Protein Surfaces Probed by Aqueous Two-phase Partitioning

Abstract: This thesis is concerned primarily with peptide and protein partitioning in aqueous two-phase systems and the connection between partitioning behavior and protein structure. The surface properties of a protein is often crucial for recognition and interaction with other molecules. Important functional residues can be identified by mutational analysis. There is a need for rapid methods to study protein surfaces and surface changes due to mutations. Partitioning in aqueous two-phase systems has the potential to be used in this respect, since protein partitioning depends on the surface properties of the protein. However, we need to know how different amino acid residues contribute to the partition coefficient of a protein. Two aqueous phases can be formed by two structurally different polymers in aqueous solution. The polymers mainly used in this work have been thermoseparating ethylene oxide-propylene oxide random copolymers and dextran. The results in this thesis have shown the influence both qualitatively and quantitatively of different amino acid residues on the partitioning of proteins. Additionally, peptide partitioning could be used to predict the contribution of surface exposed amino acid residues to the partition coefficient of a protein. The effect on partitioning could be described only taking into account solvent accessibility and type of residue. Thus, aqueous two-phase partitioning can be used as a rapid method for studying the surface exposure of charged, hydrophobic, and especially aromatic amino acid residues. The knowledge gained can also be used to design efficient peptide tags for a target protein to direct it to a desired polymer phase. This technique can be used for large-scale purification of a protein.