Native quaternary structure of bovine alpha-crystallin

Alpha-crystallin is the most important soluble protein in the eye lens. It is responsible for creating a high refractive index and is known to be a small heat-shock protein. We have used static and dynamic light scattering to study its quaternary structure as a function of isolation conditions, temperature, time, and concentration. We have used tryptophan fluorescence to study the temperature dependence of the tertiary structure and its reversibility. Gel filtration, analytical ultracentrifugation, polyacrylamide gel electrophoretic analysis, and absorption measurements were used to study the chaperone-like activity of alpha-crystallin in the presence of destabilized lysozyme. We have demonstrated that the molecular mass of the in vivo alpha-crystallin oligomer is about 700 kDa (alpha(native)) while the 550 kDa molecule (alpha(37 degrees C),diluted), which is often found in vitro, is a product of prolonged storage at 37 degrees C of low concentrated alpha-crystallin solutions. We have proven that the molecular mass of the alpha-crystallin oligomer is concentration dependent at 37 degrees C. We have found strong indications that, during chaperoning, the alpha-crystallin oligomer undergoes a drastic rearrangement of its peptides during the process of complex formation with destabilized lysozyme. We propose the hypothesis that all these processes are governed by the phenomenon of subunit exchange, which is well-known to be strongly temperature-dependent.     

Correlation between the chaperone-like activity and aggregate size of alpha-crystallin with increasing temperature

alpha-Crystallin, the major protein of the mammalian eye lens, is also found in the major tissues of the body, where one or the other of its two isoforms is characteristically expressed. Both isoform sequences are highly related to others of the small heat shock protein superfamily, leading to speculation about their functions in vivo outside of the lens. Tests of chaperone-like activity at 37 and 66 degrees C indicate that the protein can act to prevent the superaggregation of partially denatured proteins, but both alpha-crystallin aggregate size and shape are significantly altered with increasing temperature. Characterization of these changes indicates that secondary, tertiary, and quaternary structure are modified, with the latter effect especially striking above 50 degrees C. Furthermore, these changes appear to be irreversible when the temperature is returned to 25 or 37 degrees C. Functionally, the protein is effective in chaperone-like activity at all temperatures, but exhibits a somewhat increased capability after a cycle of heating and cooling. The results presented here indicate the heat-induced formation of high-molecular-weight aggregates of alpha-crystallin is a slow progressive process. The increased activity of these aggregates suggests that chaperone-like activity depends in part on the packing parameters of the aggregate and on conformation of the subunit within that aggregate.     

Proteolytic analysis of domain structure in the beta heavy chain of dynein from sea urchin sperm flagella.

The stability of different regions of the beta heavy chain of dynein has been investigated by examining the perturbing effects of methanol, temperature, salt, and nucleotide on the pattern of tryptic digestion. In standard low-salt medium, tryptic proteolysis cleaves the beta heavy chain into three principal polypeptides of 130, 215, and 110 kDa, with the 215-kDa central peptide containing the ATP binding site as well as the vanadate and iron photocleavage sites (Mocz, G., Tang, W.-J. Y., & Gibbons, I. R. (1988) J. Cell Biol. 106, 1607-1614). The 130-kDa peptide is the most stable, and its susceptibility to trypsin appears unaffected by methanol concentrations up to 25% or temperatures up to 45 degrees C, although a 5-kDa region at one end is lost in the presence of salt (greater than 20 mM NaCl). The 215-kDa tryptic peptide contains two regions of different stability: its 123-kDa portion adjoining the 130-kDa peptide is destabilized by mild heat (37 degrees C) or by 25% methanol and becomes digested away to leave the more stable region of 92 kDa that is located toward the 110-kDa peptide and retains the V1 photocleavage site and most of the ATP binding site. The 110-kDa peptide is the least stable and at 37 degrees C, or in the presence of low concentrations of methanol or salt, it rapidly digested to small peptides. The presence of ATP during digestion of the beta heavy chain retards the formation of the 130- and 215-kDa peptides and also protects the 215-kDa peptide from further digestion at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential temperature-dependent chaperone-like activity of alphaA- and alphaB-crystallin homoaggregates

