Protein and RNA folding: from bulk towards high throughput single molecule experiments

high throughput fluorescence detection;protein folding;RNA folding;ribosomal signatures;molecular dynamics;thermodynamics and kinetics;temperature jump

Sarkar, Krishnarjun

关键词: high throughput fluorescence detection;    protein folding;    RNA folding;    ribosomal signatures;    molecular dynamics;    thermodynamics and kinetics;    temperature jump;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/16991/1_Sarkar_Krishnarjun.pdf?sequence=2&isAllowed=y

美国|英语

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

【 摘 要 】

The folding energy landscapes of RNA and proteins have been studied using experiments and molecular dynamics (MD) simulations. Firstly, the folding of an RNA hairpin has been looked at with the fluorescent probe 2-aminopurine. The effect of stem and loop dynamics have been separately analyzed. Motivated by MD simulations, a global four state energy landscape for both the stem and loop mutants was found to be an ideal fit to the observed experimental data. Secondly, kinetic and thermodynamic predictions based on very long MD simulations of a variant of the β-sheet WW domain protein Fip35, were experimentally verified to be true. This provided an atomistic detail understanding of the folding landscape and will act as a key benchmark inunraveling the protein folding problem. Thirdly, protein and RNA interactions were looked at with MD simulations of the ribosomal signatures S4 and h16 along with fluorescence experiments to characterize the nature of the binding interaction between them. The results point toward a fly casting mechanism whereby the presence of the h16 helps the S4 signature to adopt its structure. Finally, a design and implementation of a high throughput time correlated single photon counting experiment is presented. Drops of ~10 μm diameter are optically trapped and interrogated by a femtosecond probe laser beam. Fluorescence photons emitted by the sample inside the drop are collected in all 4π steradians with polarization sensitivity. The instrument performs close to the single molecule limit, illustrated by the detection of ~100 Cerulean molecules on average in each drop. Further modifications that would possibly allow high throughput single molecule detection and its corresponding implications are discussed.