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Reduced Stability and Enhanced Surface Hydrophobicity Drive the Binding of Apo-Aconitase with Groel During Chaperone Assisted Refolding

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Reduced Stability and Enhanced Surface Hydrophobicity Drive the Binding of Apo-Aconitase with Groel During Chaperone Assisted Refolding The International Journal of Biochemistry & Cell Biology 42 (2010) 683–692 Contents lists available at ScienceDirect The International Journal of Biochemistry & Cell Biology journal homepage: www.elsevier.com/locate/biocel Reduced stability and enhanced surface hydrophobicity drive the binding of apo-aconitase with GroEL during chaperone assisted refolding Parul Gupta a,1, Saroj Mishra a, Tapan Kumar Chaudhuri a,b,∗ a Department of Biochemical Engineering and Biotechnology, Hauz Khas, New Delhi 110016, India b School of Biological Sciences, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India article info abstract Article history: Apo-aconitase, the Fe4S4 cluster free form of TCA cycle enzyme aconitase, binds with GroEL and dis- Received 2 September 2009 sociates itself upon maturation through insertion of the cluster. It is not clearly established as to why Received in revised form apo-protein binds with GroEL. In order to explore the possibility that stability is a factor responsible 21 December 2009 for the aggregation of apo-form at low ionic strengths and hence it associates with GroEL to avoid the Accepted 4 January 2010 unfavorable event, we carried out the unfolding studies with holo- and apo-aconitase. By probing the Available online 9 January 2010 unfolding process through the changes in secondary structural element, exposed surface hydrophobicity, and the microenvironment around tryptophan residues, we were able to establish the relevant changes Keywords: Apo-aconitase associated with the event. Apparent guanidine hydrochloride concentration required for unfolding of 50% Holo-aconitase of aconitase indicates that aconitase is destabilized in the absence of the Fe4S4 cluster. The destabilization Aconitase unfolding of the apo-aconitase was further reflected through its three times higher rate of unfolding as compared GroEL-assisted folding to the holo-protein. It was also observed that the apo-form has higher surface hydrophobicity than the Metallo protein biosynthesis holo-form. Hence, the lower ground state stability and higher solvent exposed hydrophobic surface of the apo-form makes it aggregation prone. Based on the present observation and earlier findings, we propose that binding of apo-aconitase to GroEL not only rescues it from the aggregation, but also assists in the final stage of maturation by orienting the cluster insertion site of GroEL bound apo-protein. This information sheds new light on the potential role of GroEL in the biosynthetic pathway of the metallo proteins. © 2010 Elsevier Ltd. All rights reserved. 1. Introduction around 30% of the proteins in the cell (Frausto da Silva and Williams, 1991), considerable effort has been expended in the last few years Cofactors play an important role in biological function, sta- in addressing the determinants of stability and folding properties bility and folding of various proteins (Wittung-Stafshede, 2002). of proteins, containing metal cofactors. Addressing the stability properties of proteins with bound pros- Aconitase (EC 4.2.1.3) is an 82 kDa protein that serves to cat- thetic groups may pose particular difficulties: (a) if the cofactor alyze the stereospecific conversion of citrate to iso-citrate through is covalently bound, it is likely that the unfolding peptide is able the intermediate cis-aconitate. It contains an iron–sulfur cluster, to refold spontaneously to its native state; in fact, there are sev- and the active enzyme requires the fully assembled [4Fe–4S] clus- eral available studies in which the working models are small ter. The crystal structure (Lauble et al., 1992; Robbins and Stout, monomeric proteins which contain a prosthetic group with a 1989) shows the protein to be composed of four structural domains. covalent linkage. (b) On the other hand, conformational stability The first, second and third domains form a relatively tightly packed studies on proteins containing non-covalently bound cofactors may structure connected to the fourth domain through a potentially be much more difficult to interpret, due to their complex large flexible hinge-linker peptide. This fourth domain, in the crystal multi-domain structures. In recognition of the important role of structure, is closely apposed to domains 1–3, forming a cleft that metal-containing proteins in the living systems, which make up serves as the active site of the enzyme. The iron–sulfur cluster is within the cleft and is intimately associated with amino acid residues that are involved in binding of the substrate and catalyz- ing the citrate–iso-citrate interconversion. Despite several studies Abbreviations: ANS, 1-anilino naphthalene-8-sulfonate; GdnHCl, guanidine on the biophysical and spectroscopic properties of Fe–S proteins hydrochloride; MG, molten globule; N, native; U, unfolded; PK, proteinase K. (Lippard and Berg, 1994), there are few folding and stability studies ∗ Corresponding author. Tel.: +91 11 2659 1012; fax: +91 11 2658 2282. of these proteins. There is only one study in which the thermody- E-mail addresses: [email protected] (P. Gupta), [email protected] namic stability of the apo- and holo-forms of a Fe–S protein has (S. Mishra), [email protected], [email protected] (T.K. Chaudhuri). 