Research Communication Native-like Tertiary Structure in the Mucor miehei Lipase Molten Globule State Obtained at Low pH Sadaf Fatima, Basir Ahmad and Rizwan Hasan Khan Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India Summary Studies on the acid-induced denaturation of Mucor miehei lipase (E.C. 3.1.1.3) were performed by circular dichroism (CD) spectro- scopy, fluorescence emission spectroscopy and binding of hydro- phobic dye, 1-anilino 8-naphthalenesulfonic acid (ANS). Acid denaturation of the lipase showed loss of secondary structure and alterations in the tertiary structure in the pH range 4 to 2 and 7 to 2 respectively, suggesting that the lipase exists as an acid-unfolded state *pH 2.0. A further decrease in pH (from 2.0 to 1.0) resulted in a second transition, which corresponded to the formation of both secondary and tertiary structures. The acid unfolded state at around pH 2.0 has been characterized by significant loss of secondary structure and a small increase in fluorescence intensity with a blue shift of 2 nm, indicating shift of tryptophan residues to less polar environment. Interestingly, the lipase at pH 1.0 exhibits character- istics of molten globule, such as enhanced binding of hydrophobic dye (ANS), native-like secondary structure and slightly altered tryptophanyl environments. That the molten globule of the lipase at pH 1.0 also possesses native-like tertiary structure is an interesting observation made for this lipase. IUBMB Life, 59: 179–186, 2007 Keywords Lipase; Mucor miehei; molten globule; circular dichro- ism; tryptophanyl fluorescence. INTRODUCTION Lipases are versatile biocatalysts that bring about a range of bioconversions of lipids such as hydrolysis, transesterification, alcoholysis, acidolysis and aminolysis. They also exhibit cutinase and amidase activities (1). Biotechnological versatility, high regio-/enantioselectivity and the tremendous potential of these enzymes have been harnessed in areas such as food technology, biomedical sciences, chemical and oleochemical industries, pesticides, single cell proteins, cosmetics, waste disposal, biosensor modulations, detergents, leather processing etc. (2). Mucor miehei triacylglycerol lipase, in general terms, falls into the category of parallel a/b hydrolase as defined by Richardson (3). The three-dimensional structure of this lipase has been solved at 1.9 A8 (4). A single polypeptide chain of 269 residues is folded into a central mixed beta-pleated sheet with predominantly parallel (8 major þ 1 additional) strands con- nected by a variety of hairpins, loops and helical segments. The protein also contains 5 alpha helices (5). In total, 130 amino acid residues (48.3%) occur in sheets or helices with the remaining 51.7% residues located in beta-hairpins and longer surface loops. The catalytic center of M. miehei lipase is formed by Ser-144, Asp-203 and His-257. In native and inactive forms of the enzyme, the catalytic site is shielded from the solvent by an amphipathic alpha-helix (residues 82 – 96) also called a lid or flap. Three disulfide bridges stabilize the folding of the polypeptide chain: Cys40-Cys43, Cys29- Cys268 and Cys235- Cys244. Our attempts towards understanding mechanisms by which proteins acquire their three-dimensional structure from one- dimensional amino acid sequence have been many, though still insufficient. The current literature view is that each sequence folds through a unique energy landscape that is dictated by intrinsic properties of the polypeptide and by the extrinsic influence of the folding environment (6). Kinetic refolding experiments in vitro as well as theoretical calculations suggest that protein folding is a sequential hierarchical process (7), with the existence of stable ‘molten globule’ (MG) conforma- tions between fully folded and unfolded states (8). The characteristic features of the ‘molten globule’ state are: (i) it contains extensive secondary structure; (ii) it has loose tertiary contacts without tight side-chain packing; (iii) it is less compact than the native state; (iv) it is more compact than the unfolded state. Proteins in this MG state contain high level of secondary structure, as well as rudimentary native-like tertiary topology. Recent evidence, however, supports the idea that molten globules may also possess well-defined tertiary Received 11 January 2007; accepted 13 March 2007 Address correspondence to: Rizwan Hasan Khan, Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh-202 002, India. Tel: þ 91 571 2720388. Fax: þ 91 571 2721776. E-mail: rizwanhkhan@hotmail.com; rizwanhkhan1@yahoo.com IUBMB Life, 59(3): 179 – 186, March 2007 ISSN 1521-6543 print/ISSN 1521-6551 online Ó 2007 IUBMB DOI: 10.1080/15216540701335716