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State-of-the-art Protein Structural Biology 25 and its modification using molecular dynamics to yield a class of rationally modified mutant proteins which is followed by site-specific mutagenesis to synthesize the mutant proteins. In our research on the structural biology of proteins we have successfully exploited such an integrated, hollistic approach which draws upon the salient features of both methodologíes and its symbiosis in order to derive the 3D structures of proteins and hence is able to maximize the structural information content. The approach is illustrated schematically in the flow sheet, shown in Fig. l. A number of polypeptide and protein structures upto M r = ~21 kDa have been solved using such an integrated approach. The structure of a Ca ++ sequestering protein, amelogenin, which plays a critical role in developmental biology of mammalian tooth is illustrated here as an example. The method outlined is generic and is applicable to other protein systems. 2. Bovine Amelogenin Fig. 1. Flow sheet illustrating a hollistic, integrated approach for deriving the 3D structure of a protein. teoglycans, lipoproteins, NMR remains the only avenue to determine the 3D structure in solution. The gap between known primary structures of proteins and their 3D structures is continuously increasing and hence there is an urgent need to reduce the time span in the derivation of the 3D structures of proteins. There has been a phenomenal increase in the knowledge of the protein primary structures i.e. the amino acid sequences, due to rapid advances in the gas-phase microsequencing and the derivation of the primary structure from their cDNA sequences. The secondary and tertiary structures of a few thousand proteins are known presently from X-ray and nuclear magnetic resonance (NMR) studies (Protein Data Bank, Brookhaven, New York, NY and European Molecular Biology Laboratory, Heidelberg, Germany) and therefore the gap between the two continues to increase significantly. A multi-pronged approach in the elucidation of the 3D structures offers much promise than any one method. We have recognized from the beginning that no one single physical method in structural biology can provide a complete picture of protein structure and dynamics. Our ultimate goal is to develop rationally modified mutant proteins with enhanced function and explore their potential applications in biomedical, material science, electronics (Renugopalakrishnan et al., a series of US Patents pending) and in technological areas. A rational modification of a protein begins with the experimentally derived 3D structure of the wild-type protein as the first step Amelogenin, a ~19 kD extracellular hydrophobic, phosphoprotein with an overall ellipsoidal shape (laser light seaterring studies from the author’s Harvard laboratory, unpublished), secreted by ameloblasts, from bovine tooth enamel contains a primary sequence which is unique in comparison to other known primary structures of mammalian proteins (Personal Communication, Hunt, 1989). It is characterized by a large proportion of Leu, His, Met, and Pro residues and contains a Fig. 2. A Comparison of the primary structure of bovine amelogenin (Takagi et al., 1983) with the primary structures of mouse, rat, pig, and human.
26 Rev. Soc. Quím. Méx. Vol. 43, Núm. 1 (1999) V. Renugopalakrishnan et al. single phosphorylated Ser l6 residue. A comparison of the amino acid sequence, i.e. the primary structure, of bovine amelogenin [5], with amelogenins from different species is shown in Fig. 2. Amelogenin structure function studies have been the focus of research at our Harvard Laboratory since 1984 [6]. Amelogein mutants and synthetic fragments have potential clinical use in fighting Amelogenisis imperfecta (AI), a dental disease prevalent in Mexico. 3. Commonly Occurring Secondary Structural Motifs in Proteins When the structural studies began in 1984, its primary structure was not known. Early structural studies of amelogenin were frustrating since the CD (Circular Dichroism) spectral features were not reminscent of proteins containing α-helical and/or β-sheet patterns [7]. The primary structure of calf amelogenin was derived by Edman degradation gas phase sequencing [5] and later from its cDNA sequence. The agreement between the two methods are excellent. Under normal circumstances, amelogenin would have been labelled a protein with a “random” structure e.g. lacking recognizable order. The intrepretation of its spectral features took an unexpected and surprising turn when its primary structure became availabIe [5] and it was found to contain unusual tandem repeats, especially starting from residues Gln 112 and extending upto Leu l38 and the repetitive tandem template being a tripeptide sequence, (Gin-Pro-X) and such contiguous triplets occurred scattered throughout its primary structure. Amelognin is not the only protein in nature to contain such tandem repeats, albeit, the triplet template occurring nine times consecutively suggested that repeating polypeptide segment is probably composed of a helical array of repetitive β-turns, hairpin or U- turns wound around a common helical axis. Similar tandem repeats, have also been found to occur in elastin, RNA Polymerase II and it remains to be determined whether tandem repeats serve important structural and hence functional roles. The tandem repeats in elastin, [8, 9], have been ascribed a role in its elastomeric property, like in connective tissues in lungs, where the increase in volume during respiratory cycle requires a protein framework that can quite easily expand and contract while the tandem repeats in other proteins may have other important biological functions In the case of amelogenin, the major functional role is triggering Ca ++ ion accumulation in the process of building hydroxyapatite e.g. octacalcium phosphate crystals in enamel. The well organizad mineral associated with enamel has been strikingly demonstrated by recent atomic force microscopic studies of normal and Amelogenesis imperfecta afflicted human tooth enamel from our laboratories [10]. When one examines the amelogenin primary structure, it is rather surprising that such a primary structural entity will ever be implicated in a hydroxyapatite build up process on a bewildering time scale e.g. the entire process of mineral accumulation is complete in the very early phase of mammalian tooth development and amelogenin is rapidly degraded enzymatically after initiating mineralízation of enamel. 4. Time-Resolved Picosecond Fluorescence Studies of Bovine Amelogenin We have inferred from the three different decay times, τ, observed from pico-second fluorescene spectroscopic studies of bovine amelogenin in solution [11], that either amelogenin manifests three conformers equilibrating rapidly in aqueous solution or the three Trp residues present in its primary structure (Fig. 2) are on the surface of amelogenin, contributing to the three different decay times, τ. 5. Circular Dichroism (CD) Spectroscopic Studies of Bovine Amelogenin Initial CD studies were indicative of a pattern similar to “random coil” proteins, [12] CD spectroscopy provides qualitative information on the secondary structure of a protein. CD bands arise from n – π and π – π* transitions in the electronic structure of a peptide moiety. CD spectral features of multiplet β- turn structures remain obscure even today. Furthermore, CD patterns of a β-sheet and β-turn protein with a large β-turn contribution are not available in the literature with the exception of tandem repeating segments in cereal storage proteins, tropoelastin, and RNAse polymerase II. The CD spectra of mouse amelogenin, a 179 residue hydrophobic protein a recombinant manifests a temperature sensitive CD spectra in 10 mM phosphate buffer which resembles the CD spectra of tropoelastin polypeptides [11]. 6. Infrared Red (IR) and Raman Spectroscopic Studies of Bovine Amelogenin Neverthless, CD studies were contradicted by FT-IR studies [12], and laser Raman studies from our Harvard laboratory [13], which were suggestive of a β-turn and low β-sheet segments with a low α-helical content. Amide I mode usually occurs between 1600 and 1690 cm –1 and is largely C=O stretching frequency, amide II mode consists of C–N stretching and NH bending, occurring between 1480 and 1575 cm –1 , and amide III mode, occurring between 1229 and 1301 cm –1 is similar to amide II mode. Amide II mode is sometimes, not always, absent in the Raman spectra of proteins. The amide I region of the FT-IR spectra of amelogenin as a function of pH [12] was subjected to Fourier self-deconvolution which revealed the sub-structures contributing to the broad amide I band. Fourier self-deconvolution of amide I band showed that the conformation features present in amelogenín were dominated by β-sheet and β-turn segments. Raman spectrum of amelogenin in aqueous solution [l3] revealed a quadruplet
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