Effect Of Chemical Modification And Osmolyte On Protein Folding And Stability

Protein is a very important class of biological macromolecules and plays important roles in metabolism, cell communication, individual growth and development, etc. In order to perform biological functions, proteins must fold into specific native structure. Only can properly folded proteins perform their unique functions; misfolded proteins cannot function properly and often lead to the formation of disease-related aggregates. Proteins are affected by complicated physical and chemical environments in vivo. For example, the human brain contains high concentrations of osmolytes. Despite some efforts have been directed towards understanding the effects of osmolytes on protein folding/unfolding process, most of these efforts are focused on simple two-state proteins without any detectable intermediates and their effects on intermediates are less understood. The function of proteins can also be regulated directly by ligand binding. In this thesis, we studied the effect of ligand binding on the mechanical properties of a small protein, SH3. We found that the stabilization effects upon ligand binding on the mechanical stability are dependent on the force direction. This study provides a general picture on the ligand-regulated mechanical response of proteins. Moreover, we are also interested in how chemical modifications affect the stability and folding of proteins. As chemical modifications are ubiquitous in pharmaceutical industry, this study will be helpful for rational improve the stability and function of proteins for clinic applications.The main contents of this thesis are arranged as follows:Chapter1introduces the structure of proteins and protein folding theory, and briefly reviews the history of protein folding researches. In addition, this chapter also describes the main experimental techniques used in this thesis:stopped-flow technique and single-molecule force spectroscopy. As the most widely used experimental methods for protein folding kinetics, stopped-flow technique helps people have an in-depth understanding on protein folding and unfolding process. Single-molecule force spectroscopy allows people to study the effects of mechanical force on biological molecules. Single-molecule force spectroscopy is complementary to traditional methods yet can observe rare events at the single molecule level which otherwise cannot be observed by the conventional methods. These methods allow people understanding the relationship between structure and function of biological molecules more clearly.In chapter2, we use a PEG with different length to modify SH3and measure thermodynamic and kinetic properties of mono-and multi-conjugated proteins. For the N-terminal conjugated SH3, folding and unfolding kinetics were affected slightly. However, for multi-conjugated SH3, unfolding kinetics was retarded significantly and thermodynamic stability was increased. Furthermore, increasing the molecular weight of PEG from5000to10000Da does not lead to a difference. These experimental findings inspire us to derive a physical model based on excluded volume effect, which can satisfactorily describe all experimental observations.In chapter3, we directly probed the effect of osmolyte on the unfolding pathway of BLG by stopped-flow method. Some previous researches have shown that during the unfolding of β-lactoglubulin an on-pathway kinetic intermediate state accumulates. Dobson has demonstrated that intermediate state in the folding and unfolding of BLG can become a precursor for amyloid fibril formation. A detailed characterization of the effect of osmolyte to the kinetic intermediate state of BLG will be of special interest. We found that glycerol increases its thermodynamic stability and significantly retards both fast and slow unfolding phase. However, for PEG, thermodynamic stability of β-lactoglubulin are unaltered, the slow unfolding phase is also significantly retarded but the fast phase is only slightly retarded. Due to the weak binding of PEG to the exposed hydrophobic residues of the intermediate and unfolded states, the stabilization from the excluded volume effect of these two osmolytes is compromised by such hydrophobic binding effect. However, such hydrophobic binding is not observed for glycerol.In chapter4, we studied the effect of osmolyte and viscosity on folding kinetics of proteins with similar size. Obtained experimental results show that the osmolyte effects on protein folding and unfolding depend on the original folding and unfolding rates of the protein. If the protein folds faster, osmolyte will accelerate the folding rate with a smaller degree. If the protein unfolds faster, osmolyte will decelerate the unfolding rate more significantly. Moreover, we found that the m-value also correlates with the osmolyte effects.In chapter5, we studied the effect of ligand binding on the mechanical unfolding of SH3. We used the cysteine engineering approach to construct poly-SH3proteins through the chemical crosslinking of the cysteine residues engineered to SH3. This allows us to stretching SH3from different directions that predefined by the cysteine positions. We found that ligand-binding increases the mechanical unfolding force of SH3without changing its unfolding pathway in a certain direction. In other directions, ligand binding does not show pronounced effect on the mechanical stability of SH3. Therefore, the mechanical stabilization effect on SH3is also anisotropic.In summary, in this thesis we studied different directs and indirect effects of the physical and chemical environments on the stability and folding of proteins. Our study could extend the general understanding of protein folding to more complicated systems. Many environmental factors can be considered in the general frame of protein folding theory. Finding quantitative descriptions of environmental effects on protein folding will be the ultimate goal of this research.