Conformational properties of escherichia coli SecA protein in the aqueous conditions

Abstract

It has been shown that most proteins interact with lipid bilayer strongly, only when converted into a partially unfolded structure such as molten globule. SecA protein, one of the translocation machinery of Escherichia coli, is an exceptional water soluble protein which native form can readily penetrate the membrane and lipid bilayer under physiological condition. In order to see whether the native SecA exhibits the partially unfolded characteristics, temperature- and denaturant-induced denaturation studies of SecA performed by following the changes of the mean residue ellipticity of the far-UV circular dichroism and the intensities of tryptophan and 8-anilinonaphthalene-1-sulfonic acid (ANS). Solvent accessibility of SecA was also investigated using the techniques such as the hydrogen-tritium exchange kinetics of amide-backbone hydrogens, ANS binding and quenching of tryptophan fluorescence by iodide. It appears that denaturation of SecA by heat and denaturant goes through intermediates with the characteristics of molten globule state. The solvent accessibility studies show that 60% of backbone hydrogens of SecA is exchangeable, while 75% of tryptophans are exposed to the solvent. These results are compared with the properties of native and molten globule forms of -lactalbumin (-LA) as well as those of apocytochrome c. The exposure of hydrophobic residues as judged from the ANS binding and the extent of amide hydrogen exchange of SecA were comparable to those of native -LA. On the other hand, equilibrium unfolding experiments showed that SecA is less stable than native -LA. The stable N-terminal 45-kDa fragment caused by the tryptic digestion in aqueous solution, the change of endogenous ATPase activity by guanidine-hydrochloride and the extent of quenching of Trp fluorescence which is concentrated in the C-terminal end are suggestive of the C-terminal half of SecA being more flexible than the rest of the protein. The overall conclusion is that the SecA, as a ...