Structural Dynamics of ABC Transporter update, year 1
Summary of Progress
The project got underway 10/1/2010. In the first year of activity, the experimental focus was to select and develop the expression platform. In parallel, computational analysis was initiated to characterize the dynamic modes of apo Pgp and simultaneously select sites for incorporation of spin label probes. The summary describes progress on the specific aims. The synergy with cores, which underlies the rationale for the consortium, is emphasized in projects described as research highlights (RH). The pending publication of the first crystal structure of an ABC heterodimer spurred us to initiate spin labeling studies of this subclass of transporters (RH #1). Synthesis of spin labeled Pgp substrate analogs is providing a new approach for investigation of transport (RH #3). Alternative approaches to Pgp expression are being pursued (RH #3) and novel computational tools (RH #4) are being developed to analyze distance distributions between spin labels. Many of these methodological developments will be critical to the success of this project and can be applied in other bridges and by the membrane protein community at large.
Construction and Expression of cysteine less human Mdr1 in insect cells.
In collaboration with the Membrane Protein Expression / Purification Core, we compared Pgp expression in Saccharomyces cerevisiae to that in insect cells infected with baculovirus. The results suggested the higher eukaryotic system is better suited for production of the mammalian transporter at the scales required for EPR spectroscopy. While yeast cells are easier and less expensive to grow, the difficulties in solubilizing the protein from the ergosterol-containing yeast membranes is a distinct disadvantage compared to insect cells. The Mchaourab lab cloned human P-glycoprotein gene into pFastBac HT A (Invitrogen). There is a 28 residues linker before the start of the P-gp gene that includes a 6x His tag. To generate P-gp Cless, all seven cysteines (C137, C431, C717, C956, C1074, C1125, and C1227) were mutated to alanines. Figure 1 shows the expression and purification of human Pgp detected by western blotting.
The Protein Core has further developed the expression system of the cysteine-less human P-glycoprotein. The original cysteine-less baculovirus construct produced in the Mchaourab lab was tested in different insect cell lines. The highest expression system was grown at large scale and the protein purified via its (His)6 affinity tag. Based on results from molecular dynamics simulations (see below), five double.
DEER analysis of apo Pgp structure.
We have succeeded in obtaining DEER data for the double mutant 215/858 (human sequence). Figure 1 shows that the distance distribution for this double mutant is bimodal suggesting equilibrium fluctuations at the extracellular side of the transporter. The long distance component is consistent with apo Pgp crystal structure corrected for the projection of the spin label side chain. The shorter component is suggestive of a more open conformation. Although preliminary, this data highlights the unique insight obtained from spectroscopic analysis of this transporter. Five double mutants have been constructed and are now in production stage. An initial mapping of distance changes upon ATP hydrolysis will be performed in the next year of activity.
Click to find out more about each research highlight (RH).