Protein folding and diffusion: from in vitro to live cells

protein folding;fluorescence imaging;temperature jump;anomalous diffusion;single molecule

Guo, Minghao

关键词: protein folding;    fluorescence imaging;    temperature jump;    anomalous diffusion;    single molecule;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/46611/Minghao_Guo.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Protein folding landscapes and protein-protein interaction landscapes are subject to modulation by many factors inside living cells: crowding, electrostatics, hydrophobic interactions, and even hydrodynamic phenomena.The resulting spatio-temporal fluctuations in protein folding rates, protein stability, protein diffusion, as well as protein structure, protein function, and protein interactions are the subject of a recent generation of experiments, as well as in-cell modeling.We discussed some specific examples of how the cell modulates protein folding kinetics and diffusion. We also provide a hidden Markov model for studying the folding and binding of the single- and few-molecule experiment. In the first chapter, we measured the stability and folding rate of a mutant of the enzyme phosphoglycerate kinase (PGK) in living cells as a function of temperature. To facilitate measurement in individual living cells, we developed a rapid laser temperature stepping method capable of measuring complete thermal melts and kinetic traces in about two min.We compared the temperature dependence of folding kinetics in vitro and in vivo. The temperature-dependent crowding, local viscosity, or hydrophobicity are included in the effective two-state model.In the second chapter, we measured the protein diffusion in living cells with fluorescence loss in photobleaching experiment. The temperature dependence of diffusion of proteins with different sizes, stabilities are compared. We studied the anomalous diffusion and microenvironment fluctuation in the cell by comparing the experimental and simulation results. In the third chapter, we discussed a hidden Markov model that can be applied to single- and few-molecule experiment to study the protein folding and binding kinetics. The time resolved fluorescence spectroscopy allows us to study the fluctuations in fluorescence lifetime and fluorescence anisotropy during the folding and binding transitions.