5v33

Publication Abstract from PubMed

Elucidating and clarifying the function of membrane proteins ultimately requires atomic resolution structures as determined most commonly by X-ray crystallography. Many high impact membrane protein structures have resulted from advanced techniques such as in meso crystallization that present technical difficulties for the set-up and scale-out of high-throughput crystallization experiments. In prior work, we designed a novel, low-throughput X-ray transparent microfluidic device that automated the mixing of protein and lipid by diffusion for in meso crystallization trials. Here, we report X-ray transparent microfluidic devices for high-throughput crystallization screening and optimization that overcome the limitations of scale and demonstrate their application to the crystallization of several membrane proteins. Two complementary chips are presented: (1) a high-throughput screening chip to test 192 crystallization conditions in parallel using as little as 8 nl of membrane protein per well and (2) a crystallization optimization chip to rapidly optimize preliminary crystallization hits through fine-gradient re-screening. We screened three membrane proteins for new in meso crystallization conditions, identifying several preliminary hits that we tested for X-ray diffraction quality. Further, we identified and optimized the crystallization condition for a photosynthetic reaction center mutant and solved its structure to a resolution of 3.5 A.

X-ray transparent microfluidic chips for high-throughput screening and optimization of in meso membrane protein crystallization.,Schieferstein JM, Pawate AS, Sun C, Wan F, Sheraden PN, Broecker J, Ernst OP, Gennis RB, Kenis PJA Biomicrofluidics. 2017 Apr 24;11(2):024118. doi: 10.1063/1.4981818. eCollection, 2017 Mar. PMID:28469762^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.