is a lysyl inhibitor type (i.e., the reactive site is a Lys-X bond), (2) its interaction with elastase follows a pattern similar to trypsin and chymotrypsin, and (3) the positively charged epsilon-NH2 group of lysine is essential for the maintenance of elastase inhibitory capacity.     

Folding-unfolding and aggregation-dissociation of bovine alpha-crystallin subunits; evidence for unfolding intermediates of the alpha A subunits

The aggregation and dissociation behavior of bovine alpha-crystallin as well as the folding and unfolding of its subunits were investigated by equilibrium studies using tryptophan fluorescence measurements and two isoelectric focusing techniques, viz. isoelectric focusing across a urea gradient and isoelectric focusing in two dimensions with different concentrations of urea. It was found that the alpha B chains lose their ability to aggregate and start unfolding at a lower concentration of urea than the alpha A chains. Equilibrium intermediates were found upon unfolding or refolding of alpha A subunits, which can be explained by a two-domain organization of these molecules.     

Identification by 1H NMR spectroscopy of flexible C-terminal extensions in bovine lens alpha-crystallin.

Two-dimensional 1H NMR spectroscopy of bovine eye lens alpha-crystallin and its isolated alpha A and alpha B subunits reveals that these aggregates have short and very flexible C-terminal extensions of eight (alpha A) and ten (alpha B) amino acids which adopt little preferred conformation in solution. Total alpha-crystallin forms a tighter aggregate than the isolated alpha A and alpha B subunit aggregates. Our results are consistent with a micelle model for alpha-crystallin quaternary structure. The presence of terminal extensions is a general feature of those crystallins, alpha and beta, which form aggregates.     

Reaction of ovine interstitial cell stimulating hormone with citraeonic and maleic anhydrides

The free amino groups of ovine interstitial cell stimulating hormone and its subunits are modified with citraconic and maleic anhydrides. Three lysine residues in the native hormone are not available for reaction. Introduction of negatively charged groups does not cause dissociation of the hormone into its subunits. The completely modified interstitial cell stimulating hormone-β combines with the native α subunit to give a recombinant that has biological activity, while the modified interstitial cell stimulating hormone-α is unable to form an active product with native interstitial cell stimulating hormone-β. The results suggest that the ?-NH₂ groups of the α subunit play an important role in determining biological activity.     

The reaction of alpha-crystallin with the cross-linker 3,3'-dithiobis(sulfosuccinimidyl propionate) demonstrates close proximity of the C termini of alphaA and alphaB in the native assembly

The chaperone-like activity of human lens alpha-crystallin in inhibiting the aggregation of denatured proteins suggests a role for alpha-crystallin in cataract prevention. Although a variety of techniques have generated structural information relevant to its chaperone-like activity, the size and heterogeneity of alpha-crystallin have prevented determination of its crystal structure. Even though synthetic cross-linkers have provided considerable information about protein structures, they have not previously been used to study the proximity and orientation of subunits within human alpha-crystallin. Cross-linkers provide structural insight into proteins by binding the side chains of amino acids within close proximity. To identify the cross-linked residues, the modified protein is digested and the resulting peptides are analyzed by mass spectrometry. Analysis of products from the reaction of alpha-crystallin with 3,3'dithiobis(sulfosuccinimidyl propionate), DTSSP, identified several modifications to both alphaA and alphaB. The most structurally informative of these modifications was a cross-link between lysine 166 of alphaA and lysine 175 of alphaB. This cross-link provides experimental evidence supporting theoretical structural models that place the C termini of alphaA and alphaB within close proximity in the native aggregate.     

Protein-deficient ribosomal particles from yeast 60S subunits obtained by modification with dimethylmaleic anhydride and by treatment with NH4Cl

The protein-deficient particles prepared from yeast 60S subunits by modification of lysine residues with the reversible reagent dimethylmaleic anhydride are compared with those obtained by treatment with NH4Cl. The two procedures cause selective dissociation of certain proteins. With a few exceptions, the dissociation pattern is similar in both cases. When using dimethylmaleic anhydride, a variation in the protein composition of the ribosomal cores is obtained by modification of the ribosomal subunits in the presence of any of the following ligands: elongation factor-2, ricin A, verrucarine A and puromycine.     