alpha-Crystallin, a heteromultimeric protein made up of alphaA- and alphaB-crystallins, functions as a molecular chaperone in preventing the aggregation of proteins. We have shown earlier that structural perturbation of alpha-crystallin can enhance its chaperone-like activity severalfold. The two subunits of alpha-crystallin have extensive sequence homology and individually display chaperone-like activity. We have investigated the chaperone-like activity of alphaA- and alphaB-crystallin homoaggregates against thermal and nonthermal modes of aggregation. We find that, against a nonthermal mode of aggregation, alphaB-crystallin shows significant protective ability even at subphysiological temperatures, at which alphaA-crystallin or heteromultimeric alpha-crystallin exhibit very little chaperone-like activity. Interestingly, differences in the protective ability of these homoaggregates against the thermal aggregation of beta(L)-crystallin is negligible. To investigate this differential behavior, we have monitored the temperature-dependent structural changes in both the proteins using fluorescence and circular dichroism spectroscopy. Intrinsic tryptophan fluorescence quench-ing by acrylamide shows that the tryptophans in alphaB-crystallin are more accessible than the lone tryptophan in alphaA-crystallin even at 25 degrees C. Protein-bound 8-anilinonaphthalene-1-sulfonate fluorescence demonstrates the higher solvent accessibility of hydrophobic surfaces on alphaB-crystallin. Circular dichroism studies show some tertiary structural changes in alphaA-crystallin above 50 degrees C. alphaB-crystallin, on the other hand, shows significant alteration of tertiary structure by 45 degrees C. Our study demonstrates that despite a high degree of sequence homology and their generally accepted structural similarity, alphaB-crystallin is much more sensitive to temperature-dependent structural perturbation than alphaA- or alpha-crystallin and shows differences in its chaperone-like properties. These differences appear to be relevant to temperature-dependent enhancement of chaperone-like activity of alpha-crystallin and indicate different roles for the two proteins both in alpha-crystallin heteroaggregate and as separate proteins under stress conditions.     

Identification of a region in alcohol dehydrogenase that binds to alpha-crystallin during chaperone action

alpha-Crystallin, the major eye lens protein and a member of the small heat-shock protein family, has been shown to protect the aggregation of several proteins and enzymes under denaturing conditions. The region(s) in the denaturing proteins that interact with alpha-crystallin during chaperone action has not been identified. Determination of these sites would explain the wide chaperoning action (promiscuity) of alpha-crystallin. In the present study, using two different methods, we have identified a sequence in yeast alcohol dehydrogenase (ADH) that binds to alpha-crystallin during chaperone-like action. The first method involved the incubation of alpha-crystallin with ADH peptides at 48 degrees C for 1 h followed by separation and analysis of bound peptides. In the second method, alpha-crystallin was first derivatized with a photoactive trifunctional cross-linker, sulfosuccinimidyl-2[6-(biotinamido)-2-(p-azidobenzamido)-hexanoamido]ethyl-1,3di-thiopropionate (sulfo-SBED), and then complexed with ADH at 48 degrees C for 1 h in the dark. The complex was photolyzed and digested with protease, and the biotinylated peptide fragments were isolated using an avidin column and then analyzed. The amino acid sequencing and mass spectral analysis revealed the sequence YSGVCHTDLHAWHGDWPLPVK (yeast ADH(40-60)) as the alpha-crystallin binding site in ADH. The interaction was further confirmed by demonstrating complex formation between alpha-crystallin and a synthetic peptide representing the binding site of ADH.     

