Physicochemical Properties Studies Of Platelet Integrin ?_?b?₃ And Associated Ligands Protein Tablysin-15 And CagL

The basis of multicellular life activities is cell adhesion,which is generally mediated by cell adhesion molecules?CAM?.Integrin,also known as integrins,is an important component of the family of cell adhesion molecules,composed of two subunits,? and ?.In this study,the integrin ?_?b?₃ has a high expression level on the surface of platelets.It is the main receptor of platelet membrane and responsible for mediating platelet adhesion and aggregation,participates in bidirectional signal transduction of platelet activation,and participates in thrombus formation.The arginine-glycine-aspartate?RGD?sequence polypeptide is a recognition site for attachment of the extracellular matrix to the integrin receptor,and thus this sequence is used to develop different integrin antagonists.Tablysin-15 and CagL are RGD sequence proteins with a certain conformation that specifically bind to cell adhesion receptors.Tablysin-15 is the key protein that extracted from the salivary glands of Tabanus.When the Tabanus is sucking blood,Tablysin-15 will be released around the mouthparts.Because of its high affinity with ?_?b?₃,it will bind to it and hindering blood coagulation around the mouthparts,which is conducive to Tabanus eating blood.CagL is a Helicobacter pylori outer membrane chaperone protein that interacts with integrins on the host cell membrane to exert its biological functions.At present,most of the research strategies of integrin antagonists focus on blocking the binding of physiological ligands to integrin,and by adjusting the binding of proteins containing RGD peptides to integrins,thereby regulating and affecting the function of integrins.Structural biology is a powerful technical means for determining the spatial structure and intermolecular interactions of biological macromolecules.Analyze the three-dimensional structure of integrin and its related proteins,also clarify the interaction between these proteins and their mechanisms,which will help people better understand the structural basis and molecular mechanism of integrin function.Develop new antithrombotic drugs or treatment of platelet insufficiency syndrome caused by ?_?b?₃ lesions,as well as thrombocytopenia,provide important theoretical basis and potential new drug targets.This project will use molecular biology and structural biology methods and heteronuclear multi-dimensional nuclear magnetic resonance structural determination technology to construct prokaryotic expression vectors of integrin ?_?b?₃ intracellular and transmembrane regions,and explore and optimize the expression and purification conditions of recombinants.Finally,a large number of stable,high-purity protein samples were successfully prepared.Tablysin-15 and CagL proteins were characterized by inclusion body renaturation and physicochemical properties using a variety of biophysical techniques.The one-dimensional ¹H spectrum of Tablysin-15 is near the chemical shift of 0 ppm.We see the sharp methyl peak,and with a broad distribution between chemical shifts of 6-8 ppm.It is presumed that protein folding is better and can be further passed NMR technical analysis of three-dimensional structures and study of their molecular dynamics.The one-dimensional ¹H spectrum of CagL protein has a sharp methyl peak near chemical shift of 0 ppm and a broad distribution between chemical shifts of 6-8 ppm,but the ¹H-15 N HSQC experiment shows that the peak is crowded in the middle.And the size of the peak is not uniform,indicating that the protein solution is easy to aggregate and the structure is not well folded.This means that the analysis of the three-dimensional structure by biological NMR technology also needs to continue to explore the purification conditions to help the protein fold correctly.Our results provide a basis for further clarification of the nuclear magnetic three-dimensional structure of Tablysin-15 and CagL proteins and their interaction with integrin ?_?b?₃.