The thermodynamics of association and unfolding of the 205–316 C-terminal fragment of thermolysin (original) (raw)
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Biochemistry - BIOCHEMISTRY-USA, 1996
Differential scanning calorimetry and size-exclusion chromatography have been used to characterize the dimerization and unfolding of the 205-316 C-terminal fragment of thermolysin at pH 7.5. We show that the folded fragment dimerizes at low temperature with a moderate affinity and undergoes thermal unfolding according to a N 2 a 2N a 2U model. This behavior has already been observed at acid pH, where a similar dissociation equilibrium has been found [Azuaga, A., Conejero-Lara, F., Rivas G., De Filippis, V., Fontana, A., & Mateo, P. L. (1995) Biochim. Biophys. Acta 1252, 95-102]. Nevertheless, at pH 7.5 the dimerization equilibrium slows down below about 30°C, with virtually no interconversion between the monomeric and the dimeric states of the fragment. We have studied the kinetics of interconversion between monomer and dimer by size-exclusion chromatography experiments and have shown that a very high energy barrier (83.8 kJ/mol at 26.5°C) exists between either state. A mathematical analysis of the DSC thermograms on the basis of the proposed model has allowed us to obtain the thermodynamic characterization of the dimerization and the unfolding processes of the fragment and confirms the kinetic parameters obtained in the chromatographic experiments. The thermodynamic functions for the unfolding of the fragment are compatible with some degree of disorder in the structures of both the monomer and the dimer. According to circular dichroism measurements, the dimerization of the fragment seems to be linked to some conformational change in the subunits, most probably due to a rearrangement of the existing secondary-structure elements. This fragment displays several features already observed in folding intermediates, such as the partial disorder of the polypeptidic chain, association processes, and kinetic barriers between different regions in the conformational space. Abstract published in AdVance ACS Abstracts, February 15, 1996.
Differential scanning calorimetry of thermolysin and its 255-316 and 205-316 C-terminal fragments
Reactive and Functional …, 1997
High-sensitivity differential scanning calorimetry has been applied to the study of the thermal denaturation of thermolysin from Bacillus thermoproteolyticus rokko and its 255-316 and 205316 fragments. Stability investigations into thermolysin have been extended from a previous calorimetric study at pH 7.5 [2] to different experimental conditions, which included 0.3-3.7 mg/ml of protein concentration, pH values within the range 3.0-9.0, and inhibitors such as phosphoramidon and l,lO-phenanthroline. The thermal transitions were always irreversible, kinetically ~ controlled and followed the two-state kinetic model. Autolysis of native thermolysin and/or the unfolded enzyme together with aggregation of the unfolded state in the presence of inhibitors seem to be the reasons for the irreversible debaturation. On the other hand, calorimetric studies into the concentration effects on the 255-316 and 205-316 tbermolysini fragments show the presence of dimers in solution undergoing equilibrium unfolding processes. The thermodynamic pammeters of unfolding for both fragments are consistent with a higher compact globular structure for the shorter dimeric for the larger one. Given their spontaneous folding capability, these fragments could well be considered as foldi in thermolysin.
FEBS Letters, 1994
Differential scanning calorimetry has been used to study the thermal unfolding of the 255-316 C-terminal fragment of thermolysin. The concentration effect on the calorimetric transitions of the fragment in 0.1 M NaCl and 20 mM phosphate buffer, pH 7.5, shows that it behaves as a highly stable dimer in solution, whithin the concentration range 0.194.55 mg/ml, undergoing a reversible two-state thermal unfolding process. The thermodynamic parameters of unfolding (dG = 60 f 6 kJ/(mol of dimer) at 20°C) are similar to those normally observed for small, compact, globular proteins. This and previous studies [1989, Eur. J. Biochem. 180, 513-5181 show that the 255-316 fragment folds into a stable, native-like globular structure.
Biochemistry, 1997
The solution structure of the C-terminal fragment 205-316 of thermolysin has been determined by 1 H-NMR methods. The fragment forms a dimer in which each subunit has two different regions: the largely disordered N-terminal segment 205-260 and the structurally well-defined segment 261-316. The structured part of each subunit is composed of three helices and is largely coincident with the corresponding region in the solution structure of the dimer formed by the shorter fragment 255-316, which in turn coincides with the crystallographic structure of intact thermolysin. As with the fragment 255-316, the subunit interface is highly hydrophobic and coincides topologically with the one between the segment 255-316 and the rest of the protein in the intact enzyme. A fourth helix (residues 235-246), present in the segment 205-316 of native thermolysin, is mostly disordered in the dimer formed by the fragment 205-316. The location of the fourth helix in the native structure of intact thermolysin does not allow the formation of the dimer interface observed in the solution structure of the fragment 255-316. Under the NMR conditions, dimer formation is energetically more favorable than the dissociated monomers. The latter, based on calorimetric data, was proposed to have partial structure in the region 205-254 as in native thermolysin. Thus, it appears that the assembly of the dimer would require an initial unfolding in the region 205-254 of the monomer.
