Developing Novel Fluorescent Probes And Paramagnetic Probes For Characterizing Protein Dynamics And Functions

Proteins which are involved in nearly all the physiological process in organisms,are the main participants in life process.The dynamics personalities of proteins play key roles in realizing their functions,so obtaining high-resolution structures and dynamics information are important for understanding the underlying mechanism.Nuclear magnetic resonance spectroscopy and fluorescence spectroscopy are currently the main experimental tools to investigate protein structural dynamics.The fluorescence resonance energy transfer(FRET)is the common technique based on fluorescence spectroscopy that could be applied to obtain distance restraints.Because the FRET efficiency is R6 dependent on the distance between the FRET donor and accepter,the distance information could be obtained.While protein could not fluorescent itself,fluorescence labeling must be introduced first.For labeling protein in living cells,the common way is to fusion fluorescent protein in its terminals,while due to the large size of fluorescent protein,usually 27 kDa,may disrupt the structure and function of the target protein.Another problem is that the maturation of fused protein may take hours,thus it prevent rapid labeling of the target protein.Therefore it is necessary to develop fast fluorescent labeling methods that minimize the influence on the structure and function of the protein.Based on the dipolar-dipolar interactions between unpaired electron and nuclei,the paramagnetic effects of a paramagnetic center caused on protein include paramagnetic relaxation enhancement(PRE),pseudo-contact chemical shift(PCS).With those paramagnetic effects,plenty of geometric information could be obtained to investigate protein structure and dynamics studies.Similar to FRET,the first step for taking such experiments is to site specifically label the target protein using a paramagnetic tag.The rigidity of tag directly determines the resolution of the calculated structure,so it is particularly important to develop rigid paramagnetic probes.In the development of fluorescent probes,we designed a novel cell surface protein labeling scheme based on the split-GFP strategy.From Analyzing the barrel structure composed of eleven ? strand,we split GFP into two parts.With The 10th and 11th ? strand fused into loop region of target protein,the fluorescent labeling could be fast realized by complemented with separately prepared GFP 1-9,which contain the mature chromophore of GFP.By using this novel labeling scheme,we investigated the internalization of two G protein coupled receptor,GPR17 and CysLT2R on living cells,and we found that these two GPCRs both internalized with time,migrate from cell membrane to cytoplasm.In the development of paramagnetic probes,we designed two tags,DTTA-C3 and DTTA-C4,which could be site-specifically introduced into protein via copper catalyzed azide-alkyne cycloaddition.These two tags are highly symmetric and rigid.The rigidity was optimized by change the number of carbon atoms between lanthanide metal chelating groups and the reacting group.By incorporate a unnatural amino acid(UAA)with azide group through codon expansion and azide-alkyne cycloaddition,these tags could be site specifically conjugated to protein regardless of surface exposure cysteine.We introduced the new lanthanoid tags to ubiquitin and a cysteine containing protein EIIB,and characterized the properties of the two tags,we found that DTTA-C3 tag has the best rigidity relative to all the UAA-conjugating paramagnetic tags that have been reported.Using the constraints provided by intermolecular PCSs,we calculated the structure of ubiqutin-UBAl transient protein complex.Furthermore,we studied the dynamics of RSV(Rous Sarcoma Virus)capsid protein with joint use of NMR pseudo-contact shift(PCS)and single molecule FRET(smFRET).smFRET provides population information of each species,PCS provides inter-domain constraints,with these complementary constraints we try to calculate the two states ensemble structure of RSV capsid protein.Also,we investigate the structural basis of the two states and its role in capsid assembly.In summary,the major focus of this thesis is to develop fluorescent and paramagnetic probes,and using these tools to investigate protein dynamics and functions.This work expanded the repertoire of fluorescence and paramagnetic tags that can be used to evaluate the dynamic properties of proteins.The joint use of smFRET and paraNMR on investigating RSV capsid dynamics may put insight into the mechanism of retrovirus capsid assembly.