Effects of protein microstructure on the retention time of T4 lysozyme variants in cation exchange chromatography.
The structure of a protein directly affects its function. Therefore, characterization of recombinant protein structures is important but is a challenging task. One of the important forces that play a major role in maintaining both structural and functional properties of proteins is electrostatic interactions among different amino acid residues. In this article, cation exchange chromatography was used to study how the microstructure of some charged amino acid residues may affect a protein's retention. Two sets of T4 lysozyme variants were generated. The first set included seven variants that varied in their charge distribution. These variants were obtained by replacing a charged amino acid residue at different sites on lysozyme. The second set included ten variants that varied in both net charge and charge distribution, and these variants were obtained by replacing charged and neutral amino acid residues at different sites on the protein. The microstructure was quantified by calculating the relative hydrophilicity around the replacing amino acid residue. The retention times of all variants were compared with the retention time of the respective control variant. Among the first set of variants, there was a direct correlation (R(2) = 0.93) between the relative hydrophilicity of the replaced amino acid and the protein's retention time, except for two variants (K83H and K124H) whose replacing amino acid residue was involved in intramolecular interactions. For the second set, there was a direct correlation (R(2) = 0.97) between the change in net charge (-2 to +2 units) and the retention times. However, the retention times of two variants (R76D and R76S) did not follow the correlation. We hypothesize that the structure around the replacing charged group is responsible for the deviated protein retention pattern.
Pathange
Lakshmi P
LP
Department of Biological Systems Engineering, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, USA.
Bevan
David R
DR
Zhang
Chenming
C
D019943:Amino Acid Substitution; D002852:Chromatography, Ion Exchange; D009113:Muramidase / Q000737:chemistry* / Q000302:isolation & purification / Q000648:ultrastructure; D016297:Mutagenesis, Site-Directed; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification / Q000648:ultrastructure
D020033:Protein Isoforms; D009113:Muramidase