Testing SAXS Applicability for Detection of Illumination-Driven Structural Changes in the Purple Membranes from H. salinarum

IF 1.1 Q4 CELL BIOLOGY
A. Yu. Shishkin, D. D. Kuklina, E. A. Dronova, Yu. L. Ryzhykau
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引用次数: 0

Abstract

Rhodopsins are photosensitive transmembrane proteins found in bacteria, archaea, and eukaryotes. Upon photon absorption, they undergo conformational changes critical for their physiological functions, such as ion transport and signal transduction. Microbial rhodopsins are key tools in optogenetics, with applications in biomedical research and clinical treatments. To advance optogenetic techniques, it is crucial to understand the molecular mechanisms of the rhodopsin photocycle. The key approach for studying these mechanisms, cryotrapping, has a setback associated with possible deterioration in resolution due to the unexpectedly large light-induced structural changes. To estimate the scale of these changes, small-angle X-ray scattering (SAXS) can be used. In this study, we applied SAXS to the investigation of light-induced structural changes in purple membranes (PMs) as their two-dimensional crystal organization makes them a suitable model object for studying light-induced changes in 3D rhodopsin crystals. SAXS data from illuminated and non-illuminated PM samples revealed detectable changes, including variations in membrane thickness and planar unit cell dimensions. Particularly, we observed a statistically significant increase in the radius of gyration for flat particles (Rₜ) of ~0.2 Å for the PMs with mutant HsBRE204Q, while no significant change was detected for wild-type HsBR. However, wild-type HsBR data showed a statistically significant shift in the (1, 1) Bragg peak position (Δ q ~ –10–4 Å–1; ~3% of the peak width) upon illumination. For HsBRE204Q data, the shift was two times greater; however, in this case due to the lower sample concentration εΔq was ~50%, making it difficult to compare the wild-type and mutant cases. Future experiments should aim for better signal-to-noise ratios using synchrotron radiation, higher sample concentrations, and longer exposure times.

测试SAXS在检测光照驱动紫色膜结构变化中的适用性
紫红质是在细菌、古生菌和真核生物中发现的光敏跨膜蛋白。光子吸收后,它们发生构象变化,这对它们的生理功能至关重要,如离子传输和信号转导。微生物视紫红质是光遗传学研究的重要工具,在生物医学研究和临床治疗中有着广泛的应用。为了进一步发展光遗传学技术,了解视紫红质光循环的分子机制是至关重要的。研究这些机制的关键方法——低温捕获,由于意想不到的大的光诱导结构变化而可能导致分辨率下降,因此受到挫折。为了估计这些变化的规模,可以使用小角度x射线散射(SAXS)。在本研究中,我们将SAXS应用于紫色膜(pm)的光诱导结构变化的研究,因为它们的二维晶体组织使它们成为研究三维视紫红质晶体光诱导变化的合适模型对象。照射和未照射PM样品的SAXS数据显示了可检测的变化,包括膜厚度和平面单细胞尺寸的变化。特别的是,我们观察到在HsBRE204Q突变体的pmms中,平面粒子的旋转半径(R - l)增加了~0.2 Å,而在野生型HsBR中没有发现明显的变化。然而,野生型HsBR数据显示,(1,1)Bragg峰位置发生了统计学上显著的变化(Δ q ~ -10-4 Å-1;~峰宽的3%)。对于HsBRE204Q数据,这种变化是前者的两倍;然而,在本病例中,由于样本浓度较低εΔq为~50%,因此难以比较野生型和突变型病例。未来的实验应该以更好的信噪比为目标,使用同步辐射,更高的样品浓度,更长的曝光时间。
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来源期刊
CiteScore
1.40
自引率
0.00%
发文量
28
期刊介绍: Biochemistry (Moscow), Supplement Series A: Membrane and Cell Biology   is an international peer reviewed journal that publishes original articles on physical, chemical, and molecular mechanisms that underlie basic properties of biological membranes and mediate membrane-related cellular functions. The primary topics of the journal are membrane structure, mechanisms of membrane transport, bioenergetics and photobiology, intracellular signaling as well as membrane aspects of cell biology, immunology, and medicine. The journal is multidisciplinary and gives preference to those articles that employ a variety of experimental approaches, basically in biophysics but also in biochemistry, cytology, and molecular biology. The journal publishes articles that strive for unveiling membrane and cellular functions through innovative theoretical models and computer simulations.
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