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Domain Characterization of the Carboxyl-Terminal Fragment 206-316 of Thermolysin: pH and Ionic Strength Dependence of Conformation
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Limited proteolysis of thermolysin by subtilisin: isolation and characterization of a partially active enzyme derivative.
No full textIncubation of the neutral metalloendopeptidase thermolysin at pH 9-10 in the presence of 10 mM CaCl2 for 2 days at room temperature with subtilisin at a 50:1 molar ratio leads to a derivative possessing lower (approximately 3%) but intrinsic catalytic activity. This derivative, called thermolysin S, was isolated by gel filtration in approximately 80% yield and then separated from some residual intact thermolysin by an affinity chromatographic step on Sepharose-Gly-D-Phe. It was found that thermolysin S results from a tight association of two polypeptide fragments of apparent Mr of 24000 and 10000. Dissociation of the complex was achieved under strong denaturing conditions, such as gel filtration on a column equilibrated and eluted with 5 M guanidine hydrochloride. The positions of the clip sites were defined by amino acid analysis, end-group determination, and amino acid sequencing of the isolated fragments and shown to lie between Thr-4 and Ser-5, between Thr-224 and Gln-225, and also between Gln-225 and Asp-226. Thermolysin S, which is therefore a stable complex of fragments 5-224(225) and 225(226)-316, shows a shift in optimum pH of about 1 unit toward the acid range with respect to intact thermolysin and a Km essentially unchanged, with furylacryloyl-Gly-Leu-NH2 as substrate. Inhibitors of thermolysin such as ethoxyformic anhydride and Zn2+ ions inactivate also the nicked enzyme
Domain characteristics of the cyanogen bromide fragment 121-316 of thermolysin.
No full textThe molecule of thermolysin was shown by X-ray crystallography to be composed of two structural domains of equal size comprising residues 1-157 and 158-316. In order to explore the possibility that these domains correspond to globular fragments able to refold autonomously, we have investigated the conformational and stability properties of fragment 121-316, which was obtained by limited chemical cleavage of thermolysin with cyanogen bromide. As judged by far-ultraviolet circular dichroism measurements, in aqueous solution under neutral conditions the fragment maintains a relative amount of helical structure which is comparable to that exhibited by the corresponding region in native thermolysin. The secondary structure attained by the fragment appears remarkably stable to the denaturing action of heat. By measuring the temperature dependence of the dichroic signal at 220 nm a Tm near 74 degrees was obtained. Immunodiffusion analyses indicated that the fragment recognizes and precipitates antibodies raised in rabbits using native thermolysin as immunogen. The overall conformational and immunochemical data indicate that fragment 121-316 of thermolysin is able to refold into a stable structure of native-like characteristics independently of the rest of the molecule. The results of this study complement those previously reported for fragment 206-316 (Vita, C., Fontana, A., Seeman, J.R. & Chaiken, I.M. (1979) Biochemistry 18
Folding of thermolysin fragments. Identification of the minimum size of a carboxyl-terminal fragment that can fold into a stable native-like structure.
No full textThe COOH-terminal cyanogen bromide fragment 206-316 of thermolysin has been shown to possess protein domain characteristics that are able to refold into a stable native-like structure (Fontana et al., 1982). We now report the results of limited proteolysis of this fragment with the aim of identifying the minimum size of a COOH-terminal fragment of thermolysin that is able to fold by itself. Proteolysis with subtilisin, chymotrypsin, thermolysin and trypsin allowed us to isolate to homogeneity eight different subfragments, which can be grouped in two sets of peptides, i.e. (218-222)-316 and (252-255)-316. These subfragments are able to acquire a stable conformation of native-like characteristics, as judged by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunochemical properties using rabbit anti-thermolysin antibodies. In addition, even the smallest fragment isolated (sequence 255-316) shows co-operative and reversible unfolding transitions mediated by heat (tm 65 degrees C) and guanidine hydrochloride (midpoint transition at 2.5 M denaturant), as often observed with globular proteins. From the kinetics of the proteolytic digestion and analysis of the isolated subfragments, it is concluded that proteases lead to a stepwise degradation of fragment 206-316 from its NH2-terminal region, leading to the highly helical fragment (252-255)-316, quite resistant to further proteolytic digestion. The results of this study provide evidence that it is possible to isolate stable supersecondary structures of globular proteins and correlate well with predictions of subdomains of the COOH-terminal structural domain of thermolysin
Isolation of large peptides derived by cyanogen bromide cleavage of thermolysin using fast protein liquid chromatography (FPLC).
