Analysis Of The Anti-Aggregation Chaperone Mechanism And N-terminal Domain's Motion State Of Spy

Proteins need to maintain certain structures to function properly.Abnormal aggregation of proteins may lead to a series of diseases.Ubiquitous molecular chaperones in living organisms participate in maintaining protein homeostasis in several ways:assisting nascent polypeptide to fold,binding misfolded proteins to prevent their aggregation,and even disaggregating some proteins which have already formed deposits to help them refold.Therefore,molecular chaperones have become important therapeutic targets for neurodegenerative diseases and cancers.However,the understanding of molecular chaperones is still not deep enough.The mechanism of some molecular chaperones is very complicated,making it difficult to discovery the key factors that affect chaperone activity.Spy is a small,ATP-independent chaperone found in Escherichia coli and is an ideal model to study the working mechanisms of molecular chaperones.We aimed to illustrate how Spy exerts its chaperone activity in order to gain a deeper understanding of how other complex molecular chaperones work.This paper mainly involves three aspects.In the first part,we demonstrate that electrostatic interaction is an important factor affecting chaperone activity.From E.coli,we expressed and purified four mutants of Spy with enhanced positive charge on their concave surfaces.These mutants showed at least 1.9-fold increase of chaperone activity in vitro.Subsequently,we found that increased salt concentrations reduced the chaperone activity of these mutants,making them acting like the wild-type chaperone.In the end,we mutated the four sites into negatively charged glutamate and tested the mutants' chaperone activity in vitro.All the four glutamate-substituted mutants showed weaker chaperone activities.These results collectively demonstrated that electrostatic interaction is an important factor affecting Spy's chaperone activity.Previous studies have shown that Spy' s disordered N-terminal domain(no.1-28 residues)can promote substrate release through the D26 residue,but the mechanism of release is still unclear.In the second part of the research,using Paramagnetic Relaxation Enhancement(PRE)technique,we demonstrated that the disordered N-terminus of Spy forms an electrostatic interaction with its concave,substrate-binding surface.We also showed that this interaction can competitively promote the release of substrate.Additionally,by combining mutagenesis study with PRE experiments,we identified the D26-interacting sites on the concave surface of Spy,which are the K54,R55,and R89 residues.In the third part,we established an expression and purification method for Spy heterodimer,which lays a foundation for following studies on the dynamic motion of Spy's disordered N-terminal domain using single molecule fluorescence resonance energy transfer(smFRET).