Fast Mixing Reveals Two-state Folding of Apomyoglobin Público

Burke, Kelly Suzanne (2012)

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Fast Mixing Reveals Two-state Folding of Apomyoglobin
By Kelly Burke

The development of a novel fast mixer and its application to the study of protein folding is described. Construction of the mixer is simple and inexpensive, requiring no specialized equipment or techniques. The mixer is designed to achieve three-dimensional (3D), hydrodynamic focusing of the sample by a surrounding sheath solution. This focusing promotes rapid diffusional mixing between the two solutions. To characterize the mixer's performance, a sample flow rate calibration method utilizing the fluorescence decay of excited europium (Eu) microspheres was conducted. The achievable diffusional mixing times were also determined by observing the fluorescence quenching of fluorescein experiencing a rapid pH drop within the mixer. Mixing times as low as 111 μs were recorded. The mixer was then used to study the folding kinetics of apomyoglobin (apoMb) undergoing pH changes in the absence of buffer. Previous studies have demonstrated that apoMb populates four unique conformations -native, intermediate, extended, and unfolded - during its folding pathway. Under the conditions of this experiment, however, a two-state folding mechanism was observed. This suggests that these experimental conditions alter the relative energies of apoMb's conformations such that only two of them are appreciably populated. It is possible that apoMb follows a folding pathway in which it reaches its lowest-energy native state without being trapped in relatively low-energy intermediate states.

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Table of Contents

Materials and Methods...9
Results and Discussion...22

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