Correlations between dynamic motions and function in modular proteins.

摘要

The aim of my doctoral dissertation was to study how the structure and dynamics of domains in modular repeat proteins influence their protein binding functions. Two systems were chosen that had similar sizes and number of disulfide bonds, but distinct protein binding functions.; Modules of repeated domains are found in extracellular proteins like thrombomodulin, an anticoagulant cofactor that interacts with thrombin in the clotting cascade and inactivates it. Low density lipoprotein receptor-related protein (LRP1) is also a perfect example of a modular protein, and its domain architecture allows it to clear various ligands from the extracellular space, such as apolipoprotein E.; Chapter I introduces these two systems and their biological relevance. Chapter II starts with the dynamics study by NMR relaxation experiments of the wild type two EGF-like domain fragment from thrombomodulin, TMEGF45, showing some interesting features in the dynamics of the fifth domain. Chapter III compares the dynamics of the single domains TMEGF4 and TMEGF5 to the two domain fragment in order to point out how the dynamics of one domain is affected by the presence of the other showing the close interaction mediated by the key linker residue M388. Chapter IV explores the relationship between the function and dynamics of TMEGF45 by a study of mutations at the linker residue. An ordering of key thrombin binding residues proves to be important for function.; The last chapter, focuses on narrowing down the smallest fragment of LRP1 that will still bind apoE. The production and characterization of the different repeats as well as results from binding assays on different fragments are described. Finally chemical shift perturbation experiments that show which residues within the domains are affected upon binding of apoE to the three domain fragment.