ORIGINAL PAPER A single mutation induces molten globule formation and a drastic destabilization of wild-type cytochrome c at pH 6.0 Md. Khurshid Alam Khan Æ Utpal Das Æ Md. Hamidur Rahaman Æ Md. Imtaiyaz Hassan Æ A. Srinivasan Æ Tej P. Singh Æ Faizan Ahmad Received: 31 October 2008 / Accepted: 20 February 2009 / Published online: 10 March 2009 Ó SBIC 2009 Abstract Amino acid sequences of seven subfamilies of cytochromes c show that other than heme binding residues there are only four positions which are conserved in all subfamilies: Gly/Ala6, Phe/Tyr10, Leu/Val/Phe94, and Tyr/Trp/Phe97. These residues are 90% conserved in all sequences reported and are also considered to be involved in a common folding nucleus. To determine the importance of conserved interactions offered by the side chain of Leu94, we made an L94G mutant of horse cytochrome c. Characterization of this mutant by the far-UV, near-UV, and Soret circular dichroism, intrinsic and 1-Anilino-8- naphthalene sulfonate fluorescence, and dynamic light scattering measurements led to the conclusion that the L94G mutant has all the common structural characteristics of a molten globule at pH 6.0 and 25 °C. NaCl induces a cooperative transition between the acid-denatured state and a state of L94G having all the common structural charac- teristics of a pre-molten-globule state at pH 2 and 25 °C. Thermal denaturation studies showed that the midpoint of denaturation of the mutant is 28 °C less than that of the wild-type protein. Interestingly, the structure analysis using the coordinates given in the Protein Data Bank (1hrc) also suggested that the L94G mutant would be less stable than the wild-type protein. Keywords Site-directed mutagenesis  Molten globule  Pre-molten globule  Thermal stability  Cytochrome c Introduction It is well known and widely discussed that there are fam- ilies of functionally and evolutionary related proteins which have similar overall 3D structures (folding patterns) but quite different amino acid sequences [1, 2]. This does not mean, of course, that protein 3D structures do not depend on the protein sequences. However, it implies that not all features of the protein sequence is important for its folding pattern. The usual explanation of this phenomenon is that the protein-folding pattern is determined not by all details of the amino acid sequences but by the key char- acteristics of the sequences of all proteins of a given family [3]. That is, just a few identical or conserved residues are enough to ensure the common folding patterns of these proteins. Both experiment and theory have shown that these conserved residues form a folding nucleus which is specific, i.e., it involves a definite set of native contacts, and that the residues which form the nucleus are scattered along the chain [3]. Cytochrome c, a 104-residue protein, is a member of an extended family of heme proteins involved in electron transfer functions in the mitochondrial respiratory chain and bacterial photosynthesis [4]. Despite their highly divergent amino acid sequences, these cytochromes c share some common structural features, including the covalent linkage of the heme group to two Cys residues, the axial ligands of the heme iron, and a cluster of three a-helices grouped Md. Khurshid Alam Khan  Md. Hamidur Rahaman  Md. Imtaiyaz Hassan  F. Ahmad (&) Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, New Delhi 110025, India e-mail: faizan_ahmad@yahoo.com U. Das  A. Srinivasan  T. P. Singh Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India 123 J Biol Inorg Chem (2009) 14:751–760 DOI 10.1007/s00775-009-0488-6