Quantitative Investigation Of The Structure And Function In Peptide Deformylase Studied By X-ray Absorption Spectroscopy

Metalloprotein is a generic term that describes protein which combines metal ions as cofactors. Almost40%of all proteins available in Protein Data Bank are members of this category. Metalloproteins are involved in a wide range of basic biological processes that represent important chemical reactions for metabolism. These chemical reactions can be correlated to structural changes around the metal ions binding sites which are meanful to be precisely determined. X-ray absorption spectroscopy is a very powerful tool to determine the fine structure around a metal ion binding site with subatomic-resolution in particular in biological systems. Samples can be investigated in a variety of forms, such as single crystals, solutions in frozen or room-temperature or as proteins inserted in membranes. It is very important to characterize local structural information around the metal site in metalloproteins.Prion-related protein (PrP), a cell-surface copper-binding glycoprotein, is considered to be responsible for a number of transmissible spongiform encephalopathies (TSEs). The structural conversion of PrP from the normal cellular isoform (PrPC) to the post-translationally modified form (PrPSc) is thought to be relevant to Cu2+binding to histidine residues. Because of the structural characteristics of the rabbit prion protein (RaPrPC) itself, rabbits are one of the few mammalian species that appear to be resistant to TSEs. Here we determined the three-dimensional local structure around the C-terminal high-affinity copper-binding sites using x-ray absorption near-edge structure combined with ab initio calculations in the framework of the multiple-scattering (MS) theory. Result shows that two amino acid resides, Gln97and Met108, and two histidine residues, His95and His110, are involved in binding this copper(Ⅱ) ion.Although limited, this preliminary study may be useful to understand the roles of copper in prion conformation conversions, and of the molecular mechanisms of prion-involved diseases. To this pourpose, we purified four mutants of RaPrP, H95A, H110A, H139A, H186A, as well as hPrP to determine the binding affinity of copper ions to the wild type and mutant proteins by ITC. Also, the radii of RaPrP and hPrP were measured after adding copper ions using DLS. Meanwhile, We determined the three-dimensional fine structure of C-terminal high-affinity copper binding site in recombinant human prion91-231(hPrP91-231) by the same method as RaPrP. Results show that amino acid residues have closer distances from the copper ion comparing with that of the rabbit prion, in a good agreement with the results of ITC and DLS. This result may partly explain why different species have different immunities to TSE.Peptide deformylase (PDF) is a prokaryotic enzyme that catalyzes the deformylation of nascent peptides generated during protein synthesis. We investigated the local atomic environment of Zn2+/Co2+ion binding in the active site of LiPDF and EcPDF by X-ray absorption spectroscopy (XAS). Moreover, we monitored functional and structural changes of this protein at different pH values and with different metal ions via X-ray absorption near edge spectroscopy (XANES) and ab initio calculations. Results show that a correlation occurs between pH values/metal ions and enzymatic activity via the bond length of the nearest coordinating water (watl) to the metal ion. Combining experimental and theoretical data we may claim that PDF exhibits a high enzymatic activity when the watl is located within a limited range of distance (2.15-2.55A) with the metal ion, while a lower enzymatic activity occurs outside this values.