20418 Phys. Chem. Chem. Phys., 2011, 13, 20418–20426 This journal is c the Owner Societies 2011 Cite this: Phys. Chem. Chem. Phys., 2011, 13, 20418–20426 Protein unfolding and subsequent refolding: a spectroscopic investigation Uttam Anand, Chandrima Jash and Saptarshi Mukherjee* Received 31st May 2011, Accepted 19th September 2011 DOI: 10.1039/c1cp21759c The mechanism by which the protein Bovine Serum Albumin (BSA) undergoes unfolding induced by Guanidine Hydrochloride (GdHCl) and then the subsequent refolding brought in by many-fold dilution was studied by steady-state fluorescence, anisotropy, time resolved measurements and Circular Dichroism (CD) spectroscopy. CD data reveal that the protein attains a degree of extra rigidity at low concentrations of the denaturant, GdHCl, and this observation was correlated with other techniques used in this present work. The unfolding and refolding of BSA appear to proceed through intermediates and both the processes are sequential in nature. The intrinsic fluorescence from the tryptophan amino acid residue of BSA and another external fluorophore Nile Red was made use of in order to investigate the mechanisms of unfolding and refolding and we have conclusively proved that both these processes follow a reversible mechanism. Introduction Unraveling the mechanisms through which the most intricate components of molecular structures self-assemble is one of the greatest challenges of modern science. 1 The folding of proteins into their three-dimensional complex structures is one of the most fundamental examples of such self-assembly. Although such self-aggregation has drawn considerable research inter- ests of late, 2–12 the mechanism by which a polypeptide chain folds to form specific three-dimensional protein structures has not yet been clearly understood. The conformation of a protein in solution is generally a function of electrostatic, hydrogen-bonding, van der Waals, and hydrophobic inter- actions among the amino acid residues that all typically favor a folded conformation, overcoming the entropic penalty asso- ciated with this folding of the protein into a compact state. 9 However, when proteins fold incorrectly (misfold), there can be serious consequences leading to Alzheimer’s disease and cancer. 13 Protein unfolding can be induced by a variety of external conditions such as changes in pH, temperature, pressure and through the addition of chemical denaturants. In addition to the methodologies mentioned above, proteins can unfold in response to surfactants and drug molecules. 14–25 At a high surfactant concentration, the three dimensional structure of a protein is destroyed and the protein is said to be denatured. However, in 1948 Duggan and Luck 23 showed that the urea induced denaturation of proteins may be prevented by the addition of a small amount of SDS. Moriyama and Takeda 19a proposed that the secondary struc- ture of HSA remains more or less unchanged up to the addition of 200 mM of SDS. Panda and co-workers also studied the unfolding process of the three domains of HSA using covalently bound fluorescence probes and opined that the unfolding of these domains, induced by guanidine hydro- chloride (GdHCl), is sequential in nature. 25b Protein folding involves very small (B1–15 kcal mol 1 ) changes in energy, whereby the protein transforms from its denatured state into its native state possibly via some inter- mediate states. It is a real challenge to characterize these ‘‘intermediate’’ states as their existence depends on the experi- mental conditions used. The denatured state of a protein is a heterogeneous collection of rapidly inter-converting structures, some of which have flickering native-like elements. 10 However, the protein attains a denatured structure via some intermediate states which need proper characterizations. Also, once dena- tured, proteins can be made to regain their almost native-like structure. Such renaturation processes are kinetically compe- titive between folding and aggregation, and suppressing such aggregation is the key point for enhancing the protein renaturation yield. Among the several proteins studied, Bovine Serum Albumin (BSA) is perhaps one of the most widely used and best characterized proteins mainly because of its relatively large number of charged amino acids on the surface (BSA has a net charge of 18 at neutral pH). 15 The primary structure of BSA consists of nine loops held together by 17 disulfide bonds, resulting in three domains (I, II and III) each consisting of two sub-domains (A and B). BSA shares 76% sequence homology with another albumin protein, Human Serum Albumin (HSA), and houses two tryptophan (Trp) amino acid residues, one at 213 and another at 134 position of the amino acid sequence. In spite of the complexity in shape and size, the Department of Chemical Sciences, Indian Institute of Science Education and Research Bhopal, ITI Campus (Gas Rahat) Building, Govindpura, Bhopal 462 023, Madhya Pradesh, India. E-mail: saptarshi@iiserbhopal.ac.in PCCP Dynamic Article Links www.rsc.org/pccp PAPER Downloaded by Indian Institute of Science Education and Research – Bhopal on 19 April 2012 Published on 13 October 2011 on http://pubs.rsc.org | doi:10.1039/C1CP21759C View Online / Journal Homepage / Table of Contents for this issue