Stepwise modification of lysine residues of glucose oxidase with citraconic anhydride

Structural properties of modified forms of glucose oxidase made by stepwise specific modification have been investigated. By a single step modification, one of the modified forms resulted in the conversion of native structure of glucose oxidase to molten globule like form [S. Hosseinkhani, B. Ranjbar, H. Naderi-Manesh, M. Nemat-Gorgani, FEBS Lett. 561 (2004) 213–216]. Chemical modification of lysine residues in glucose oxidase was carried out using different concentration of citraconic anhydride. Modification brought about changes in the tertiary structure with some degree of alteration in secondary structure. FTIR, far and near-UV CD spectropolarimetry, intrinsic and extrinsic fluorescence spectroscopy showed structural changes of glucose oxidase in a concentration dependent manner. This was supported by comparative study of secondary and tertiary structure.     

Specific dissociation of alpha B subunits from alpha-crystallin

Exposure of bovine alpha-crystallin to 0.1 M glycine at pH 7 decreases the average molar mass of the protein from 700 to 420 kDa. When the pH is lowered to 2.5, in the same buffer, the alpha B chains specifically dissociate from the aggregates, leaving a particle of 290 kDa containing only alpha A chains. The decrease in the molar mass corresponds to the mass of the alpha B chains in the original aggregate. The pH-dependent dissociation is fully reversible. Similar changes were observed with rat and kangaroo alpha-crystallins but the dogfish protein was not affected. Sedimentation velocity analyses and fluorescence spectroscopy yielded a pK, for the dissociation, of 3.7 for alpha-crystallin and 4.0 for a homopolymer constructed from purified alpha B2 polypeptides. An alpha A2 homopolymer was virtually unaffected by the lowering of pH. The products from the dissociation were isolated and their properties studied by sedimentation analysis and acrylamide quenching of tryptophan fluorescence. The alpha B chains were found to be completely denatured, whereas the structure of the alpha A chains, in the 290 kDa, particle, were only slightly altered. Comparisons of the sequences of the various proteins examined suggested that decreased ionization of aspartic acid 127 in the alpha B chain was responsible for the specific dissociation of this polypeptide.     

Impairment by covalent modification of the ability of Phaseolus vulgaris phytohemagglutinin to stimulate cultured human lymphocytes: a comparison of different forms of covalent modification

Phytohemagglutinin (PHA) isolated from Phaseolus vulgaris has been modified by treatment with various chemical reagents and the modified proteins have been tested for their ability to stimulate peripheral lymphocytes from two healthy human donors, in vitro. Reaction of PHA with citraconic anhydride, S-methyl isothiourea, or 2-hydroxy-5-nitrobenzyl bromide produced derivatives which retained the ability to stimulate lymphocytes, at low concentrations. Acylation of the lectin with acetic anhydride or masking of the carboxyl side chains by reaction with glycinamide-carbodiimide impaired stimulation. When PHA was treated with N-bromosuccinimide or with tetranitromethane, the derivatives were ineffective as lymphocyte stimulants. Chemical modifications affected, in some cases, the quaternary structure of the lectin. Glycinamide-, homoarginine-, and nitro-PHA were tetramers whereas acetyl-, citraconyl-, and N-bromosuccinimide-treated lectin were dimers. Antinative lectin antiserum cross-reacted with all the modified proteins, except in the case of the N-bromosuccinimide derivative. The results show that, in the human lymphocyte transformation assay, the mitogenic property of PHA may depend on intact aspartic, glutamic, and tyrosine residues whereas lysine residues do not appear to be essential.     

