Journal of Biochemistry Advance Access originally published online on February 16, 2007
Journal of Biochemistry 2007 141(4):573-583; doi:10.1093/jb/mvm057
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© 2007 The Japanese Biochemical Society.
Different Molten Globule-like Folding Intermediates of Hen Egg White Lysozyme Induced by High pH and Tertiary Butanol
1Department of Biotechnology, University of Kashmir, Hazratbal, Srinagar 190006; and 2Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India
*To whom correspondence should be addressed. Tel: +91-571-2720388, Fax: +91-571-2721776, E-mail: rizwanhkhan{at}hotmail.com, rizwanhkhan1{at}yahoo.com
Received December 9, 2006; Accepted February 7, 2007
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Abstract |
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We have provided evidence that hen egg white lysozyme (HEWL) existed in 
helical and ß 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 (
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 ß sheets. On further increasing the t-butanol concentration helix was found to reform. We have proposed that formation of both helical and ß sheet dominated intermediate may be possible in the folding pathway of + ß protein.
Key Words: alkali induced unfolding, circular dichroism, fluorescence quenching, lysozyme, molten globule
Abbreviations: ANS, 8-anilinonaphthalene-1-sulphonic acid; CD, circular dichroism; GnHCl, guanidinium hydrochloride; HEWL, hen egg white lysozyme; MG, molten globule; MRE, mean residual ellipticity; PFI, partially folded intermediate; pI, isoelectric point; RFI, relative fluorescence intensity; t-butanol, tertiary butanol; TFE, tri-fluoro ethanol; Ualk, alkali unfolded state; UV, ultra violet