Amyloid protofilaments from the calcium-binding protein equine lysozyme: formation of ring and linear structures depends on pH and metal ion concentration

The calcium-binding equine lysozyme has been found to undergo conversion into amyloid fibrils during incubation in solution at acidic pH. At pH 4.5 and 57 degrees C, where equine lysozyme forms a partially unfolded molten globule state, the protein forms protofilaments with a width of ca. 2 nm. In the absence of Ca(2+) the protofilaments are present as annular structures with a diameter of 40-50 nm. In the presence of 10 mM CaCl(2) the protofilaments of equine lysozyme are straight or curved; they can assemble into thicker threads, but they do not appear to undergo circularisation. At pH 2.0, where the protein is more destabilised compared to pH 4.5, fibril formation occurs at 37 degrees C and 57 degrees C. At pH 2.0, both ring-shaped and linear protofilaments are formed, in which periodic repeats of ca 35 nm can be distinguished clearly. The rings constitute about 10% of all fibrillar species under these conditions and they are characterised by a larger diameter of 70-80 nm. All the structures bind Congo red and thioflavine T in a manner similar to fibrils associated with a variety of amyloid diseases. At pH 2.0, fibril formation is accompanied by some acidic hydrolysis, producing specific fragmentation of the protein, leading to the accumulation of two peptides in particular, consisting of residues 1-80 and 54-125. At the initial stages of incubation, however, full-length equine lysozyme represents the dominant species within the fibrils. We propose that the ring-shaped structures observed here, and in the case of disease-associated proteins such as alpha-synuclein, could be a second generic type of amyloid structure in addition to the more common linear fibrils.     

Cause of abnormal acidity of lysozyme ionogenic active center groups at cell wall lysis

pH-Dependence of the kinetic parameters of Micrococcus lysodeicticus cell lysis under the action of the protein hen egg lysozyme at the pH 6.9-10.0 at 25 and 37 degrees C has been investigated. The pKb effective values for the lysozyme catalytic activity controlling group have been calculated. The DeltaHion value indicates that this group is the carboxyl one though its pK (9.15 at 25 degrees C) is found far for the limit of the carboxyl groups pK values. The cause of this abnormal pK values is supposed to be the strong negative charge of the bacterial cell wall. As a result the enzyme that catalyzes the hydrolysis ofcopolymer N-acetylglucosamine--N-acetylmuramic acid acts in the high acidity microenvironment.     

The interaction between apolipoprotein serum amyloid A and high-density lipoprotein

Serum amyloid A (SAA) is a small apolipoprotein that binds to high-density lipoproteins (HDLs) via its N-terminus. The murine isoform SAA2.2 forms a hexamer in solution and the N-terminus is shielded from the solvent. Therefore, it is unclear how the SAA2.2 hexamer might bind HDL. In this study, the binding of SAA2.2 to murine HDL was investigated by glutaraldehyde cross-linking and polyacrylamide gel electrophoresis. The hexamer did not bind HDL significantly at 20 degrees C. However, at temperatures between 25-30 degrees C, SAA2.2 became destabilized and its monomeric form bound to HDL. SAA2.2 binding did not significantly replace Apo A-I in HDL particles. At 37-45 degrees C SAA2.2 binds less to HDL, suggesting that its binding is weak and sensitive to physiological and pathological temperatures, and thereby, potentially modulated, in vivo, by other factors.     

Thermo- and photoinduced aggregation of alpha-crystallin

Some properties of bovine alpha-crystallin, in particular its thermo- and photoaggregation, were studied by fluorescent spectroscopy of tryptophan residues and the probe 8-anilino-1-naphthalenesulfonate and light scattering. The effective diameter of a globule of native alpha-crystallin was 90 A, as estimated from the data on the polarization and lifetime of 8-anilino-1-naphthalenesulfonate using the Levshin-Perren equation, and increases at an aggregation of no less than 140 A. The increase in the intensity of tryptophan fluorescence of alpha-crystallin during its thermo- and photodenaturation with the formation of aggregates is due to local conformational changes in the surroundings of tryptophan residues and light scattering. Tryptophan residues are buried in the interior of the aggregates. The thermoaggregation of the protein occurs not only at high temperatures. By approximating the experimental time dependence of slow spontaneous aggregation to the range of large times, the time of denaturation aggregation t(e) was found. For alpha-crystallin (at a concentration of 0.8 mg/ml in phosphate buffer at pH 8.4), t(e) at 70 degrees C is 100 h. This approach can be used in finding t(e) for any protein during its thermal treatment or long-term storage.     

