Different molten globule-like folding intermediates of hen egg white lysozyme induced by high pH and tertiary butanol.
We have provided evidence that hen egg white lysozyme (HEWL) existed in alpha helical and beta structure dominated molten globule (MG) states at high pH and in the presence of tertiary butanol, respectively. Circular dichroism (CD), intrinsic fluorescence, ANS binding and acrylamide-induced fluorescence quenching techniques have been used to investigate alkali-induced unfolding of HEWL and the effect of tertiary butanol on the alkaline-induced state. At pH 12.75, HEWL existed as molten globule like intermediate. The observed MG-like intermediate was characterized by (i) retention of 77% of the native secondary structure, (ii) enhanced binding of ANS (approximately 5 times) compared to native and completely unfolded state, (iii) loss of the tertiary structure as indicated by the tertiary structural probes (near-UV, CD and Intrinsic fluorescence) and (iv) acrylamide quenching studies showed that MG state has compactness intermediate between native and completely unfolded states. Moreover, structural properties of the protein at isoelectric point (pI) and denatured states have also been described. We have also shown that in the presence of 45% tertiary butanol (t-butanol), HEWL at pH 7.0 and 11.0 (pI 11.0) existed in helical structure without much affecting tertiary structure. Interestingly, MG state of HEWL at pH 12.7 transformed into another MG state (MG2) at 20% t-butanol (v/v), in which secondary structure is mainly beta sheets. On further increasing the t-butanol concentration alpha helix was found to reform. We have proposed that formation of both alpha helical and beta sheet dominated intermediate may be possible in the folding pathway of alpha + beta protein.
Hameed
Mahrukh
M
Department of Biotechnology, University of Kashmir, Hazratbal, Srinagar, India.
Ahmad
Basir
B
Fazili
Khalid Majid
KM
Andrabi
Khurshid
K
Khan
Rizwan Hasan
RH
D020106:Acrylamide; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D005453:Fluorescence; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000737:chemistry*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D020002:tert-Butyl Alcohol / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D020106:Acrylamide; C421864:hen egg lysozyme; D009113:Muramidase; D020002:tert-Butyl Alcohol