LCP-FRAP Assay for Pre-Screening Membrane Proteins for in Meso Crystallization.

Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 USA.
Crystal Growth & Design (Impact Factor: 4.89). 02/2008; 8(12):4307-4315. DOI: 10.1021/cg800778j
Source: PubMed


Fluorescence recovery after photobleaching was used to study the diffusion of two integral membrane proteins, bacteriorhodopsin and beta2-adrenergic receptor, in lipidic cubic phase (LCP). We found that the diffusion properties within the LCP matrix strongly depend on the protein construct and applied screening conditions. Common precipitants often induce restriction on diffusion of proteins in LCP and thereby impede their chances for crystallization. A high protein mobile fraction and a fast diffusion rate correlate very well with known crystallization conditions. Using this knowledge, one can now pre-screen precipitant conditions with microgram quantities of material to rule out conditions that are not conducive to diffusion, nucleation, and crystal growth. The results of this assay will narrow membrane protein crystallization space by identifying suitable protein constructs, stabilizing compounds and precipitant conditions amenable to in meso crystallization. Crystallization pre-screening will significantly increase the chances of obtaining initial crystal hits, expediting efforts in generating high-resolution structures of challenging membrane protein targets.

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Available from: Jeffrey Liu, Apr 30, 2014
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    • "Fluorescence intensity was corrected by dividing the value of the integrated intensity ROI 1 in the bleached spot by the average integrated intensity of the ROI 2 . As described by Cherezov et al.[7]. The fractional fluorescence recovery curves, F(t), were calculated according[1]. "
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    • ") . In a second approach , since used to determine the majority of nonrhodopsin GPCR structures to date , a T4 lysozyme fusion was introduced within the third cyto - plasmic loop of heterologously expressed ␤ 2 - adrenergic receptor to aid crystallization in a modified lipid cubic phase matrix and resulted in high - order diffracting crys - tals ( Cherezov et al . , 2008 ; Hanson et al . , 2008 ; Roth et al . , 2008 ) . In addition to the high - resolution 2 . 4 - Å cara - zolol - bound structure ( PDB ID 2RH1 ) , a series of adren - ergic receptor structures have been solved that con - tained a variety of antagonist and inverse agonist compounds ( Hanson et al . , 2008 ; Roth et al . , 2008 ; Wacker et"
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