No full textThe recently introduced fast protein liquid chromatography (FPLC) system of Pharmacia (Uppsala, Sweden) was employed to isolate rather large peptides derived from thermolysin by selective chemical fragmentation at methionine in positions 120 and 205 of the polypeptide chain of 316 amino acid residues. Thermolysin was cleaved under conditions of limited fragmentation in order to produce, besides fragments 1-120, 121-205 and 206-316, the overlapping fragments 1-205 and 121-316. These polypeptides were separated employing prepacked Mono Q or Mono S columns (quaternary ammonium and sulfonic acid support, respectively). The columns were equilibrated with acetate-7 M urea buffer, pH 5.0 or 6.0, and eluted with a gradient of sodium chloride or acetate. Separations were achieved in 10-20 min and were carried out also at a semi-preparative level (1-3 mg per run). All five protein fragments were isolated in homogeneous form, as judged by amino acid analysis and electrophoresis. Considering that protein fragmentation with cyanogen bromide is the most commonly used procedure to achieve selective chemical fragmentation of a polypeptide chain, these results indicate that FPLC with ionic exchangers can be usefully employed to isolate rather large protein fragments especially suitable for automatic sequence analysis with the sequenator
Independent folding of the carboxyl-terminal fragment 228-316 of thermolysin.
No full textThe COOH-terminal fragment 206-316 of thermolysin was shown previously to maintain a stable folded structure in aqueous solution comparable to that of the corresponding region in native thermolysin and thus to possess protein domain characteristics [Fontana, A., Vita, C., & Chaiken, I. M. (1983) Biopolymers 22, 69-78]. In order to study the effect of polypeptide chain length on folding and stability of an isolated domain, the 111 amino acid residue fragment was shortened on the NH2-terminal side by removal of a 22-residue segment. Treatment of fragment 206-316 with hydroxylamine under alkaline conditions permitted selective cleavage of the Asn227-Gly228 peptide bond, and from the reaction mixture fragment 228-316 was isolated in homogeneous form. This fragment appeared to attain in aqueous solution the folding properties of the corresponding segment in the intact protein, as indicated by quantitative analysis of secondary structure from far-ultraviolet circular dichroism spectra and immunological properties. Thus, double-immunodiffusion analyses showed that fragment 228-316 is able to recognize and precipitate anti-thermolysin antibodies raised in rabbits with native thermolysin as immunogen. The fragment displayed fully reversible and cooperative conformational transitions mediated by pH, heat, and guanidine hydrochloride (Gdn.HCl), as expected for a globular protein species. Thermal denaturation of the fragment in aqueous solution at pH 7.8 showed a Tm of 66 degrees C and the Gdn.HCl-mediated unfolding a midpoint transition at 2.2 M denaturant concentration
Thermolysin and Bacillus subtilis neutral protease. Conformation and stability of two homologous neutral metalloendopeptidases.
No full textA comparative study on thermolysin from Bacillus thermoproteolyticus and neutral protease from Bacillus subtilis involving (far- and near-ultraviolet circular dichroism (CD) and immunological techniques is reported. These enzymes are homologous metalloendopeptidases, having similar size, kinetic behaviour, substrate specificity and susceptibility to inhibitors. The far-ultraviolet CD spectrum of each protein shows a minimum at 208 and a shoulder near 220 nm; differences in the extent of ellipticity, however, have been observed. Estimates of secondary structure obtained by quantitation of the far-ultraviolet CD spectra indicated a higher helicity of neutral protease relative to thermolysin. In the presence of ethylenediaminetetraacetic acid, which removes calcium and the functional zinc ion from the metalloenzymes, neutral protease is immediately denatured, whereas thermolysin maintains a globular structure, although thermolabile. On the other hand, the zinc-specific chelating agent tetraethylenepentamine does not have measurable effects on the conformation and conformational stability of either protein. Marked higher stability to temperature and guanidine hydrochloride were observed for thermolysin as compared with neutral protease, as indicated by monitoring conformational transitions with CD measurements at 220 nm. Antisera prepared in rabbits using thermolysin as immunogen do not cross-react with neutral protease, indicating differences of surface structure between the two proteins. On the basis and limitations of the techniques employed, it is proposed that the two sequentially and functionally homologous metalloendopeptidases may have similar conformations at specific regions (active and binding sites, at least) of the polypeptide chain essential for biological function, while some variability in the structure of other regions may be tolerated
Fluorescence properties of neutral protease from Bacillus subtilis.
No full textThe fluorescence properties of neutral protease from B. subtilis have been investigated under a variety of conditions and the results compared with those previously reported for the homologous metalloendopeptidase thermolysin (Fontana et al., 1977). In the pH range 5-9 neutral protease displayed a quite unusual fluorescence emission spectrum with a maximum near 320 nm, when excitation was at 295 nm. At this wavelength the protein fluorescence is due to tryptophan residues only, which, considering their blue-shift emission, appear rather buried in the hydrophobic protein interior. Specific removal of the functional zinc ion from the enzyme with tetraethylenepentamine does not lead to alteration of the microenvironment around tryptophan residues. On the other hand, removal of both zinc and calcium with ethylenediaminetetraacetic acid brings these residues in full contact with the aqueous solvent medium. Fluorescence quenching measurements were also used to determine the exposure of tryptophan residues in the native enzyme as well as in the presence of chelating agents and protein denaturants. Melting profile experiments carried out by monitoring the fluorescence intensity at 320 nm indicated a cooperative transition at 60-70 degrees. Temperature effects were also determined under conditions perturbed with respect to pH, guanidine hydrochloride and chelating agents. The results reveal differences in the fluorescence properties of the tryptophanyl residues of B. subtilis neutral protease relative to those of thermolysin, which are interpretable considering the location of these residues in the sequences of the two homologous proteins
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