DESIGN AND SYNTHESIS OF ARTIFICIAL METALLOPROTEINS

Abstract

Natural proteins are super molecules with excellent structures and functions that cannot be realized by non-proteinaceous compounds. Several groups are working in the development of new methodology to design artificial proteins with native-like properties and de novo protein design is an important approach to elucidate the principles of protein structure and function. Metalloprotein account for about one-third of structurally characterized proteins and about one-half of all protein. They play important roles in biological processes whether it is in catalysis or molecular recognition. Metalloproteins are very efficient and selective in their function and only few other natural molecules have this type of quality. For this reason, the study of protein structure and function has been the focus of many years of research. Natural proteins structure is much obscured generally. However, closer look at native metalloprotein reveals that they use only a small subunit of ligands and of metal containing prosthetic groups for example, there are only 20 natural amino acids, less than half of which are capable of coordinating to metal ions and the number of metal-containing factors is also limited. It is the matter of curiosity why the structural uniqueness of metalloprotein is essential for its biological activity. Peoples are using the field of metalloprotein designing to minimize structural complexity and to maximize visualization of functional relationship with it. The design of metalloproteins provides an attractive approach to test the essential features required for folding, electron transfer and catalysis. The artificial proteins can be designed either through the introduction of novel metal binding sites into naturally occurring proteins or through de novo protein design. De novo protein design is an essential approach to elucidate the principles of protein structure and has potential applications to produce novel molecules for medical and industrial purposes. This method has also been used by several workers for the design and synthesis of various artificial heme proteins as the analogues for cytochromes and myoglobins. Costanzo et al. reported the de novo design and synthesis of catalytically active helix bundle by engineering an active site cavity into DF1 that can coordinate non active site Mn(II) ions. Schnepf et al. reported de novo design and characterization of copper centers in synthetic four helix bundle proteins. De novo proteins with zinc bindiing sites are also available in literature. Due to recent interest in metalloprotein design, several model proteins using peptides and apoprotein with manganese, iron and copper have been prepared and their spectroscopic and catalytic properties have been studied in the present thesis. For the sake of the convenience, the work embodied in the thesis is presented in the following chapter: The first chapter of the thesis is the general introduction and present an up to date and systematic survey of literature to artificial design of metalloproteins. The different kind of metalloproteins related to the present research have been posed in the context of the cited work. The design and synthesis of de novo peptide for managnese binding are presented in chapter two. The de novo peptide with 63-residues (MHB) has been synthesized biochemically by using E. coll In new peptide, MHB, the leucine of the prototype peptide dAl was replaced by His27 and Asp41 to provide the binding site for Mn(II) ion. The binding of Mn(II) ion was studied by electron paramagnetic resonance (EPR) spectroscopy and was found that the MHB contains binding site for Mn(II) ion with KD value of about 36.10 \xM. The circular dichroism (CD) studies indicated that there is no change in helical contents of the secondary structure evenafter the binding of Mn(II) ion with peptide. The SOD activity of Mn-peptide complex was tested using NBT method and the IC50 values was found to be 8.08 j^M. The chapter three deals with the construction of artificial cytochrome monooxygenases on the basis of P450 BM-3 from Bacillus megaterium and their catalytic activity. Artificial cytochrome P450 MB-3s were prepared by replacing the native heme with various 2, 4-substituted hemes: proto-, meso-, deutero- and diacetlydeuteroheme. The natural heme was removed from the hemedomainof cytochrome P450 BM-3 by treatment with 1.0 N HC1 and it was found that under carefully controlled conditions, heme can be incorporated into the apoprotein to obtain a fully reconstituted heme domain with spectroscopic, substrate-binding and catalytic properties very similar to the native heme domain. The absorption spectra of the artificial BM-3 containg meso-, deutero-, and diacetyldeutero- were similar to those native P450 BM-3 except for shift in absorption maxima and variation in the ration of low spin/ high spin. In unnatural BM-3, the monooxygenase activity first increased and then decreased as the electron withdrawing capacity decreased; the protoheme-containing BM-3 showed the highest, while diacetyl-, deuteron- and meso-heme containing enzymes had considerably lower catalytic activities. The oxidation of meristic acid with NADPH were tested with meso-, deutero-, proto- and in diacetyl deutero-heme substituted heme domain and product formation were observed with reasonable turnover numbers. The chapter four deals with the synthesis of HisAibGly (tripeptide), HisGlyGlyTrp (tetrapeptide) and their complexation with copper (II). The interaction of these peptides with Cu(II) were studied by electron spray ionization-mass spectrometry (ESI-MS), circular dichroism (CD), absorption (UV-Vis) and electron paramagnetic resonance (EPR) spectroscopic methods. The formation of different species in aqueous solution were studied by potentiometric in conjunction with spectrophotometric method and studies were performed at 25 + 0.1 °C with constant ionic strength (u. = 0.1 MNaNOs) using Bjerrum- Calvin's pH-titration technique as adopted by Irving and Rossotti for binary systems. The species distribution curves indicated that the complexation occurred from 3-11 pH and three nitrogen coordinated species predominates at 8-9 whereas the four nitrogen coordinated species was formed in between pH 9-11. The resultant copper-peptide complex was tested for SOD activity using xanthine-xanthine oxidase - nitroblue tetrazolium (NBT) methods and was found that the complex has SOD activity with the IC50 value of 1.07 uM. DNA cleavage by copper (II)-GlyAibHis, a tripeptide complex based on ATCUN peptide motifs is described in chapter five of the thesis. Development of new chemical nucleases is a matter of great concern because of their extensive use in biotechnology and as therapeutic agents. The ATCUN (amino terminal Cu(II) and Ni(II) binding) are the peptide motifs that occur naturally in the serum albumins. The similar peptide motif (GlyAibHis) having unnatural amino acid Aib (a-aminoisobutyric acid) was synthesized iv and its Cu(II) complex was characterized by spectrophotometry and ESI-MS studies. The Cu(II)-GlyAibHis shows the DNA cleavage only in presence of mild oxidizing agents like ascorbate by the oxidative mechanismrather than hydrolytic and it was found to follow the pseudo first order kinetics (Arobs = 0.085 min"1). Non hydrolytic mechanism was further supported by the hydrolysis of pNPP which followed the pseudo first order kinetics (A:obs = 1.98x10"2 min"1) with negligible pH effect.