A Unique Molten Globule State Occurs during Unfolding of Cytochrome c by LiClO 4 Near Physiological pH and Temperature: Structural and Thermodynamic Characterization ² Beenu Moza, ‡ Shabir Hussain Qureshi, ‡ Asimul Islam, ‡ Rajendrakumar Singh, ‡ Farah Anjum, ‡ Ali Akbar Moosavi-Movahedi, § and Faizan Ahmad* ,‡ Department of Biosciences, Jamia Millia Islamia, Jamia Nagar, New Delhi, 110 025, India, and Institute of Biochemistry and Biophysics, UniVersity of Tehran, Tehran, Iran ReceiVed NoVember 18, 2005; ReVised Manuscript ReceiVed February 13, 2006 ABSTRACT: We have carried out denaturation studies of bovine cytochrome c (cyt c) by LiClO 4 at pH 6.0 and 25 °C by observing changes in difference molar absorbance at 400 nm (∆ǫ 400 ), mean residue ellipticities at 222 nm ([θ] 222 ) and difference mean residue ellipticity at 409 nm (∆[θ] 409 ). The denaturation is a three-step process when measured by ∆ǫ 400 and ∆[θ] 409 , and it is a two-step process when monitored by [θ] 222 . The stable folding intermediate state has been characterized by near- and far-UV circular dichroism, tryptophan fluorescence, 8-anilino-1-naphthalene sulfonic acid (ANS) binding, and intrinsic viscosity measurements. A comparison of the conformational and thermodynamic properties of the LiClO 4 -induced molten globule (MG) state with those induced by other solvent conditions (e.g., low pH, LiCl, and CaCl 2 ) suggests that LiClO 4 induces a unique MG state, i.e., (i) the core in the LiClO 4 -induced state retains less secondary and tertiary structure than that in the MG states obtained in other solvent conditions, and (ii) the thermodynamic stability associated with the LiClO 4 -induced process, native state T MG state, is the same as that observed for each transition between native and MG states induced by other solvent conditions. The process of protein folding, while critical and funda- mental to virtually all of biology, remains a mystery. It is important to elucidate the hierarchy of interactions that stabilize the native state. Characterization of folding inter- mediates provides an insight into the understanding of how and when different forces come into play to direct protein folding. The development of a broad range of techniques has led to the identification and characterization of stable intermediates in several proteins (1-4). One such stable intermediate state termed the molten globule (MG 1 ) has been shown to be a compact collapsed structure that has pro- nounced secondary structure but largely disordered tertiary structure (5, 6). Some structural similarities between the MG and native states of proteins also seem to have a significant bearing on understanding the protein folding problem (7). The molten globule state of cytochrome c (cyt c) can be induced at low pH values (called acidic MG) by reducing the electrostatic repulsions (8, 9) and near neutral pH upon unfolding by weak salt denaturants (10). The acidic MG state has been extensively investigated, and, therefore, the con- formation, stability, and mechanism of stabilization have been understood in great detail (1-9). A broad body of information on conformation of MG state of various proteins reveals that this intermediate has a large pool of conforma- tional variations (5, 6, 8, 11, 12). This has raised new questions regarding how many intermediates can be covered by the term MG. Contrary to the data available on acidic MG, there are very few reports available in the literature about the MG state induced at or near physiological pH (10, 13, 14). It may be noted that the MG state of a protein is also shown to be present in the living cells where they are involved in many physiological processes (5, 6). Thus, the MG generated at a low pH condition may bear little relevance to the MG state found inside cells at physiological pH. It is therefore necessary to characterize extensively the MG states near neutral pH and understand the interactions that stabilize them at physiological pH. This will enhance our understand- ing not only of protein folding in vitro and in vivo but also of many cellular processes such as protein translocation or protein recognition by chaperones. In an attempt to characterize the molten globule states generated at physiological pH, we have been carrying out systematic studies of the denaturation of cyt c by weak salt denaturants. In our earlier communication (10), we have shown that MG states generated by LiCl and CaCl 2 have similar conformational and thermodynamic properties. In this study we have investigated the denaturation of cyt c by LiClO 4 at pH 6.0 and compared the results with those obtained from the LiCl-induced and CaCl 2 -induced de- ² This work was supported by Grant 37(1078) 01/EMR-II from the Council of Scientific and Industrial Research, India. * Corresponding author. Tel: +91-11-2698 1733. Fax: +91-11- 2698 1232. E-mail: faizan_ahmad@yahoo.com. ‡ Jamia Millia Islamia. § University of Tehran. 1 Abbreviations: CD, circular dichroism; ǫ, molar absorption coef- ficient; ∆ǫ400, difference molar absorbance at 400 nm; [θ]222, mean residue ellipticity at 222 nm; ∆[θ]409, difference mean residue ellipticity at 409 nm; ANS, 8-anilino-1-naphthalene sulfonic acid; cyt c, cyto- chrome c; MG, molten globule; yN, optical property of the native state; yD, optical property of the denatured state; GdnHCl, guanidine hydrochloride. 4695 Biochemistry 2006, 45, 4695-4702 10.1021/bi052357r CCC: $33.50 © 2006 American Chemical Society Published on Web 03/18/2006