Structure And Function Of Proteins CGL2373 And TGIF1 Based On Nuclear Magnetic Resonance Spectroscopy

Protein carries out its biological function in cell,which is determined by its structure.In the living biological cell,protein function means many levels,including recognition of client ligands,catalyzation,switching for regulation,and structural elements.Accordingly,protein structures are various,existing as either structure domain with stable folding,or intrinsic disorder protein?IDP?.Investigations on protein structure-function relationship remain current hot spot in the research fields of biochemistry,molecular biology and structure biology.On one hand,protein structure study can lead to prediction and confirmation of its function;on the other hand,protein function study can benefit further understanding of its structure features.Nuclear Magnetic Resonance?NMR?spectroscopy is a powerful tool used for studying protein structure and its interaction with other biological biomolecules,especially for proteins in solution.In this thesis,protein CGL2373 from Corynebacterium glutamicum?predicted to be structurally folding?and human protein TGIF1-RD2?predicted to be an IDP?are picked up as study targets for their structure and function,using NMR spectroscopy.Our research details and results are as follows:1. Study on the structure and function of protein CGL2373 from Corynebacterium glutamicum.Through gene sequence determination and comparison,Bioinformatics study indicates that protein CGL2373 belongs to a big protein family Bet v-1,which mainly plays roles in cellular stress responses.After successful design of plasmid construct,recombinant protein CGL2373 were expressed and purified.Then,a series of 2D and 3D NMR spectra were collected on the ¹⁵N-¹³C sample for protein structure determination.Solution NMR structure of CGL2373 consists of three?-helices,one 3¹⁰ helix and seven?-strands,while a large hydrophobic cavity forms in the middle.Structure similarity analysis using Dali 8 showed that CGL2373 exhibits highly structural similarity to two protein families-ABA receptor protein and polyketide cyclase family.Further comparison by structure details showed that CGL2373might belong to the family of polyketide cyclase,but it is not a typical polyketide cyclase protein,a typical class,which needs further experimental evidence.2. Study on the structure and function of the RD2 domain from human protein TGIF1.The transforming growth-interacting factor,TGIF1 is an essential regulatory protein for cell differentiation,which plays an important role in fetal facial and brain development,hematopoiesis,bone formation and energy metabolism.Our group previous investigation has accomplished the structure-function relationship of TGIF1 HD domain,which functions on specific DNA recognition.In this study,further study is focused on RD2 domain,which plays a role in interacting with many client proteins.Protein structure prediction based on amino acid sequence indicates that RD2 could be an intrinsic disorder protein.By recombinant protein technique,TGIF1-RD2?256-375?protein sample was successfully obtained from E.coli expression strains.NMR resonance assignments for backbone and side chain atoms were finished,which further confirmed that this domain belongs to IDP based on the distribution of chemical shifts.In addition,previous in-cell studies revealed that TGF-?signaling protein Smad2 interacts with TGIF1-RD2 through its MH2 domain,and inhibits the signal transmission of TGF-?signal pathway in vivo.However,in this thesis,many experiments including NMR titration,GST pull down,Co-IP and Y2H,were carried out,and no direct interaction was found between TGIF1-RD2 and Smad2-MH2 under the conditions,which suggests that other unknown biomolecules may be involved into mediating the interaction in vivo.In this study,new insights have been provided on the structure-function relations of protein CGL2373 from Corynebacterium glutamicum and human protein TGIF1-RD2,Nevertheless,further investigations are still needed to fully understand their function mechanisms.