Effects of amino group modification in discoidal apolipoprotein A-I-egg phosphatidylcholine-cholesterol complexes on their reactions with lecithin:cholesterol acyltransferase

Discoidal complexes of human apolipoprotein A-I-egg phosphatidylcholine-cholesterol were prepared by the sodium cholate dialysis procedure and were reacted to varying extents with the amino group reagents citraconic anhydride, diketene, and formaldehyde in the presence of sodium borohydride. Modification of positive lysine residues with negative or neutral groups (citraconic anhydride and diketene, respectively) resulted, for extensively reacted complexes (90%), in structural alterations and in a marked decrease in reactivity with purified human lecithin:cholesterol acyltransferase. The structural and kinetic effects were partially reversible by removal of the modifying groups or by increased ionic strength. Similar extents of modification (84%) with retention of positive charge and introduction of two methyl groups (reductive methylation) had no effect on the structure or the reactivity of the complexes. These results, together with kinetic data at variable complex concentrations or at variable temperatures, indicate that specific lysine residues of apolipoprotein A-I are not involved in the lecithin:cholesterol acyltransferase activation process; instead, charge interactions and structural changes are responsible for the observed decrease in activating capacity. In terms of kinetic parameters, intrinsic K*m values and probably enzyme-substrate particle dissociation constants are affected, but the activation energies remain the same upon chemical modification.     

Packing-induced conformational and functional changes in the subunits of alpha -crystallin

The heteroaggregate alpha-crystallin and homoaggregates of its subunits, alphaA- and alphaB-crystallins, function like molecular chaperones and prevent the aggregation of several proteins. Although modulation of the chaperone-like activity of alpha-crystallin by both temperature and chaotropic agents has been demonstrated in vitro, the mechanism(s) of its regulation in vivo have not been elucidated. The subunits of alpha-crystallin exchange freely, resulting in its dynamic and variable quaternary structure. Mixed aggregates of the alpha-crystallins and other mammalian small heat shock proteins (sHSPs) have also been observed in vivo. We have investigated the time-dependent structural and functional changes during the course of heteroaggregate formation by the exchange of subunits between homoaggregates of alphaA- and alphaB-crystallins. Native isoelectric focusing was used to follow the time course of subunit exchange. Circular dichroism revealed large tertiary structural alterations in the subunits upon subunit exchange and packing into heteroaggregates, indicating specific homologous and heterologous interactions between the subunits. Subunit exchange also resulted in quaternary structural changes as demonstrated by gel filtration chromatography. Interestingly, we found time-dependent changes in chaperone-like activity against the dithiothreitol-induced aggregation of insulin, which correlated with subunit exchange and the resulting tertiary and quaternary structural changes. Heteroaggregates of varying subunit composition, as observed during eye lens epithelial cell differentiation, generated by subunit exchange displayed differential chaperone-like activity. It was possible to alter chaperone-like activity of preexisting oligomeric sHSPs by alteration of subunit composition by subunit exchange. Our results demonstrate that subunit exchange and the resulting structural and functional changes observed could constitute a mechanism of regulation of chaperone-like activity of alpha-crystallin (and possibly other mammalian sHSPs) in vivo.     

The location of sulphydryl groups in alpha-crystallin

The microenvironments of the sulphydryl groups in the multimeric protein, alpha-crystallin, were studied by examining: the rate of the reaction of the groups with DTNB; the effect of increasing urea concentrations on their accessibilities; and the quenching of a fluorescent probe. In foetal bovine alpha-crystallin (1 SH/alpha A subunit) both kinetic and quenching studies indicated that over 90% of the sulphydryl groups fell into a single buried class; the remainder was exposed. In the human protein (2 SH/alpha A subunit), half of the groups were buried and the other half exposed. Accessible sulphydryl groups increased gradually as the urea concentration was increased, with complete exposure at about 4.0 M. Sedimentation velocity analyses revealed that no significant dissociation of the aggregates into subunits occurred below 3.5 M urea, at which point over 80% of the sulphydryl groups were exposed. An age-dependent increase (3-35%) was found in the proportion of exposed sulphydryl groups in bovine alpha-crystallin and a decrease in the urea concentration required to expose the remainder. It was concluded that the single cysteine is buried in the newly synthesized protein, but becomes solvent-exposed as a result of age-related conformational changes. Our observations are consistent with a quaternary structure in which all alpha A subunits occupy equivalent sites.