Application Of Unnatural Amino Acid ONBY In Protein Structure And Function Studies

It is well known that proteins are playing essential functional roles in living organisms.Unnatural amino acid(UAA)was reported to be site-specifically incorporated into proteins and through their special chemical or spectroscopy properties,UAA labeled proteins have become a preferred method to biological studies,which is superior than traditional ways in solving biological problems.The application of a photo-sensitive unnatural amino acid O-(2-nitrobenzyl)-L-tyrosineis will be described in this thesis.In Chapter one,the UAA labelling for proteins and its applications are introduced.Besides natural 20 amino acids that constitute natural proteins,UAAs are chemically synthesized with desired functional groups.Then,the site-specific incorporation of unnatural amino acids will provide an important tool for protein structural and functional studies.There are several ways to incorporate UAAs into proteins.Genetic code expansion is the most common way to incorporate unnatural amino acids into proteins in site-specific manner.In this way,an aminoacyl-tRNA synthetase/tRNA pair is needed to specifically insert the unnatural amino acid in response to an amber stop codon(TAG).The amber stop codon is located at a defined site in DNA of interest.Native chemical ligation will take an active part in the site-specific incorporation.Unnatural amino acids can also be incorporated residue-specifically throughout the proteome by selective pressure incorporation.The only difference is this will lead to an overall incorporation of unnatural amino acids,rather than site-specific incorporation.In Chapter two,the method optimization for chemical synthesis and experimental conditions of ONBY are introduced.Among natural 20 amino acids,tyrosine residues play important roles in protein function.Thus,tyrosine has been intensively studied.ONBY is a tyrosine derivative,and it was first synthesized and applied for neuron ion channel investigation by Henry A.Lester in 1988 with low synthetic yield around 20%.After light stimulation,ONBY in aqueous solution is decomposed to tyrosine and water-insoluble o-nitrobenzaldehyde.These make ONBY an important tool in regulating protein function.Despite of such important function,the synthetic yield of ONBY is quite low.Then we tried to optimize the ONBY synthetic approach to improve its final yield.Then,we will investigate the molecular optical disassemble property of ONBY,which might be useful for following protein studies.In Chapter 3,ONBY is incorporated into a water-soluble protein and a membrane protein.A series of experiment such as protein expression,purification,light-irradiation and NMR detection are described in details.Then site-specific incorporation of ONBY was implemented.In Chapter 4,the ONBY related experiment and the prospect of ONBY application are summarized.In previous structural-biology studies,it is focused on protein expression,extraction and purification to investigate protein structure,function and kinetic process.Site-specifically introduce ONBY into proteins provide an easy,harmless and reversible way for protein labeling.Combined with NMR detection,in situ observation and detection of protein study could be conducted.The combinations of ONBY incorporation into proteins and other detection methods will become a powerful tool for protein studies.