Subunit exchange demonstrates a differential chaperone activity of calf alpha-crystallin toward beta LOW- and individual gamma-crystallins

The chaperone activity of native alpha-crystallins toward beta(LOW)- and various gamma-crystallins at the onset of their denaturation, 60 and 66 degrees C, respectively, was studied at high and low crystallin concentrations using small angle x-ray scattering (SAXS) and fluorescence energy transfer (FRET). The crystallins were from calf lenses except for one recombinant human gamma S. SAXS data demonstrated an irreversible doubling in molecular weight and a corresponding increase in size of alpha-crystallins at temperatures above 60 degrees C. Further increase is observed at 66 degrees C. More subtle conformational changes accompanied the increase in size as shown by changes in environments around tryptophan and cysteine residues. These alpha-crystallin temperature-induced modifications were found necessary to allow for the association with beta(LOW)- and gamma-crystallins to occur. FRET experiments using IAEDANS (iodoacetylaminoethylaminonaphthalene sulfonic acid)- and IAF (iodoacetamidofluorescein)-labeled subunits showed that the heat-modified alpha-crystallins retained their ability to exchange subunits and that, at 37 degrees C, the rate of exchange was increased depending upon the temperature of incubation, 60 or 66 degrees C. Association with beta(LOW)- (60 degrees C) or various gamma-crystallins (66 degrees C) resulted at 37 degrees C in decreased subunit exchange in proportion to bound ligands. Therefore, beta(LOW)- and gamma-crystallins were compared for their capacity to associate with alpha-crystallins and inhibit subunit exchange. Quite unexpectedly for a highly conserved protein family, differences were observed between the individual gamma-crystallin family members. The strongest effect was observed for gamma S, followed by h gamma Srec, gamma E, gamma A-F, gamma D, gamma B. Moreover, fluorescence properties of alpha-crystallins in the presence of bound beta(LOW)-and gamma-crystallins indicated that the formation of beta(LOW)/alpha- or gamma/alpha-crystallin complexes involved various binding sites. The changes in subunit exchange associated with the chaperone properties of alpha-crystallins toward the other lens crystallins demonstrate the dynamic character of the heat-activated alpha-crystallin structure.     

The influence of isolation conditions on the molecular weight of bovine alpha-crystallin

The molecular weight of bovine alpha-crystallin, isolated at 37 degrees C, was studied and found to be about 800,000. This contrasts with the results of Thomson and Augusteyn (Thomson, J. A., and Augusteyn, R. C. (1983) Exp. Eye Res. 37, 367-377) who isolated a species of about half this molecular weight. We show here that this form of alpha-crystallin can only be isolated under unphysiological conditions with regard to buffer pH and ionic strength.     

A kinetic study of the competition between renaturation and aggregation during the refolding of denatured-reduced egg white lysozyme

The recovery of proteins following denaturation is optimal at low protein concentrations. The decrease in yield at high concentrations has been explained by the kinetic competition of folding and "wrong aggregation". In the present study, the renaturation-reoxidation of hen and turkey egg white lysozyme was used as a model system to analyze the committed step in aggregate formation. The yield of renatured protein for both enzymes decreased with increasing concentration in the folding process. In addition, the yield decreased with increasing concentrations of the enzyme in the denatured state (i.e., prior to its dilution in the renaturation buffer). The kinetics of renaturation of turkey lysozyme were shown to be very similar to those of hen lysozyme, with a half-time of about 4.5 min at 20 degrees C. The rate of formation of molecular species that lead to formation of aggregates (and therefore fail to renature) was shown to be rapid. Most of the reaction occurred in less than 5 s after the transfer to renaturation buffer, and after 1 min, the reaction was essentially completed. Yet, by observing the effects of the delayed addition of denatured hen lysozyme to refolding turkey lysozyme, it was shown that folding intermediates become resistant to aggregation only much more slowly, with kinetics indistinguishable from those observed for the appearance of native molecules. The interactions leading to the formation of aggregates were nonspecific and do not involve disulfide bonds. These observations are discussed in terms of possible kinetic and structural aspects of the folding pathway.     

