DAS, PRANAB

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Crystallins are the major proteins of a mammalian eye lens. The topologically similar eye lens proteins, beta- and gamma-crystallins, are the prototype and founding members of the betagamma-crystallin superfamily. betagamma-Crystallins have until recently been regarded as structural proteins. However, the calcium-binding properties of a few members and the potential role of betagamma-crystallins in fertility are being investigated. Because the calcium-binding elements of other member proteins, such as spherulin 3a, are not present in betaB2-crystallin and other betagamma-crystallins from fish and mammalian genomes, it was argued that lens betagamma-crystallins should not bind calcium. In order to probe whether beta-crystallins can bind calcium, we selected one basic (betaB2) and one acidic (betaA3) beta-crystallin for calcium-binding studies. Using calcium-binding assays such as (45)Ca overlay, terbium binding, Stains-All and isothermal titration calorimetry, we established that both betaB2- and betaA3-crystallin bind calcium with moderate affinity. There was no significant change in their conformation upon binding calcium as monitored by fluorescence and circular dichroism spectroscopy. However, (15)N-(1)H heteronuclear single quantum correlation NMR spectroscopy revealed that amide environment of several residues underwent changes indicating calcium ligation. With the corroboration of calcium-binding to betaB2- and betaA3-crystallins, we suggest that all beta-crystallins bind calcium. Our results have important implications for understanding the calcium-related cataractogenesis and maintenance of ionic homeostasis in the lens.It has been established that many heterologously produced proteins in E. coli accumulate as insoluble inclusion bodies. Methods for protein recovery from inclusion bodies involve solubilization using chemical denaturants such as urea and guanidine hydrochloride, followed by removal of denaturant from the solution to allow the protein to refold. In this work, we applied on-column refolding and purification to the second crystallin domain D2 of Yersinia crystallin isolated from inclusion bodies. We also purified the protein from the soluble fraction (without using any denaturant) to compare the biophysical properties and conformation, although the yield was poor. On-column refolding method allows rapid removal of denaturant and refolding at high protein concentration, which is a limitation in traditionally used methods of dialysis or dilution. We were also able to develop methods to remove the co-eluting nucleic acids during chromatography from the protein preparation. Using this protocol, we were able to rapidly refold and purify the crystallin domain using a two-step process with high yield. We used biophysical techniques to compare the conformation and calcium-binding properties of the protein isolated from the soluble fraction and inclusion bodies.Betagamma-crystallin is a superfamily with diverse members from vertebrate lens to microbes. However, not many members have been identified and studied. Here, we report the identification of a putative exported protein from Yersinia pestis as a member of the betagamma-crystallin superfamily. Even though calcium has been known to play an important role in the physiology and virulence of the Yersinia genus, calcium-binding proteins have not yet been identified. We have studied the calcium-binding properties of two of the three crystallin domains present in this putative exported protein designated "Yersinia crystallin." These two domains (D1 and D2) have unique AA and BB types of arrangement of their Greek key motifs unlike the domains of other members of the betagamma-crystallin superfamily, which are either AB or BA types. These domains bind two calcium ions with low and high affinity-binding sites. We showed their calcium-binding properties using various probes for calcium and the effect of calcium on their secondary and tertiary structures. Although both domains bind calcium, D1 underwent drastic changes in secondary and tertiary structure and hydrodynamic volume upon calcium binding. Domain D1, which is intrinsically unstructured in the apo form, requires calcium for the typical betagamma-crystallin fold. Calcium exerted an extrinsic stabilization effect on domain D1 but not on D2, which is also largely unstructured. We suggest that this protein might be involved in calcium-dependent processes, such as stress response or physiology in the Yersinia genus, similar to its microbial relatives and mammalian lens crystallins.BetaB2-crystallin, the major subunit of beta-crystallins, is difficult to purify either from lens homogenate or from betaH-or betaL-crystallins. It has been prepared by heterologous expression in Escherichia coli. Most often, the methods used for purifying a recombinant globular protein employ the combination of ion-exchange with gel filtration chromatography. In the case of betaB2-crystallin too, different approaches have been used to obtain the purified protein, majority of which use a combination of ion-exchange and gel filtration chromatography. We present a new approach to purify betaB2-crystallin using hydrophobic interaction chromatography. In this method, the protein is bound to the hydrophobic matrix in the presence of high concentration of a non-chaotropic salt and eluted by decreasing the salt concentration. The method that we have used for the purification of this globular protein has definite advantages over the earlier methods in its simplicity and efficiency. The most noted advantage of this procedure is the rapid purification with a relatively purified product and a comparatively high yield (>20 mg/L of culture). Over all, the present protocol provides a rapid, efficient and simplified procedure for the preparation of betaB2-crystallin in large yield, sufficient for structural and functional studiesChapter 1: Introduction	This chapter introduces the crystallin proteins of eye lens with focus on the -crystallin superfamily. -Crystallin