1 Present address: Characterization Services, Biologics Development centre, Dr. been directly compared. The study showed that when the cluster Reddy’s Laboratories Ltd., Bachupally, R R District, Andhra Pradesh, India. is removed from adrenodoxin, the transition temperature (Tm) and 1357-2725/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biocel.2010.01.002 684 P. Gupta et al. / The International Journal of Biochemistry & Cell Biology 42 (2010) 683–692 the unfolding enthalpy change is considerably reduced; the holo- aconitase can be recruited to GroEL, it is not subject to misfolding ◦ ◦ form has a Tm of 51 C whereas the apo-form has a Tm of 37 C. and aggregation in its absence. In addition, the apo-protein is much more susceptible to protease One approach towards the understanding of cluster stabiliza- digestion than the holo-protein (Burova et al., 1995). tion and its involvement in protein stability in iron sulfur proteins In spite of the accumulation of a large number of experimen- is to investigate the involvement of the intermediate structures or tal studies, protein folding remains one of the most challenging to highlight the role of particular structural elements in cluster sta- subjects in structural biology. Characterization of folding inter- bilization. In order to highlight the structural role that the Fe–S mediates is considered an important strategy for the elucidation cluster is likely to play in holding the various domains of aconitase of the mechanism of protein folding. A common intermediate, core together and to investigate the mechanism of aconitase fold- the “molten globule” (MG) state, has been detected between the ing, the formation of intermediates at different stages of unfolding native (N) and the fully unfolded (U) states for many proteins (Fink, were characterized. The present study describes the measurements 1995; Ptitsyn, 1995). The MG state is characterized by pronounced of the GdnHCl-induced unfolding of holo- and the apo-forms of secondary structure, compact globularity, exposed hydrophobic the yeast mitochondrial aconitase, monitored by intrinsic fluo- surface, and the absence of rigid side-chain packing (Fink, 1995; rescence, far UV CD, and binding with the extrinsic fluorescence Kuwajima, 1989, 1996; Ptitsyn, 1987, 1995). However, most pro- probe, 1-anilino-naphthalene-8-sulfonate (ANS). The kinetics of teins for which a MG intermediate has been well characterized the unfolding process and the effect of Fe–S cluster on the rate are small, monomeric, single domain proteins (Arai and Kuwajima, of aconitase unfolding were further studied by stopped-flow tech- 1996; Kay and Baldwin, 1996; Kim and Baldwin, 1990; Matthews, niques. The role of co-chaperonin GroES during reconstitution of 1993; Ogasahara and Yutani, 1994). Multi-domain, oligomeric pro- holo-aconitase from apo-aconitase was investigated by partial pro- teins or metallo proteins remain relatively little explored (Jeanicke, teolytic digestion of apo aconitase-GroEL binary complex. 1991). Since the folding/unfolding of such proteins is accompa- nied by the association/dissociation of subunits or incorporation 2. Results of metal cluster and are aggregation prone, the processes are much more complicated than that of monomeric proteins. 2.1. Conformation and secondary structure of recombinant holo- The efficient folding for most proteins has been shown to occur and apo-aconitase inside the cavity of the cis ring of GroEL that houses the unfolded or partially folded protein, following encapsulation or capping of the The recombinant yeast mitochondrial aconitase was expressed cis ring by dome-shaped heptameric GroES in the presence of Mg- in E. coli as soluble, biologically active enzyme. The folded protein ATP (Weissman et al., 1995). The subsequent binding of Mg-ATP to was purified and its enzymatic activity estimated. The metal clus- the unoccupied or the trans ring of GroEL, results in the collapse ter free form was generated by stripping of the iron–sulfur cluster of the cis ring assembly with the concomitant release of GroES and from the holo-form, in order to obtain apo-aconitase. The pep- partially or completely folded protein from the cis cavity. It has been tide CD spectra of the recombinant holo-aconitase was measured generally accepted that the unfolded proteins of ∼57 kDa or smaller under native conditions (0 M GdnHCl at 25 ◦C), and compared with in size can fit inside the cavity encapsulated by GroES, as shown by the CD spectra of the apo-aconitase (Fig. 1A). There was a signif- studies both in vivo (Ewalt et al., 1997; Houry et al., 1991) and in icant difference in the CD spectra between the two forms of the vitro (Sakikawa et al., 1999). For certain proteins larger than this protein in the peptide regions, indicating differences in their sec- size, for example, 75 kDa methylmalonyl-CoA mutase (Weissman ondary structures.
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