Studies of alpha- and betaL-crystallin complex formation in solution at 60 degrees C

Studies of molecular mechanisms of chaperone-like activity of alpha-crystallin became an active field of research over last years. However, fine interactions between alpha-crystallin and the damaged protein and their complex organization remain largely uncovered. Complexation between alpha- and betaL-crystallins was studied with thermal denaturation of betaL-crystallin at 60 degrees C using small-angle X-ray scattering (SAXS), light scattering, gel-permeation chromatography and electrophoresis. A mixed solution of alpha- and betaL-crystallins in concentrations about 10 mg/ml incubated at 60 degrees C was found to contain their soluble complexes with mean radius of gyration approximately 14 nm, mean molecular weight approximately 4000 kDA and maximal size approximately 40 nm. In pure betaL-crystallin solution, complexes were not observed at 60 degrees C. In SAXS studies, transitions in the alpha-crystallin quaternary structure at 60 degrees C were shown to occur and result in a double increase of the molecular weight. It suggests that during the temperature-induced denaturation of betaL-crystallin it binds with modified alpha-crystallin or, alternatively, alpha-betaL-crystallin complexation and alpha-crystallin modifications are concurrent. Estimates of the alpha-betaL-crystallin dimensions and relative contents of alpha- and betaL-crystallins in the complex suggest that several alpha-crystallin molecules are involved in complex formation.     

An aggregation-prone intermediate species is present in the unfolding pathway of the monomeric portal protein of bacteriophage P22: implications for portal assembly

The head of the P22 bacteriophage is interrupted by a unique dodecameric portal vertex that serves as a conduit for the entrance and exit of the DNA. Here, the in vitro unfolding/refolding processes of the portal protein of P22 were investigated at different temperatures (1, 25, and 37 degrees C) through the use of urea and high hydrostatic pressure (HHP) combined with spectroscopic techniques. We have characterized an intermediate species, IU, which forms at 25 degrees C during unfolding or refolding of the portal protein in 2-4 M urea. IU readily forms amorphous aggregates, rendering the folding process irreversible. On the other hand, at 1 degrees C, a two-state process is observed (DeltaGf = -2.2 kcal/mol). When subjected to HHP at 25 or 37 degrees C, the portal monomer undergoes partial denaturation, also forming an intermediate species, which we call IP. IP also tends to aggregate but, differently from IU, aggregates into a ring-like structure as seen by size-exclusion chromatography and electron microscopy. Again, at 1 degrees C the unfolding induced by HHP proved to be reversible, with DeltaGf = -2.4 kcal/mol and DeltaV = 72 mL/mol. Interestingly, at 25 degrees C, the binding of the hydrophobic probe bis-ANS to the native portal protein destabilizes it and completely blocks its aggregation under HHP. These data are relevant to the process by which the portal protein assembles into dodecamers in vivo, since species such as IP must prevail over IU in order to guarantee the proper ring formation.     

Temperature effect on inclusion body formation and stress response in the periplasm of Escherichia coli

We previously characterized a defective-folding mutant of maltose-binding protein of Escherichia coli, MalE31, which formed periplasmic inclusion bodies. Here, we show that MalE31 aggregation does not affect bacterial growth at 30 degrees C but is lethal at 37 degrees C. Surprisingly, under mild heat shock conditions at 42 degrees C, inclusion bodies are degraded and bacterial growth is restored. One physiological consequence for the cells overproducing MalE31 was to induce an extracytoplasmic stress response by increasing the expression of the heat shock protease DegP via the CpxA/CpxR two-component signalling pathway. Furthermore, we show that the Cpx response is required to rescue the cells from the toxicity mediated by MalE31. Finally, expression of highly destabilized MalE variants that do not aggregate in the periplasm also induces the Cpx pathway, indicating that inclusion body formation is not necessary to activate this specific extracytoplasmic stress regulatory system.     

