Imaging Ca2+ Concentration and pH in Nanopores/Channels of Protein Crystals.
Imaging Ca2+ Concentration and pH in Nanopores/Channels of Protein Crystals.
复制标题
蛋白质晶体纳米孔/通道中的 Ca2 浓度和 pH 值成像。
DOI：
10.1021/acs.jpcb.8b07099
复制
发表时间：
2018
期刊：
影响因子：
--
通讯作者：
B. Kuhn
中科院分区：
文献类型：
--
作者：
K. Mori;B. Kuhn
蛋白质晶体是纳米多孔材料。尽管有这一重要特征，但人们对毛孔中的情况知之甚少，毛孔也被称为通道。在这里，我们描述了一种方法来研究索马汀和溶菌酶晶体纳米孔中的钙浓度和pH。我们在晶体纳米孔上加载荧光指示剂，然后在晶体中灌流不同钙浓度和pH的溶液，同时用共聚焦显微镜读出晶体的荧光强度。通过对荧光信号的标定，我们可以确定纳米孔中的钙离子浓度和pH值。对于用比率pH传感器snarf-1测量的thaumatin纳米孔中的pH，我们发现与溶液中的pH相比，pH漂移了-0.7，对应的质子浓度增加了五倍。这类似于在溶菌酶纳米孔中发现的-0.3 pH漂移。利用单波长探针，我们发现索马菌素晶体纳米孔中的钙离子浓度与镀液中的相同，而溶菌酶纳米孔中的钙离子浓度比溶液中的低0.24倍。综上所述，我们的实验表明，蛋白质晶体纳米孔中的钙离子浓度和pH值与镀液中的钙离子浓度和pH值有明显的偏差。总体而言，所描述的方法不仅可以用比例法，而且可以用单波长荧光指示剂来测试透明纳米多孔材料中大范围的离子或小分子浓度。
Protein crystals are nanoporous materials. Despite this important characteristic, little is known about the conditions in the pores, also called channels. Here, we describe a method to study the calcium concentration and pH in the nanopores of thaumatin and lysozyme crystals. We load the crystal nanopores with fluorescent indicators and then perfuse the crystals with solutions of different calcium concentrations and pH while reading out the crystal's fluorescence intensity with confocal microscopy. By calibrating the fluorescence signal, we can determine the calcium concentration and pH in the nanopores. For the pH in thaumatin nanopores measured with the ratiometric pH sensor SNARF-1, we find a -0.7 pH shift compared to the bath pH corresponding to a fivefold higher proton concentration. This is similar to the -0.3 pH shift found in lysozyme nanopores. With single-wavelength probes, we find that the calcium concentration in thaumatin crystal nanopores is the same as in the bath, whereas it is 0.24 times lower in lysozyme nanopores. Summarizing, our experiments show that calcium concentration and pH in the nanopores of protein crystals can deviate significantly from that in the bath. In general, the described method can be applied for testing a wide range of ion or small-molecule concentrations in transparent nanoporous materials not only with ratiometric but also with single wavelength fluorescent indicators.
影响因子：
6.1
作者：
I. Khan;R. Gillilan;I. Kriksunov;R. Williams;W. Zipfel;U. Englich
通讯作者：
I. Khan;R. Gillilan;I. Kriksunov;R. Williams;W. Zipfel;U. Englich
蛋白质晶体缓慢冷却。
影响因子：
6.1
作者：
Warkentin,Matthew;Thorne,RobertE
通讯作者：
Thorne,RobertE