and taxon-specific crystallins are the other major components of eye lens of vertebrates. The founding members - and -crystallins have been described which were classified separately in the early days. The basis of double crystallin-type Greek key motifs and their types in -crystallin proteins have been described. -Crystallin members isolated from bacteria, amphibians and yeast have been described. The calcium-binding properties and three-dimensional structures determined for some of these proteins have been mentioned. Effect of calcium in tissues or microorganisms where these proteins are expressed and their possible roles are also described. Observations about this superfamily of proteins leading to a hypothesis behind this work are described in the last section of this chapter.Chapter 2: Experimental Procedures	All the experimental procedures used in this work are described in this chapter excluding the cloning, overexpression and purification procedures of individual proteins, which have been mentioned in their respective chapters. A set of common biochemical, spectroscopic and calorimetric techniques applied to this study are described.Chapter 3: Calcium-binding properties of lens -crystallins	Calcium plays an important role in the pathophysiology of cataractogenesis. It is known that free calcium rises several fold in the cataractous lens compared to normal lens. This free calcium could activate calpains, calcium-dependent proteases and transglutaminases leading to precipitation of proteins, and covalent crosslinking in the lens thereby increasing the light scattering resulting in blurred vision. In spite of many studies, calcium-binding targets in lens are not known. Since crystallins are the major proteins in lens, they could act as calcium-binding agents. -Crystallin is known to function as molecular chaperone whereas -crystallins’ function are not yet known. I hypothesized that -crystallins could be calcium-binding proteins. -Crystallins are known to bind calcium whereas concrete evidences of such property of -crystallins are lacking. It was shown that L-crystallin fraction from bovine eye lens binds calcium with micromolar affinity. Since L fractions consists of several subunits of -crystallins, calcium-binding ability of individual subunits needs to be investigated. In this chapter, I have addressed the issue of calcium-binding to individual subunits namely bovine B2- and A3-crystallins as representative members of -crystallins. I have used recombinantly produced protein for a homogenous protein preparation using hydrophobic interaction chromatography. Different calcium-binding assays such as Stains-all, terbium binding and calcium-45 overlay were used to establish calcium-binding to these proteins. Calcium-binding affinity for B2-crystallin was determined using isothermal titration calorimetry (ITC) which revealed four calcium-binding sites with low to moderate affinity. However, this technique was not suitable for determining very low binding affinity of A3-crystallin for calcium instead terbium, a calcium mimic was used. Effect of calcium-binding on the conformation of B2- and A3-crystallin was probed using fluorescence and CD spectroscopy, which indicated no significant change in the apo to holo transition of these -crystallins. Holo form of B2-crystallin does not have significantly different chemical or thermal stability compared to apo form. Since all -crystallins share high sequence identity, these studies clearly prove that all -crystallins are calcium-binding proteins with low to moderate affinity for the cation. These studies fill a missing gap in our understanding of the calcium induced cataractogenesis and the functional role played by -crystallins in general.Chapter 4: Calcium-binding properties of two single -crystallin domains from Yersinia pestis	Studies on -crystallins in the previous chapter indicated that -crystallins bind calcium. However, they lack any of the known calcium-binding motifs of EF-hand, annexin, C2 domain or gelsolin type. It is important to identify the calcium-binding motif of -crystallins because few more proteins of this superfamily are now known to bind calcium such as -crystallins, Proteins S and Spherulin 3a. I hypothesize that -crystallin superfamily is a novel calcium-binding superfamily and calcium binds at the Greek key motif. In order to establish this, I identified many new putative protein sequences containing this motif mostly from microbes described in the following chapters. From the genome sequence of Yersinia pestis, I identified a pair of paralogous proteins containing the characteristic Greek key motifs. I identified four Greek key motifs organized as two -crystallin domain in one of the proteins which I named as “Yersinia crystallin”. The nucleic acid sequence corresponding to these two domains were cloned into an expression vector and overexpressed in E. coli as single domain for the production of recombinant proteins. These isolated domains were named as D1 and D2. D1 was isolated from the soluble fraction whereas D2 was refolded and purified to homogeneity from inclusion bodies using on-column refolding technique for various biochemical and biophysical assays for calcium-binding. Sequence analysis of these domains revealed the unique nature of these proteins due to the presence of one type of Greek key motif combination AA and BB in D1 and D2, respectively. These proteins tested positive for calcium-binding using the conventional methods of Stains-all, terbium binding and calcium-45 overlay assays. I then looked at the conformational changes upon calcium-binding using florescence and CD spectroscopy. Surprisingly, these domains are intrinsically unstructured proteins (IUP) in apo form. Calcium addition changes the random coil content to -sheet conformation in D1, whereas D2 retains its predominantly unstructured nature even in holo form. Intrinsically unstructured nature of the proteins was further confirmed by analytical gel filtration. As expected for an IUP, apo D1 elutes at a volume higher than expected of a monomer whereas