Lens proteasome shows enhanced rates of degradation of hydroxyl radical modified alpha-crystallin

Proteasome, a high molecular weight protease complex (HMP, approximately 600 kDa) was isolated from bovine eye lens epithelium tissue. In contrast with prior reports, lens proteasome degraded the major lens protein alpha-crystallin and S-carboxymethylated bovine serum albumin at 37 degrees C, mostly to trichloroacetic acid precipitable polypeptides. The proteasome, thus isolated, was labile at 55 degrees C. As indicated by the ability of p-chloromercuribenzoate and N-ethylmaleimide to block activity, a thiol group is required for activity. Alpha-crystallin was oxidized by exposure to 60Co-irradiation under an atmosphere of N2O (1-50 kilorads). This dose delivered 0.1-5.7 mol of hydroxyl radicals per mol of crystallin. Irradiation resulted in increased heterogeneity, aggregation, and fragmentation of the crystallin preparation. The proteolytic susceptibility of alpha-crystallin to the lens HMP was enhanced by the irradiation in a dose-dependent manner up to 20 kilorads (.OH concentration up to 2.3 mol per mol of alpha-crystallin). When 50 kilorads (5.7 mol .OH per mol of alpha-crystallin) was used, there was extensive aggregation and no enhancement in proteolysis over the unirradiated sample. The data indicate that the lens HMP can degrade mildly photooxidized lens proteins, but proteins which are extensively damaged are not degraded and may accumulate. This may be related to cataract formation.     

Amyloid fibril formation and seeding by wild-type human lysozyme and its disease-related mutational variants

Wild-type human lysozyme and its two stable amyloidogenic variants have been found to form partially folded states at low pH. These states are characterized by extensive disruption of tertiary interactions and partial loss of secondary structure. Incubation of the proteins at pH 2.0 and 37 degrees C (Ile56Thr and Asp67His variants) or 57 degrees C (wild-type) results in the formation of large numbers of fibrils over several days of incubation. Smaller numbers of fibrils could be observed under other conditions, including neutral pH. These fibrils were analyzed by electron microscopy, Congo red birefringence, thioflavine-T binding, and X-ray fiber diffraction, which unequivocally show their amyloid character. These data demonstrate that amyloidogenicity is an intrinsic property of human lysozyme and does not require the presence of specific mutations in its primary structure. The amyloid fibril formation is greatly facilitated, however, by the introduction of "seeds" of preformed fibrils to the solutions of the variant proteins, suggesting that seeding effects could be important in the development of systemic amyloidosis. Fibril formation by wild-type human lysozyme is greatly accelerated by fibrils of the variant proteins and vice versa, showing that seeding is not specific to a given protein. The fact that wild-type lysozyme has not been found in ex vivo deposits from patients suffering from this disease is likely to be related to the much lower population of incompletely folded states for the wild-type protein compared to its amyloidogenic variants under physiological conditions. These results support the concept that the ability to form amyloid is a generic property of proteins, but one that is mitigated against in a normally functioning organism.     

