Crystallization of the large membrane protein complex photosystem I in a microfluidic channel.
Crystallization of the large membrane protein complex photosystem I in a microfluidic channel.
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DOI：
10.1021/nn402515q
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发表时间：
2013-12-23
期刊：
影响因子：
17.1
通讯作者：
Ros, Alexandra
中科院分区：
文献类型：
--
作者：
Abdallah, Bahige G.;Kupitz, Christopher;Fromme, Petra;Ros, Alexandra
传统的大尺度蛋白质结晶是通过探索一系列蛋白质浓度和溶液中的缓冲液来完成的，直到获得合适的组合。这种方法费时费力，阻碍了蛋白质结构的确定。当结晶像光系统I(PSI)这样的大的膜蛋白复合体时就会出现更多的困难，因为它们的大的单位晶胞主要是溶剂和复杂的特性，这需要更严格的缓冲液要求。为这些难以结晶的蛋白质量身定做的结构确定技术，如飞秒纳米晶体技术正在开发中，但仍需要特定的晶体特征。在这里，我们展示了一种简单而稳健的方法，在微流控设备中筛选低离子强度的蛋白质结晶条件。这是在一个使用低样本量的微流控实验中实现的，不像传统方法那样，每个溶液条件都是单独设置的。通过手性晶体的二阶非线性成像的二次谐波产生显微镜(SONICC)被用于检测微通道中的纳米和微米级的PSI晶体。为了绘制结晶相图，用SONICC成像的晶体在特定的通道位置与蛋白质和盐的浓度相关联，这些浓度是通过数值模拟沿通道的随时间变化的扩散过程确定的。我们的方法表明，PSI结晶相图的一部分可以与传统方法确定的结晶条件非常一致地重建。我们推测，这种方法可以被用来有效地研究和优化到目前为止还知之甚少的一系列蛋白质的结晶条件。
Traditional macroscale protein crystallization is accomplished non-trivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time consuming and resource intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these ‘difficult to crystallize’ proteins such as femtosecond nanocrystallography are being developed, yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via Second Order Nonlinear Imaging of Chiral Crystals (SONICC) was applied for the detection of nanometer and micrometer sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date.
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影响因子：
4.3
作者：
Fromme, P;Witt, HT
通讯作者：
Witt, HT
影响因子：
4.8
作者：
McPherson, A
通讯作者：
McPherson, A
影响因子：
4.6
作者：
Feng, YY;Zhou, ZY;Xiong, HJ
通讯作者：
Xiong, HJ
DOI：
10.1107/s1744309106002648
发表时间：
2006-03-01
影响因子：
0.9
作者：
Gavira, JA;de Jesus, W;García-Ruiz, JM
通讯作者：
García-Ruiz, JM
DOI：
10.1107/s0907444902014609
发表时间：
2002-10-01
影响因子：
2.2
作者：
Gavira, JA;García-Ruiz, JM
通讯作者：
García-Ruiz, JM