Monitoring and Optimizing Detergent Concentration For Membrane Protein Crystallization While Following Protein Homogeneity

Abstract

Detergent concentration is critical to growing quality membrane protein crystals. Since the optimal detergent concentration lies just below the detergent phase boundary, the starting detergent concentration must be minimized for initial crystallization trials. However, the majority of Pure, Homogenous and Stable targets contain excessive levels of detergent micelles upon concentration using molecular weight cut-off filters. Size Exclusion Chromatography (SEC) and a Tetra Detector Array (refractometer, viscometer, light scattering, and UV detectors; TDA) are now being successfully utilized to monitor and optimize detergent concentration while assaying PDC homogeneity during purification and concentration for crystallization. In doing so, the oligomeric state, size, shape and the detergent:protein ratio of the Protein Detergent Complex (PDC) is measured.Five different membrane proteins using 3 different detergents (OG, DDM, and FC14) and 4 different methods will be presented where detergent was successfully minimized while maintaining PDC homogeneity. Methods utilized where ultra filtration (centrifugal and high-pressure molecular weight cut-off filters) plus SEC and dialysis, changing detergent isomer, Ni-NTA and ion exchange chromatography.Detergent micelle SEC retention volume, dn/dc, Rh, IV, mass and behavior on different molecular cut-off filters and formats are all being measured using TDA. As expected, there is a direct correlation of measured excess micelle concentration to crystal phase separation and diffraction quality. Unexpectedly, free micelles in the presence of PDCs tend to be highly retained on cut-off filters which would freely pass a pure detergent micelle system; therefore, when measuring whether a micelle is retained or passed through by specific molecular cut-off filters and formats, it must be measured using a PDC system and not just buffered detergent controls.