Formation of amyloid fibrils by bovine carbonic anhydrase Anshul Rana, Teemish Praveen Gupta, Saurabh Bansal, Bishwajit Kundu ⁎ Department of Biochemical Engineering and Biotechnology, Indian Institute of Technology, Hauz Khas. New Delhi 110016, India article info abstract Article history: Received 13 December 2007 Received in revised form 25 February 2008 Accepted 26 February 2008 Available online 18 March 2008 Amyloids are typically characterized by extensive aggregation of proteins where the participating polypeptides are involved in formation of intermolecular cross β-sheet structures. Alternate structure attainment and amyloid formation has been hypothesized to be a generic property of a polypeptide, the propensities of which vary widely depending on the polypeptide involved and the physicochemical conditions it encounters. Many proteins that exist in the normal form in-vivo have been shown to form amyloid when incubated in partially denaturing conditions. The protein bovine carbonic anhydrase II (BCA II) when incubated in mildly denaturing conditions showed that the partially unfolded conformers assemble together and form ordered amyloid aggregates. The properties of these aggregates were tested using the traditional Congo-Red (CR) and Thioflavin-T (ThT) assays along with fluorescence microscopy, transmission electron microscopy (TEM), and circular dichroism (CD) spectroscopy. The aggregates were found to possess most of the characteristics ascribed to amyloid fibers. Thus, we report here that the single-domain globular protein, BCA II, is capable of forming amyloid fibrils. The primary sequence of BCA II was also analyzed using recurrence quantification analysis in order to suggest the probable residues responsible for amyloid formation. © 2008 Elsevier B.V. All rights reserved. Keywords: Amyloidogenesis Recurrence quantification analysis Aggregation Bovine carbonic anhydrase 1. Introduction Amyloid fibrils are highly ordered structures formed by aggregation of polypeptide chains, based upon long range repetitive intermolecular interactions [1]. Initially amyloids were discovered as fibrillar protein aggregates associated with neurodegenerative diseases like Alzhei- mer's and Parkinson's disease in humans and prion diseases in animals [2–5]. Amyloid fibrils have also been found in many systemic diseases like primary systemic amyloidosis [6]. Recently, amyloid formation has been hypothesized to be a property common to all polypeptide chains [2,6,7]. This hypothesis is based on the lack of any apparent structural, functional or sequential similarity between the observed amyloido- genic proteins [8]. It is further strengthened by the observation of amyloid formation, under suitable conditions, by several proteins unrelated to any known diseases [9–11]. Despite significant differences in the parent proteins involved, the fibrils formed display a high de- gree of orderliness and show remarkable similarities in a number of physicochemical, morphological and structural properties [12–14]. The formation of ordered aggregates by unrelated and dissimilar proteins points to some intrinsic uniqueness. Evidence for similarity in the lag- times and growth rates ratio for unrelated proteins has also been reported [15]. It also suggests that such structured aggregation may proceed by a general mechanism [16–18]. The actual mechanism has thus far eluded researchers, though there have been some advances in understanding fiber elongation and growth processes [19,20] as well as structural changes accompanying amyloid formation [21]. The process of fiber formation becomes more important because it has been shown that oligomers formed in the preaggregation stage are toxic to the cells rather than the fibrils [22–25]. Furthermore, the non- crystalline and insoluble character of amyloid fibrils makes them poor candidates for X-ray crystallography and solution NMR. Recently, solid state NMR methods have helped in understanding the structural characteristics of several amyloid proteins [26,27]. However, research- ers have often had to depend on computational and predictive methods [28] resulting in postulates to explain amyloidogenicity of proteins. None of these methods unfortunately have been able to explain all the observations related to amyloid formation satisfactorily. Carbonic anhydrase (CA) is a protein found in almost all animals and photosynthesizing organisms. In animals, its major function is to catalyze the reversible conversion of carbon dioxide to carbonic acid in red blood cells where it is found in abundance. CA performs other physiological functions such as acid secretion in stomach, pH maintenance of alkaline pancreatic secretions and saliva, osmoregula- tion in kidneys and eyes [29,30]. As a subject of enzyme research, CA is important for its extremely high turnover rate [31]. BCA II has no disulphide linkages [32], and complete denaturation of the protein happens at a guanidinium hydrochloride (GdnHCl) concentration of 4.0 M. Apart from its biological and scientific significance as mentioned above, BCA II emerged as a favored molecule for amyloidogenic studies owing to some additional characteristics; i) All known CAs and their isozymes are structurally similar containing a 10-stranded twisted β-sheet with a few small helices [33,34]. ii) It was shown that Biochimica et Biophysica Acta 1784 (2008) 930–935 ⁎ Corresponding author. Tel.: +9111 26591037; fax: +9111 26582282. E-mail addresses: kundudr@yahoo.com, bxk@dbeb.iitd.ernet.in (B. Kundu). 1570-9639/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2008.02.020 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbapap