SDS induced molten globule state of heynein; a new thiol protease: Evidence of domains and their sequential unfolding

Abstract

The molten globule state can be an intermediate in the protein-folding pathway and its detailed description can help understand the protein folding and an insight into the molecular structure of a protein. Sodium dodecyl sulfate (SDS), an anionic surfactant is known to induce molten globule sate in some proteins. SDS-induced changes in heynein were monitored by CD, fluorescence, 8-anilino-1-napthalenesulfonic acid (ANS) binding and proteolytic activity measurements. An enhancement in the α-helicity of protein with increasing concentration of SDS along with exposure of tryptophans is seen. At a concentration of SDS (∼2 mM) heynein loses activity and rigid tertiary structure but possesses considerable amount of secondary structure along with strong ANS binding, indicating the presence of an intermediate state, which is like molten globule state seen in the case heynein. Chemical and temperature induced unfolding of SDS-induced state of heynein is non-cooperative contrary to the protein in the absence of detergent. Further, the cooperative unfolding transition curve of heynein in the absence of SDS intersects at a point where the second transition of SDS-induced state starts suggesting that the molecule of heynein consist of at least two structural domains which are stabilized differentially and unfolds sequentially. Enhancement of α-helicity of heynein in the presence of SDS suggests the α-rich domain of the protein was stabilized and unfold later as compared with β-rich domain during temperature and chemical induced denaturation. Equilibrium unfolding pathway of heynein in SDS-induced state provides knowledge of the structure and stability of this plant cysteine protease at domain level.