Organic Osmolyte Mediated Kinetic Capillary Electrophoresis for Study of Protein−DNA Interactions

Abstract

An organic osmolyte mediated kinetic capillary electrophoresis is developed and applied to study the binding of Escherichia coli RecA protein to single-stranded (ss) DNA. The complex of RecA and ssDNA can be significantly enhanced and stabilized by 20% glycerol over 5.6 times. Kinetic capillary electrophoresis study reveals that glycerol may function in the preservation of RecA native conformation, enhancement of specific binding affinity (20 times), and decrease in dissociation rate constant (1 time). The combined laser-induced fluorescence polarization (LIFP) study further indicates a tightened assembly of RecA on ssDNA induced by glycerol. The binding of RecA to ssDNA was also enhanced by two additional protecting organic osmolytes, sucrose and PEG 400. In contrast, a denaturing organic osmolyte, urea, even at a concentration of 110 mM can prevent the formation of the RecA-ssDNA complexes. The detection of the binding of E. coli single-stranded binding protein (SSBP) to ssDNA was also improved by the same method. By combining the protection of organic osmolytes with low-temperature storage, a method for preservation of protein-DNA complex up to 43 h is further demonstrated. By use of a long capillary and protecting glycerol together an enhanced kinetic CE analysis was also developed, providing a better separation and analysis of the undissociated complex, the dissociated complex, and the unbound probe. The results demonstrate the wide applicability of the method to the study of various protein-DNA binding.