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6th semester-2006 Project Proposal Laurent Duroux “Construction of plasmid vectors to tag proteins for universal light-induced protein immobilization on surfaces” Background: A method of light-induced immobilization of proteins(1,2) on chemically treated surfaces has been successfully developed over the past years in the group, by Teresa Petersen and colleagues. It essentially involves a chemical energy/electron transfer from a light-excited tryptophan (or tyrosine) residue to a neighbouring disulfide bridge. This results in the generation of highly reactive charged cysteine residue which immediately cross-link to free sulfhydryl groups. The method is currently used to generate nano-arrays of technologically relevant proteins such as immunoglobulins, for medical diagnosis. The main drawback of the technology is that it is only limited to these proteins presenting a triad of amino acids, Cys-Cys / Trp (or Tyr), close neighbours in 3D space and surface-exposed. Thus, it excludes a large number of potentially interesting proteins for future applications in nano-biotechnology. The goal of the project is to propose a solution to this limitation and to extend the concept developed in the group. Project description: The general aim of the project is to generate plasmid vectors for the production of tagged proteins, which would be immobilized via a light-induced method. The idea is to build a system for recombinant production of any protein with a “tag” at its N- or C-terminal end, which would allow for light-induced immobilization, irrespective of the structural features of the target protein. The concept of tag implicates that the peptide fused to the target protein should be as neutral as possible regarding its folding and its biological activity. To achieve this, the tag should be of limited size, typically no more than 30 amino-acid residues. The basic assumption is that the smaller the tag, the lesser the probability that it interacts with the target protein. Of course, this peptide tag will have to carry the essential CysCys / Trp triad necessary for light-induced immobilization. This is where the first (and most likely main) challenge resides in this project. Here, the target protein used as model would be a fatty-acid binding protein (FABP) from human(3), for which we have a good deal of practical expertise. Proposed strategy /methods: (1) Designing the N-term (and/or C-term) peptide with the Cys-Cys / Trp triad. The most likely secure route to achieve success is (again) to observe nature’s ways. A number of dedicated specific databases for disulfide bridges already exist(4, 5, 6) and should be used as a starting point. In particular, a class of peptides called “Knottin”(7) appear to be suited for the purpose (http://knottin.cbs.cnrs.fr/Main.html), see figures below. Figure: Disulfide connectivity in the Knottin family Figure: 3D model of a typical Knottin (2) Design of PCR primers for molecular cloning of the peptide(s) into a plasmid vector (pET series), Fusion to hb-FABP. Recombinant expression in E. coli. (3) Protein purification, stability analysis (CD, fluorescence), activity analysis (fluorescence) (4) Immobilization to functionally activated glass plates (-SH chemistry), light treatment (laser) (5) Structural analysis of the chimera protein (NMR) References: (1) Neves-Petersen MT, Gryczynski Z, Lakowicz J, Fojan P, Pedersen S, Petersen E & Bjorn Petersen S. 2002. High probability of disrupting a disulphide bridge mediated by an endogenous excited tryptophan residue. Protein Sci. 11:588-600. (2) Snabe T, Roder GA, Neves-Petersen MT, Buus S & Petersen SB. 2005. Oriented coupling of major histocompatibility complex (MHC) to sensor surfaces using light assisted immobilisation technology. Biosens. Bioelectron. In press. (3) Balendiran, G. K.; Schnutgen, F.; Scapin, G.; Borchers, T.; Xhong, N.; Lim, K.; Godbout, R.; Spener, F. & Sacchettini, J. C. 2000. Crystal structure and thermodynamic analysis of human brain fatty acid-binding protein. J. Biol. Chem. 275:27045-27054. (4) Herman W. T. van Vlijmen, Abhas Gupta, Lakshmi S. Narasimhan & Juswinder Singh. 2003. A Novel Database of Disulfide Patterns and its Application to the Discovery of Distantly Related Homologs. J. Mol. Biol. 335:1083–1092. (5) http://research.i2r.a-star.edu.sg/CysView/ (6) http://www.ncbs.res.in/~faculty/mini/dsdbase/dsdbase.html (7) Gelly JC, Gracy J, Kaas Q, Le-Nguyen D, Heitz A & Chiche L. 2004. The KNOTTIN website and database: a new information system dedicated to the knottin scaffold. Nucleic Acids Res. 32:D156-159. Important note: the project is ambitious and could be important for the scientific development of the group. It is not the purpose in this project to achieve all mentioned points (6 th semester is limited in time), but rather to lay the fundament of an overall project that will be hopefully followed-up. The different points mentioned in the strategy are there to outline the overall project (beyond this sole 6th semester). For example, it is obvious that more proteins will have to be included to “prove” the “universality” of the concept.