PO01

Refolding of Lysozyme by Quasi-static and Direct Dilution Reaction Paths:

A First-Order-Like State Transition

 

Xu-Cheng Yeh1,2 , Hui-Ting Lee1, Po-Yen Lin1,2, and Lou-Sing Kan2* ,Chia-Ching Chang1*

1: Department of Physics, National Dong Hwa University, Hualien, Taiwan 97401

2: Institute of Chemistry, Academia Sinica, Nankang, Taipei, Taiwan 11529

1, Sec 2, Da-Hsueh Rd., Shou-Feng, Hualien, 974 Taiwan

 

A first-order-like state transition model is considered to be a global reaction mechanism to directly fold protein from unfolded state to its native form.  In order to verify the general applicability of this mechanism, we used lysozyme as a model protein.  It was fully unfolded by 4.5 M urea, 0.1 M dithiothreitol (DTT) in pH 3 and refolded to its native form by way of an overcritical reaction path (a quasi-static process) or directly crossing transition boundary path (a directly dilution process).  In addition to the two states coexisted in direct folding path, the lyzosyme might be trapped into a glassy state.  However, it can escape from the glassy state by concentration twice.  This indicates the existence of a state transition line or boundary in direct folding reaction.  However, the lysozyme can continuously fold from unfolded to native by an overcritical reaction path.  During the overcritical path, four stable folding intermediates and native lysozyme were obtained.  Secondary structures, particle size distributions, thermal stabilities, and oxidation state of disulfide bonds of folding intermediates were analyzed by circular dichroism (CD) spectra, dynamic light scattering (DLS), differential scanning calorimetry (DSC), and Raman spectra, respectively.  According to the data, the intermediates of both the overcritical reaction and the direct crossing transition boundary paths can be described by a common concept pertaining to a model that undergoes collapse, sequential and first-order-like state transition.  This indicated that protein folding by way of different reaction paths might follow a similar folding mechanism i.e., a mechanism of overcritical folding of intermediates.  A protein folding reaction diagram is postulated and discussed.  In spite of a global interaction mechanism the α-helix is formed prior to the β-sheet, which may indicate that the protein folding is initiated by local interactions.

 

  

PO02

How fast is protein stable from unfolded to folded state?

Po-Yen Lin1,2 , Xu-Cheng Yeh1,2, and Lou-Sing Kan2,Chia-Ching Chang1*

1: Department of Physics, National Dong Hwa University, Hualien, Taiwan 97401

2: Institute of Chemistry, Academia Sinica, Nankang, Taipei, Taiwan 11529

1, Sec 2, Da-Hsueh Rd., Shou-Feng, Hualien, 974 Taiwan

 

Proteins folding problem is an important topic in both experimental and theoretical studies. Conventional, reaction kinetics of protein folding is determined by specific structure forming. However the global stability of protein may not directly relate to partial structure formation. In this study, we directly investigated protein folding stability and its mechanism by both experimental approach and molecular simulation. During the experiment unfolded protein was drop directly into native solution environment. Both aggregated and soluble protein can be obtained. Meanwhile a molecular simulation study indicated that proteins followed random walk process and then aggregated when they collided to each other. However, proteins did not aggregate when they reached to stable state. This indicated that protein folding is an antagonistic reaction between diffusion-limited aggregation (DLA) and spontaneously folding. By changing the mean-free path of unfolded protein, the fraction of aggregated protein decay followed the function of logistic function, a limited growth model. The correlation time of protein aggregation indicated that the protein is stable as fast as sub- microsecond time scale.