Expression、Purification And Folding Study Of The PGK And Knotted Protein YibK、YbeA

A growing number of proteins containing topological knots have been identified over thelast few decades.But how such proteins fold remains a mystery.It is thought impossible that apolypeptide chain could “knot” itself to form a functional protein. Their formation does not fitany current folding models or mechanisms, and therefore represents an important piece of theprotein-folding puzzle. YibK and YbeA belong to the SPOUT class of methyltransferases.Proteins in this group all display a unique topological feature; the backbone polypeptide chainfolds to form a deep trefoil knot. Their knotted structure as compared to some complicatedknotted proteins, are relatively simple, and their molecular weight are moderate, makingthem an ideal candidate for study. We have established the stability of them with chemicaldenaturation by measuring fluorescence and CD. Thermodenaturation was also carried out ona CD instrument. Meanwhile we labeled the protein through the cysteine residues with AlexaFluor488and Alexa Fluor647as the donor and acceptor fluorophores，investigating thefolding pathway by using single-molecule fluorescence resonance energy transfer (smFRET).PGK (Phosphoglycerate kinase) is not only a key enzyme in glycolysis but also anecessary enzyme for each organism to live. The deficiency of this enzyme can causeorganisms metabolism dysfunction.It mainly consists of two spherical domains.Uponcombination with substrates it takes a significant conformational change and realizes itscatalysis functions. Study on the structure, function and folding of PGK is very important fordiscovering the folding mechanism of proteins and for understanding how PGK realizes itsfunctions, which would be necessary for rational protein engineering. In this thesis, we haveestablished a way for the expression of PGK in E. Coli and following purification. PGK wascharacterized systematically, including:(1)PGK was studied with circular dichroism (CD) andthe CD spectrum showed typical pattern for-helix, which was consistent with its structurereported；(2)Thermodenaturation was also carried out in the presence and absence of Al3+on a CD instrument.(3)We investigate the folding pathway by using single-molecule fluorescenceresonance energy transfer (smFRET).