Expression and equilibrium denaturation of cardiac troponin I: stabilization of a folding intermediate during denaturation by urea.
Human cardiac troponin I has been expressed at high level in Escherichia coli as a fusion protein by using the expression vector Ptac114. The expressed protein forms primarily intracellular inclusion bodies that are solubilized in the presence of 8 M urea. The purified troponin I is recognized by anti-(human cardiac troponin I) monoclonal antibodies. Equilibrium denaturation of recombinant human troponin I and bovine troponin I is compared by monitoring changes in the protein's fluorescence and CD characteristics. At pH 7.5 the equilibrium denaturation of both proteins by urea occurs in two distinct steps involving at least three major conformational states: native, intermediate and fully denatured. The biphasic profile in the presence of urea is observed by both fluorescence and CD spectroscopy. In the intermediate state the native tertiary structure is largely disrupted and 40% of the secondary structure is conserved, as suggested by near-UV and far-UVCD respectively. Thermal denaturation of troponin I, as followed by fluorescence, shows a loss in the signal that is not reversible after heating to 90 degrees C. In the presence of a constant amount of urea (not greater than 0.5 M) the thermal denaturation becomes biphasic, suggesting the accumulation of an intermediate species that is stabilized by urea. The fluorescence of 1-anilino-8-naphthalenesulphonate produced on binding troponin I decreases in the presence of increasing concentrations of urea up to 3 M; at higher urea concentrations no further change in the remaining signal is observed. Kinetic studies show at least two phases of renaturation for troponin I previously denatured with 8 M urea, whereas only a single phase is detected for the renaturation process in the presence of 3 M urea. The results suggest the occurrence of a stable folding intermediate, the formation of which might be related to the two-domain architecture of troponin I.
Chemistry Research and Development, Dade Behring, P.O. Box 520672, Miami, FL 33152-0672, USA.
N
N
Morjana
R
R
Tal
D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D002417:Cattle; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D009206:Myocardium / Q000737:chemistry*; D010206:Papain / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013696:Temperature; D019210:Troponin I / Q000737:chemistry*; D014508:Urea / Q000494:pharmacology*
D000817:Anilino Naphthalenesulfonates; D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D019210:Troponin I; C027132:1-anilino-8-naphthalenesulfonate; D014508:Urea; D010206:Papain