holo D1 behaves as a globular monomer. Even though apo D1 elutes at a higher volume, it was a monomer as seen by glutraldehyde crosslinking. Thus, these studies challenge the conventional notion that -crystallin domains are compact rigid globular proteins. These studies also indicate that minor sequence variations can affect the globular nature of these proteins. The only prevalent nature of these proteins is the calcium-binding property. It has been known for a long time that calcium regulates the virulence of Yersinia species which are pathogenic to humans and animals. In spite of several studies, presence of a calcium sensor has not been identified in Yersinia species. I suggest that yersinia crystallin is a potent candidate for such calcium sensing function because of the conformational transitions from unstructured form to beta-sheet upon binding calcium. However, further in vivo studies are required for deciphering the function of this protein in Yersinia species.Chapter 5: Calcium-binding and stability of an archaeal -crystallin domain from Methanosarcina acetivorans	In the earlier chapter 4, the detailed studies on pathogenic bacterial -crystallin domains have been described. In continuation of above studies, I have for the first time identified a -crystallin domain in the genome of Methanosarcina acetivorans, a methanogenic archaebacteria. I have named it as “M-crystallin”. Using a number of methods, I established that M-crystallin is a calcium-binding protein. Unlike Yersinia crystallin, calcium-binding does not cause significant changes in the secondary or tertiary structure as probed by CD and fluorescence spectroscopy. Each molecule binds two calcium ions with micromolar affinity as determined by isothermal titration calorimetry (ITC). However, calcium-binding significantly enhances the stability of the domain against chemical and thermal denaturation unlike its lens homologues. Its solution structure was solved both in apo and in holo form. This single domain calcium-binding protein shows high structural similarities to mammalian lens -crystallins from the lens. It is clear from these studies on the ancient homologue of mammalian -crystallins that even though both share high sequence similarity, their calcium-binding properties and effects are significantly different.  Since archaebacteria are thought to be the oldest of all living organisms on this planet, this domain could be a direct ancestor of lens -crystallin domains. Extrinsic stabilization of this protein by calcium could help this methanogen counter stress, which is usually found in extreme environmental conditions.Chapter 6: Calcium-binding to paralogous -crystallin domains from Caulobacter crescentus	Our studies on the diverse -crystallin domains from mammals, bacteria and archaea described in previous chapters clearly established that calcium-binding is a prevalent property of the proteins of this superfamily. To further validate our hypothesis that -crystallin domains bind calcium, I selected a pair of paralogous proteins from the genome of an eubacterium, Caulobacter crescentus.  Both proteins contain a single -crystallin domain in the C-terminal end and an unusually long N-terminal extension. These proteins named as “caulollin1 and caullolin2” were over expressed in E. coli. Full length caullolin2 was not expressed instead its domain part was selected for overexpression and purified. Each of these proteins binds two calcium ions with micromolar affinity. CD and fluorescence spectroscopy indicate that caulollin2 is partly unstructured in apo form and gains -sheet conformation upon binding calcium.  Analytical gel filtration also confirms this observation. Caullollin1 on the other hand shows no significant conformational changes upon binding calcium. Based on these results, it can be decidedly concluded that -crystallin is a novel calcium-binding superfamily.Chapter 7: Conclusions and future prospectus: -Crystallins is a novel calcium-binding superfamilyChapter 7 concludes the thesis work by emphasizing on novel findings of this work. This work identified and characterized several new and unique domains from organisms spanning extremes of the spectrum of life. Low affinity calcium-binding to mammalian -crystallins fills a vital missing gap in our understanding of calcium homeostasis, disturbances of which results in cataractogenesis. Of general importance is the calcium-binding motif in these proteins, which appears to have very few members. Identification of more members is boasted by the various genome sequencing projects, using which I identified several new and diverse members and characterized their calcium-binding properties. These include the -crystallin domains from a pathogenic bacterium, Yersinia pestis and the only known domain from an archaebacteria, Methanosarcina acetivorans. I have also for the first time characterized a pair of paralogous -crystallin domains from Caulobacter crescentus. Calcium plays an important role in structural stabilization and conformation of many of these domains. Based on this and the regulation exerted by calcium on the phenotype exhibited by some of these organisms, the possible physiological role of these proteins looks important. Although the exact role can further be ascertained by in vivo studies, the most important finding of these studies is that the function of -crystallin proteins irrespective of the source should be seen in the context of calcium. These results can be summarized as the discovery of a novel calcium-binding superfamily with crystallin type Greek key motif as the calcium-binding motif.In addition, this work has demonstrated that various domains exhibit different conformational and thermodynamic properties with respect to calcium-binding, which are likely due to the diversity of their sequences. Therefore, more structural studies need to be directed on the diverse members of -crystallin domains. This would add further knowledge to our understanding of the calcium related functions of these diverse proteins.

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