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PRE-CRYSTALLIZATION SCREENING home > research > pre-crystallization screening

Pre-crystallization screening (PCS) refers to experiments conducted before crystallization experiments are performed. The intent of PCS for transmembrane proteins (TMPs) is to provide ‘quality-control’ data prior to set-up and evaluation of thousands (or tens to hundreds of thousands) of crystallization experiments. If the protein-detergent complex (PDC) going into crystallization is somehow ‘wrong’, then crystallization experiments will be futile. The goal is to reduce the very large crystallization space to a more tractable number of experiments and to eliminate, if possible, experiments that will not be productive.

Formulation of a ‘broad-spectrum’ detergent panel.  One of the most important requirements for successful TMP crystallization is the identification of a compatible detergent.  The Wiener laboratory has compiled a list of 94 detergents suitable for TMP studies and is exploring rapid, inexpensive methods of screening for suitable protein-detergent pairs. The criteria used in selecting the detergents were (1) a critical micelle concentration (CMC) value between 0.03 and 40mM, (2) moderate solubility in water, and (3) a detergent that is either zwitterionic or non-ionic. This panel facilitates the testing of novel detergents for stubborn TMPs, such as those that are unstable in the small, traditional set of detergents and thus not amenable for structural studies. It allows a wide exploration of “detergent-space” for any given TMP. The final concentration of detergent used is based on each detergent’s CMC value as discussed by Wiener [1].
Creation of a detergent stability assay. To efficiently explore detergent-space, the Wiener laboratory has developed a high-throughput detergent stability assay called the “Prompt Assay of Stability and Size” (PASS). PASS simultaneously screens the 94-detergent panel for the ability to maintain the solubility of a given TMP. This method utilizes a standard buffer exchange technique that has been successfully implemented to screen detergents for the ability to extract proteins from their membrane environment (2). Initially in a single detergent, the protein to be tested is bound to an affinity resin and divided into the wells of a filter microplate.  The resin in each well is extensively washed and eluted with a solution containing one component of the detergent panel. The eluants are then subjected to ultrafiltration sequentially using a 0.2μm filter plate, a 300kDa molecular weight cutoff (MWCO) filter plate, and a 100kDa MWCO filter plate. The sequential use of these plates provides a high-throughput, simple substitute for gel filtration analysis of the eluted protein. For example, the nominal 300kDa MWCO plate has a cutoff similar to that of a GE Biosciences Superdex 200 column. Plates with different molecular mass cutoffs are used so that analysis of the 300kDa plate filtrate reports primarily on stability (defined here operationally as propensity to aggregate) and the ratio of the 100kDa and 300kDa plate filtrates reports on PDC size. The sizing information from PASS correlates well with traditional size exclusion chromatography, and results can be obtained in approximately two hours using only a few hundred micrograms of protein. Using a conjugated primary antibody specific to the affinity tag and a fast Western approach, the MWCO filter plate elutions can be visualized and quantified in under an hour. Detergents that maintain the solubility of a TMP produce a dot on the blot with the dot intensity directly related to stability, while detergents that are incompatible with the TMP do not produce any dot due to precipitation of the protein on the affinity resin. Loss of TMP stability is manifested as the presence of irreversible aggregates that prevent the TMP from passing through the ultrafiltration membranes. [ Learn More ]

References

1. Wiener, M.C., A pedestrian guide to membrane protein crystallization. Methods, 2004. 34(3): p. 364-72.   (PMID: 15325654)

2. Niegowski, D., M. Hedren, P. Nordlund, and S. Eshaghi, A simple strategy towards membrane protein purification and crystallization. Int J Biol Macromol, 2006. 39(1-3): p. 83-7. (PMID: 16546251)

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