Human immunodeficiency virus type 1 Gag assembly through assembly intermediates

Human immunodeficiency virus Gag protein self-assembles into spherical particles, and recent reports suggest the formation of assembly intermediates during the process. To understand the nature of such assembly intermediates along with the mechanism of Gag assembly, we employed expression in Escherichia coli and an in vitro assembly reaction. When E. coli expression was performed at 37 degrees C, Gag predominantly assembled to a high order of multimer, apparently equivalent to the virus-like particles obtained following Gag expression in eukaryotic cells, through the formation of low orders of multimer characterized with a discreet sedimentation value of 60 S. Electron microscopy confirmed the presence of spherical particles in the E. coli cells. In contrast, expression at 30 degrees C resulted in the production of only the 60 S form of Gag multimer, and crescent-shaped structures or small patches with double electron-dense layers were accumulated, but no complete particles. In vitro assembly reactions using purified Gag protein, when performed at 37 degrees C, also produced the high order of Gag multimers with some 60 S multimers, whereas the 30 degrees C reaction produced only the 60 S multimers. However, when the 60 S multimers were cross-linked so as not to allow conformational changes, in vitro assembly reactions at 37 degrees C did not produce any higher order of multimers. ATP depletion did not halt Gag assembly in the E. coli cells, and the addition of GroEL-GroES to in vitro reactions did not facilitate Gag assembly, indicating that conformational changes rather than protein refolding by chaperonins, induced at 37 degrees C, were solely responsible for the Gag assembly observed here. We suggest that Gag assembles to a capsid through the formation of the 60 S multimer, possibly a key intermediate of the assembly process, accompanied with conformational changes in Gag.     

Complex effects of molecular chaperones on the aggregation and refolding of fibroblast growth factor-1

Fibroblast growth factor one (FGF-1) exists in a molten globule (MG)-like state under physiological conditions (neutral pH, 37 degrees C). It has been proposed that this form of the protein may be involved in its atypical membrane transport properties. Macromolecular chaperones have been shown to bind to MG states of proteins as well as to be involved in protein membrane transport. We have therefore examined the effect of such proteins on the aggregation and refolding of FGF-1 to evaluate whether they might play a role in FGF-1 transport. The proposed chaperone alpha-crystallin was found to strongly inhibit the aggregation of the MG state of FGF-1. Curiously, two other proteins of similar size and charge (thyroglobulin and a monoclonal IgM immunoglobulin) with no previously reported chaperone properties were also found to have a related effect. In contrast, the chaperone GroEL/ES induced further aggregation of MG-like FGF-1 but had no effect on the native conformation. Both chaperones stimulated refolding to the native state (25 degrees C) but had no detectable effect when FGF-1 was refolded to the MG state (37 degrees C). This suggests that disordered intermediates are present in the folding pathways of the native and MG-like FGF conformations which differ from the MG-like state induced under physiological conditions. FGF-1 does, therefore, interact with molecular chaperones, although this may involve both the MG and the native states of the protein.     

Capsular polysaccharide surrounds smooth and rugose types of Salmonella enterica serovar Typhimurium DT104

The biofilms and rugose colony morphology of Salmonella enterica serovar Typhimurium strains are usually associated with at least two different exopolymeric substances (EPS), curli and cellulose. In this study, another EPS, a capsular polysaccharide (CP) synthesized constitutively in S. enterica serovar Typhimurium strain DT104 at 25 and 37 degrees C, has been recognized as a biofilm matrix component as well. Fluorophore-assisted carbohydrate electrophoresis (FACE) analysis indicated that the CP is comprised principally of glucose and mannose, with galactose as a minor constituent. The composition differs from that of known colanic acid-containing CP that is isolated from cells of Escherichia coli and other enteric bacteria grown at 37 degrees C. The reactivity of carbohydrate-specific lectins conjugated to fluorescein isothiocyanate or gold particles with cellular carbohydrates demonstrated the cell surface localization of CP. Further, lectin binding also correlated with the FACE analysis of CP. Immunoelectron microscopy, using specific antibodies against CP, confirmed that CP surrounds the cells. Confocal microscopy of antibody-labeled cells showed greater biofilm formation at 25 degrees C than at 37 degrees C. Since the CP was shown to be produced at both 37 degrees C and 25 degrees C, it does not appear to be significantly involved in attachment during the early formation of the biofilm matrix. Although the attachment of S. enterica serovar Typhimurium DT104 does not appear to be mediated by its CP, the capsule does contribute to the biofilm matrix and may have a role in other features of this organism, such as virulence, as has been shown previously for the capsules of other gram-negative and gram-positive bacteria.