Site-specific Isotope Labelling Of Protein Through Unnatural Amino Acid Incorporation And NMR Analysis

Proteins are the most important biomolecules in life,constituted by 20 kinds of natural amino acids and hold great varieties and functionalities.In the past few dacades,modification of proteins through unnatural amino acids(Uaa)incorporation become a popular strategy in protein science.With different Uaa modifications,we have a better understanding on protein structure and interactions,protein related kinetics and dynamics,and so on.There are chapters in this thesis.The synthesis approaches,classifications and incorporation are introduced in chapter one.Organic synthsis is an effective way to obtain Uaa with milligram to gram scale.Enzymatic synthesis with PTL and its' mutations of some tyrosine analogue Uaa are good supplementary approaches.According to the functions of Uaas,they are sorted as chemical reactive Uaa,post-translation modification Uaa,biophysical probe Uaa and metal chelating Uaa etc.Through description of previous reports,the importance of Uaas are carified.At Last,the incorporation methods of Uaas through residue-specific and site-specific into proteins are introduced.In chapter two,the application of photo-caged amino acids are descrided.This type of Uaa are natural amino acid(such as tyrosin,serine,lysine and cysteine)modified with UV-cleavable ptotecting groups(o-nitrobenzyl derivatives in most cases)on the reactive side chains.After genetically incorporated into proteins in either eukaryotic or prokaryotic cells to obtain caged proteins,the photolysis generates the natural amino acid thus natural proteins and restored the native protein functions.So that lots of biological process could be optically controlled.In the second part of this chapter,the site-specific incorporation development of tyrosine derivatived photo-sensitive Uaa oNBTyr was discussed.As tyrosin is an important residue for many ezyme and protein.oNBTyr could be of great value of tyrosin related protein function studys.In the third chapter,the approaches of nuclear magnetic resonance(NMR)for biological studys are briefly describe.But in some cases,the NMR signal analysis of only one residue is adequate,but the the crowd NMR spectra of fully labeled protein often represent assignment or analysis challenges.Aiming to simplified the NMR spectrum,we assume that photo-caged amino acid,as their advanced feature previously described,would be a good candicate for site-specific isotope labelling of protein.With improved chemical synthesis of 15N-oNBTyr(previously reported synthesis yield 21%,our yield 81%),we demonstrate a isotope labelling method for protein tyrosine site through 15N-oNBTyr incorporation of GB1 and PYL10.This method proved to be feasible and might be a good application example for other like-wise unnatural amino acids.In the forth chapter,the recent synthesis and applications of fluorescent unnatural amino acids(fUaa)are described,with special focus on the Anap development and synthesis of D,L-Anap.The synthesis method of D,L-Anap was reported by Peter Schultz in 2009.The synthesis pathway was complicated and the overall yield was low.Most important,the final product was a mixture of racemics.Here,we developed an enantiospecific synthesis rought for L-Anap.With 8 steps,2-naphthyl bromide and N-trityl-serine-methylester as starting material,we were able to obtain L-Anap with 15.3%yield.The key step is to take advantage of Mitsunobu reaction to attach the fluorophore with the side chain of a-amino acid.