Abstract
BackgroundStructural studies of integral membrane proteins (IMPs) are hampered by inherent difficulties in their heterologous expression and in the purification of solubilized protein-detergent complexes (PDCs). The choice and concentrations of detergents used in an IMP preparation play a critical role in protein homogeneity and are thus important for successful crystallization.ResultsSeeking an effective and standardized means applicable to genomic approaches for the characterization of PDCs, we chose 1D-NMR spectroscopic analysis to monitor the detergent content throughout their purification: protein extraction, detergent exchange, and sample concentration. We demonstrate that a single NMR measurement combined with a SDS-PAGE of a detergent extracted sample provides a useful gauge of the detergent's extraction potential for a given protein. Furthermore, careful monitoring of the detergent content during the process of IMP production allows for a high level of reproducibility. We also show that in many cases a simple sedimentation velocity measurement provides sufficient data to estimate both the oligomeric state and the detergent-to-protein ratio in PDCs, as well as to evaluate the homogeneity of the samples prior to crystallization screening.ConclusionThe techniques presented here facilitate the screening and selection of the extraction detergent, as well as help to maintain reproducibility in the detergent exchange and PDC concentration procedures. Such reproducibility is particularly important for the optimization of initial crystallization conditions, for which multiple purifications are routinely required.
Highlights
Structural studies of integral membrane proteins (IMPs) are hampered by inherent difficulties in their heterologous expression and in the purification of solubilized protein-detergent complexes (PDCs)
Step 1: Protein extraction from cell membranes Following protein expression in Escherichia coli (E. coli), a crude membrane fraction containing an overexpressed IMP is solubilized by a detergent of choice as the first step of IMP purification
The importance of monitoring the detergent content and homogeneity of the PDC during IMP preparation is illustrated by the behavior of detergents during extraction (Figure 1 and 2) and detergent exchange (Figure 3)
Summary
Structural studies of integral membrane proteins (IMPs) are hampered by inherent difficulties in their heterologous expression and in the purification of solubilized protein-detergent complexes (PDCs). There are over 43,000 structure entries for water-soluble proteins deposited in the public Protein Data Bank [3,4], in contrast to fewer than 800 IMP entries (corresponding to less than 200 unique structures). This discrepancy is largely due to the fact that the crystal structure determination of an IMP is often experimentally hampered by difficulties associated with its (page number not for citation purposes). We sought an effective and relatively fast means to characterize PDCs, which could be applied in combination with genomic strategies
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