Toward probing the protein folding kinetics using the nanosecond time-resolved temperature-jump technique

Abstract

Understanding the protein folding mechanism has been the interesting problem through biology, physics, medicine, and chemistry. To unravel a lot of questions about such folding events, the diverse probing tools like as NMR, IR, and Raman spectroscopy were tried with the complete proteins like the barstar, engrailed homeodomain, and β-hairpin fragment. ‘Mixing’ of the denatured protein and buffer favoring folding was the first technique and allowed the diverse combinations with the probe ways. It took, however some mili- to micro-seconds for mixing and limited the observation of the initiation stage of folding. So, a faster technique was required and laser-induced temperature-jump (LIT) experiment was introduced. As a non-mixing way, LIT initiates the state change by the sudden temperature-jump by laser so that it gives the better time-resolution of several nanoseconds and completes the folding process within a few microseconds. After LIT was first done by Flynn et al., 1972, many researchers including Hofrichter, Eaton, Fersht, Gruebele, and Hagen have been going on the active studies with this technique. Moreover, the de novo designed protein sample, Trp-cage reported by Neidigh et al. led to the meaningful result, the cooperative faster folding within 4μs. This time exceeds the previously reported folding rates of other proteins. With this small peptide, more researches have been developed, like as varying the solution viscosity. Now, my study is on the way for the protein folding kinetics by observing the fluorescence intensity and lifetime of Trp-cage and its applied peptide sample using laser-induced temperature-jump. Here, the setup section will be described in detail, as showing my state-of-the-art for this study. Based on this layout, the folding kinetics of Trp-cage by LIT experiment will be demonstrated with the tryptophan fluorescence intensity and lifetime. In addition, Trp-cage attached to gold particle will be fluoresced to investigate the effect...