All known proteins contain charged amino acid residues, and charged residues are considered an essential part of almost all protein properties. The charged residues are assumed vital for both the catalytic activity and solubility of proteins, and they are also thought to affect the folding, structure and interactions of proteins by long-range and complex charge networks. The importance of charge-charge interactions for the overall stability of proteins is still a matter of debate, but they can indeed enhance the stability of designed proteins. The strength of electrostatic interactions is often inferred from changes in either stability or pKa-values upon mutation of charged residues. Both the stability and pKa-values of a protein are typically the result of multiple charge-charge interactions as well as several other important factors, which makes it very difficult to measure the contributions of the individual interactions.
In this project, we focused on making a protein without any charged residues and use this protein as a simplified model protein in which individual electrostatic interactions can be examined. Our charge-free protein was shown to be soluble, functional, correctly folded and stabile, and redefines the limits of what is needed in order to design a well behaving protein. Single or pairs of charged residues were introduced in the charge-free protein in order to investigate their influence on basic physicochemical protein properties as stability and solubility. In addition, these variants were used to examine the interaction between pairs of charged residues, when no other charges are present to affect the results. Finally, the uncharged protein was used to examine SDS-protein interaction and unfolding, since the importance of charges in this process is a long-standing discussion.