A small molecule chemical chaperone optimizes its unfolded state contraction and denaturant like properties

OPEN SUBJECT AREAS: SMALL MOLECULES BIOLOGICS SINGLE-MOLECULE BIOPHYSICS A small molecule chemical chaperone optimizes its unfolded state contraction and denaturant like properties Sunny Sharma1, Suparna Sarkar1, Simanta Sarani Paul1, Syamal Roy2 & Krishnananda Chattopadhyay1 PROTEIN AGGREGATION 1 Received 24 June 2013 Accepted 29 November 2013 Published 17 December 2013 Correspondence and requests for materials should be addressed to K.C. () Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032, 2Infectious diseases and Immunology Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032. Protein aggregation is believed to occur through the formation of misfolded conformations. It is expected that, in order to minimize aggregation, an effective small molecule chaperone would destabilize these intermediates. To study the mechanism of a chemical chaperone, we have designed a series of mutant proteins in which a tryptophan residue experiences different local environments and solvent exposures. We show that these mutants correspond to a series of conformationally altered proteins with varying degree of misfolding stress and aggregation propensities. Using arginine as a model small molecule, we show that a combination of unfolded state contraction and denaturant like properties results in selective targeting and destabilization of the partially folded proteins. In comparison, the effect of arginine towards the folded like control mutant, which is not aggregation prone, is significantly less. Other small molecules, lacking either of the above two properties, do not offer any specificity towards the misfolded proteins. P rotein misfolding has been implicated in different neurodegenerative diseases1. Nature is equipped with complex biological machines to counter the misfolding threats in the form of molecular chaperones, which repair and rescue conformationally aberrant proteins2,3. Recent progress in chemical biology and biotechnology has necessitated the search for possible small molecule alternatives of the molecular chaperones, which would inhibit or reverse protein aggregation4,5. In addition, the use of small molecule chaperones has attracted enormous interests for their potential pharmaceutical roles and applications in the manufacturing and formulation development of protein based drugs6. Since protein aggregation often occurs through the formation of partially folded intermediate states with exposed hydrophobic surface, an ideal small molecule chemical chaperone is expected to target them and minimize their populations. In this paper, we hypothesize that; an efficient small molecule chemical chaperone may contain two properties: first, it should contract the unfolded state and second, it should also behave like a denaturant. The rational for the unfolded state contraction comes from the fact that the contracted unfolded state would favor the folded state. The requirement for the denaturant like properties comes from the necessity to unfold the misfolded intermediates providing them with more opportunities to fold correctly. Although it is controversial, it is often believed that a denaturant like guanidinium hydrochloride (Gdn.HCl) or urea binds to the native state affecting the side chains. To put it differently, this hypothesis assumes that the ideal small molecule should affect not only the backbone (the unfolded state), but also the side chain (the folded state). The first property (unfolded state contraction) may be conveniently satisfied by a group of small molecules popularly known as protecting osmolytes7,8. These are typically used as protein stabilizers and include free amino acids (like glycine or proline), carbohydrates (like sucrose, sorbitol or trehalose) and methylamines (such as betaine and trimethyl amine N oxide or TMAO). It has been established that the protecting osmolytes preferentially destabilize the unfolded states (affecting protein backbone), and have significantly less effect on the native folded state (or the side chains)8–10. While these osmolytes are expected to contract the unfolded state11 favoring indirectly the folded state, they may not necessarily offer any specificity towards the aggregation prone partially folded intermediates. The second property (denaturant like) can be satisfied using any common denaturant, like Gdn.HCl or urea. In a small screening experiment involving twenty small molecules (mostly protecting osmolytes), we have found an additive, arginine, to be the most successful in its ability to minimize aggregation of two different recombinant proteins. Arginine can be used as a convenient model small molecule to test the above hypothesis SCIENTIFIC REPORTS | 3 : 3525 | DOI: 10.1038/srep03525 1 www.nature.com/scientificreports because it contains a glycine backbone (colored green in Figure 1a) and hence it may behave like a protecting osmolyte. In addition, the denaturant like property of arginine would arise from its guanidine side chain (colored red in Figure 1a). Interestingly, arginine has found tremendous applications in biotechnology because of its roles in increasing protein solubility and refolding yields12–15. The mechanism of arginine’s action remains controversial. While it has been shown that arginine can follow protecting osmolyte like behavior16, it has also been shown to unfold proteins, albeit partially17. The dual roles of arginine has been suggested by previous reports18. In this paper, we aim to show that the efficiency of arginine as an aggregation inhibitor arises due to its ability to combine unfolded state contraction and its property to unfold aggregation prone partially folded proteins. To achieve that, we have prepared different mutant proteins (Table S1, Supporting Information) of varying aggregation propensities using a combination of computational biology, bioinformatics and tryptophan scanning mutagenesis. Tryptophan is used as the probe since its conformation and surface accessibility can be determined easily and conveniently using steady state fluorescence. A small (92 amino acids) protein, Kinetoplastid Membrane Protein-11 or KMP-11, found in Leishmania parasites is used as the template for designing these mutants. The protein KMP-11 is being developed as a potential vaccine candidate against visceral leishmaniasis19. Another reason why this protein has been chosen is because it does not have any tryptophan residue in its native folded state and hence the tryptophan probe can be inserted at different regions conveniently. Moreover, KMP11 does not have any proline or cysteine residue ruling out complications of proline related misfolding and cysteine-cysteine bond formation. In addition, a key prediction has been verified using an additional protein system, namely, the intestinal fatty acid binding proteins (IFABP). Contraction of the unfolded states of the mut (...truncated)