Nonlinear Optical Imaging of Integral Membrane Protein Crystals in Lipidic Mesophases

被引:55
|
作者
Kissick, David J. [1 ]
Gualtieri, Ellen J. [1 ]
Simpson, Garth J. [1 ]
Cherezov, Vadim [2 ]
机构
[1] Purdue Univ, Dept Chem, W Lafayette, IN 47907 USA
[2] Scripps Res Inst, Dept Mol Biol, La Jolla, CA 92037 USA
关键词
AUTOMATIC CLASSIFICATION; CRYSTALLIZATION; PHASE;
D O I
10.1021/ac902139w
中图分类号
O65 [分析化学];
学科分类号
070302 ; 081704 ;
摘要
Second-order nonlinear optical imaging of chiral crystals (SONICC) is explored for selective detection of integral membrane protein crystals grown in opaque and turbid environments. High turbidity is a hallmark of membrane protein crystallization due to the extensive use of detergent and/or lipids that often form various mesophases. Detection of crystals in such media by conventional optical methods (e.g., intrinsic LTV fluorescence, birefringence, bright-field image analysis, etc.) is often complicated by optical scattering and by the small sizes of the crystals that routinely form. SONICC is shown to be well-suited for this application, by nature of its compatibility with imaging in scattering media and its high selectivity protein crystals. Bright second harmonic generatioiri (SHG) (up to 18 million counts/s) was observed from; even relatively small crystals (5 pin) with a minimal background due to the surrounding lipid mesophase (similar to 1 thousand counts/s). The low background nature of the resulting protein crystal images permitted the use of a relatively simple, particle counting analysis for preliminary scoring. Comparisons between a particle counting analysis of SONICC images and protocols based on the human expert analysis of conventional bright-field and birefringence images were performed.
引用
收藏
页码:491 / 497
页数:7
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