Folding of thermolysin fragments. Hydrodynamic properties of isolated domains and subdomains
European Journal of Biochemistry, 1989
Sedimentation analysis in the analytical ultracentrifuge has been used to characterize the size and shape of thermolysin and a number of its fragments obtained by chemical or enzymatic cleavage of the protein. Four fragments (121 -316, 206-316, 225/226-316 and 255-316) originate from the C-terminal domain, and two (1 -155 and 1-205) from the N-terminal domain of the intact molecule. In aqueous solution at neutral pH the hydrodynamic properties of the C-terminal fragments, except 255 -316, are consistent with compact homogeneous monomers. Fragment 255-316 is a monomeric species below 0.08 mg/ml concentration and forms a dimer above this concentration. Dimerization does not lead to changes in fragment conformation, as determined by farultraviolet circular dichroic measurements, but to an increase of 5.6"C (to 68.2"C at 1.0 mg/ml) in the temperature for thermal unfolding and a corresponding increase of 4.6 kJ/mol in the free energy of unfolding. Fragments derived from the N-terminal domain show a strong tendency to form high-molecular-mass aggregates. Previous experiments utilizing circular dichroic measurements and antibody binding data suggested that the C-terminal fragments listed above are able to refold in aqueous solution at neutral pH into a stable conformation of nativelike characteristics J. Mot. B i d . 182,. Present data establish that all these C-terminal fragments are globular monomeric species in solution (at concentrations M 0.1 mg/ml) and thus represent 'isolated' domains (or subdomains) with intrinsic conformational stability typical of small globular proteins.
European Journal of Biochemistry, 1993
The propensity of the peptide fragments 233-248, 245-260, 258-276, 279-298 and 299-316 from the thermolysin C-terminal domain to form non-random structures has been examined by CD and two-dimensional NMR spectroscopy. The conformational properties of these fragments have been studied in aqueous solution and in the mixed solvent trifluoroethanol/H,O (3 : 7 by vol.). Small but detectable populations of helical structures (up to 10-20%) in aqueous solution have been found for the fragments 233-248,279-298 and 299-316. These populations are remarkably enhanced (50-70%) in the more hydrophobic mixed solvent, where the fragment 258-276 also forms a comparable helical population. These four fragments are helical in the native crystal structure and the spanning of the corresponding helices in the isolated peptides in solution matches very closely the ones in the native structure. In contrast, the fragment 245-260, an D-loop in the crystal, remains unstructured in both solvents. Medium-range NOE between protons in sidechains indicate the adoption of preferred sidechain conformations accompanying helix formation. Results are in agreement with the framework model of folding, in which native elements of secondary structure are formed first and folding follows from the collapse of these structural elements.
Domain characteristics of the cyanogen bromide fragment 121–316 of thermolysin
International Journal of Peptide and Protein Research, 2009
The 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 74d̀ was obtained. Immunodiffusion analyses indicated that the fragment recognizes and precipitates a...
International Journal of Peptide and Protein Research, 2009
The 21-residue fragment Tyr-Gly-Ser-Thr-Ser-Gln-Glu-Val-Ala-Ser-Val-Lys-Gln-Ala-Phe-Asp-Ala-Val-Gly-Val-Lys, corresponding to sequence 296–316 of thermolysin and thus encompassing the COOH-termi-nal helical segment 301–312 of the native protein, was synthesized by solid-phase methods and purified to homogeneity by reverse-phase high performance liquid chromatography. The peptide 296–316 was then cleaved with trypsin at Lys307 and Staphylococcus aureus V8 protease at Glu302, producing the additional fragments 296–307, 308–316, 296–302, and 303–316. All these peptides, when dissolved in aqueous solution at neutral pH, are essentially structureless, as determined by circular dichroism (CD) measurements in the far-ultraviolet region. On the other hand, fragment 296–316, as well as some of its proteolytic fragments, acquires significant helical conformation when dissolved in aqueous trifluoroethanol or ethanol. In general, the peptides mostly encompassing the helical segment 301–312 in the native thermolysin show helical conformation in aqueous alcohol. In particular, quantitative analysis of CD data indicated that fragment 296–316 attains in 90% aqueous trifluoroethanol the same percentage (∼58%) of helical secondary structure of the corresponding chain segment in native thermolysin. These results indicate that peptide 296–316 and its subfragments are unable to fold into a stable native-like structure in aqueous solution, in agreement with predicted location and stabilities of isolated subdomains of the COOH-terminal domain of thermolysin based on buried surface area calculations of the molecule
Micro-environment effect on the structure of synthetic substrates of thermolysin
Letters in Peptide Science, 1997
The conformations of thermolysin synthetic substrates in H20/D20 (9/1) and glycerol-d5 (5 M) are investigated using two-dimensional nuclear magnetic resonance spectroscopy and molecular modeling. The structures obtained from molecular modeling and NMR studies are compared. Comparisons of these structures with bound inhibitor in the active site of thermolysin are also discussed.