Theoretical Studies of Protein Folding: Experimental and Molecular Dynamics Insights into the Design of Phenomenological Models

Analyzing experimental data of thermodynamic properties of proteins reveals large discrepancies between data obtained using different experimental methods, for example, those obtained by spectroscopy methods and those by calorimetry. The interpretation is that some experimental methods probe local or site-specific properties of proteins, while others probe global properties. However, the theoretical foundation of this probe-dependent issue has not yet been established. The aim of this thesis attempts to investigate the phenomenological models, which are designed according to insight from experimental results. These models aim to explain the different results from different experimental methods from a statistical mechanics point of view.
In the first part of the thesis, the thermodynamic properties (e.g., the free energy change) of cytochrome c (Cyt c) were analyzed using the thermodynamic two-state model via absorption spectroscopy (Abs), circular dichroism (CD), fluorescence spectroscopy (Flu) and differential scanning calorimetry (DSC). These results showed that the thermodynamic properties measured by spectroscopic methods are local properties, and those measured by DSC are, however, global properties. This also confirms the existence of macro-units in Cyt c, identified as foldons by Englander’s group using hydrogen exchange (HX) experiments. A simplified zipper-like model was accordingly proposed to describe the folding-unfolding thermodynamics and kinetics of Cyt c. In addition, molecular dynamics (MD) simulations were performed to detect the macro- (or foldon) behavior of Cyt c from microscopic molecular motions. The result was in line with a thermodynamically observed sequential folding mechanism, supporting that Cyt c folding occurs in accordance with the classical pathway concept.
In the second part of the thesis, the thermodynamic behavior and its related properties (e.g., the folding fraction and free energy change) of a β-hairpin peptide (GB1 C-terminal β-hairpin) were investigated in order to understand the folding of the basic structural motif. I also defined the foldon behavior from a microscopic point of view. Based on this view, I proposed a modified Wako-Saito-Munoz-Eaton (WSME) model, designed with site-dependent properties of proteins in mind. A mathematical technique (contact-pair treatment) was accordingly developed for facilitating the calculation of the partition function. In addition, I performed MD simulations and the results showed the same site-dependent characteristics. Our results showed that the proposed model provides a statistical mechanical foundation for the foldon behavior, and may be generally useful in the interpretation of site-dependent properties of proteins as well as the study of protein folding.