Cite this: RSC Advances, 2013, 3, 8016 Dendrons and dendrimers as pseudochaperonins for refolding of proteins3 Received 7th January 2013, Accepted 15th March 2013 DOI: 10.1039/c3ra00061c www.rsc.org/advances Priyanka Dubey, Saurabh Gautam, P. P. Praveen Kumar, Sandhya Sadanandan, V. Haridas* and Munishwar N. Gupta* Peptide dendrimers are screened for ‘‘artificial chaperone’’ (protein refolding) activity by a sensitive fluorescence based assay. The refolding with largest dendrimer is found to help in recovering biological activity of >90% in the case of unfolded lipases and amylases. The refolding yields decrease down to 14% with a decrease in the complexity and hydrophobicity of the dendron/dendrimer. CD spectroscopy confirms the correct refolding in terms of secondary structure contents of the proteins. The DLS data indicates that presence of the dendrons/dendrimers facilitates protein refolding by preventing the aggregation of proteins. Introduction Protein refolding consists of recovery of biologically active proteins from their inactive forms, often in the presence of small molecular weight compounds 1–4 or polymers. 5–10 The inactive forms of the proteins mostly results from the formation of misfolded structures. The first step in the refolding process is complete unfolding of these misfolded forms. The second step is to facilitate refolding of this unfolded form to the correct native and active conformation. The side chains of the hydrophobic amino acids are more exposed in their unfolded form and show high tendency to aggregate. The appropriate additives during this step can bind to these hydrophobic residues, prevent aggregation and hence facilitate refolding to the correct structure. 1–10 Molecular chaperones assist proteins to fold into a correct native conformation. 11,12 The self-assembled structure of chaperones coupled with their efficiency and precision are envy of chemists. Chaperones have hydrophobic and hydro- philic surfaces and are efficient molecular machines for protein folding and as disaggregase. 13 The folding of unfolded protein to its native state is the first step towards under- standing the mechanism of protein folding. Therefore, the study of chaperone is considered as one crucial step for understanding the folding of proteins. Many efforts have been done and are already underway to elucidate the mechanism of chaperone function, but a desirable and highly warranted step would be a totally synthetic chaperone. In order to design a simple chemical model for a chaperone, we envisioned that dendrimers, in particular peptide dendrimers, are a much suited candidate for this purpose. Recently, use of click reactions in synthesis of dendrons/ dendrimers was reported. 14–17 The attractive attribute of dendrons/dendrimers are that structural motifs can be repeated in a controlled manner leading to their tailoring of applications in diverse areas like sensors, catalysis, therapeu- tics, and drug delivery. 18–21 Higashi et al. 22 had reported that use of a dendrimer template increased the helicity of an oligopeptide consisting of c-benzyl-L-glutamic acid. It was thought that if a series of dendrons of increasing complexity can be created, one can screen various candidates for their interaction with a given protein. The idea was to attempt creating a platform technology so that ‘‘hit and trial’’ approach in search for refolding various proteins becomes unneces- sary. 1–3 Also, given the peptide nature, these structures can serve as simple models to mimic action of biological chaperones. 11,12 In recent years, thermal shift assay has been used to evaluate interaction of proteins with other molecules successfully. 23,24 This sensitive technique was adopted to screen the suitability of the dendrons/dendrimers for protein refolding purposes. To validate the approach, three industrially useful lipases [TLL (Thermomyces lanuginosus lipase), CAL B (Candida antarctica lipase B) and RML (Rhizomucor miehei lipase)] and two amylases [BLA (a-amylase from Bacillus licheniformis) and BAA (a-amylase from Bacillus amyloliquefaciens)] were chosen. In each case, actual refolding was also studied by denaturing the proteins either thermally or by adding 8 M solution of urea. The latter condition is often used to solubilize inclusion bodies before refolding. 3 Attempts were made to refold the denatured proteins in the presence of dendrons and dendri- mers. Circular dichroism (CD) spectroscopy was used to confirm the secondary structure contents of the refolded Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016, India. E-mail: appliedbiocat@yahoo.co.in; h_haridas@hotmail.com; Fax: +91-11-2658 1073; Tel: +91-11-2659 1503 3 Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3ra00061c RSC Advances PAPER 8016 | RSC Adv., 2013, 3, 8016–8020 This journal is ß The Royal Society of Chemistry 2013 Published on 20 March 2013. Downloaded by Indian Institute of Technology New Delhi on 19/11/2014 12:27:21. View Article Online View Journal | View Issue