Leiden Institute of Chemistry, Leiden University, 2333 CC Leiden, The Netherlands.
Department of Physics and Astronomy, Vrije Universiteit Amsterdam, 1081 HV Amsterdam, The Netherlands.
Int J Mol Sci. 2020 Dec 15;21(24):9553. doi: 10.3390/ijms21249553.
HU is a nucleoid-associated protein expressed in most eubacteria at a high amount of copies (tens of thousands). The protein is believed to bind across the genome to organize and compact the DNA. Most of the studies on HU have been carried out in a simple in vitro system, and to what extent these observations can be extrapolated to a living cell is unclear. In this study, we investigate the DNA binding properties of HU under conditions approximating physiological ones. We report that these properties are influenced by both macromolecular crowding and salt conditions. We use three different crowding agents (blotting grade blocker (BGB), bovine serum albumin (BSA), and polyethylene glycol 8000 (PEG8000)) as well as two different MgCl conditions to mimic the intracellular environment. Using tethered particle motion (TPM), we show that the transition between two binding regimes, compaction and extension of the HU protein, is strongly affected by crowding agents. Our observations suggest that magnesium ions enhance the compaction of HU-DNA and suppress filamentation, while BGB and BSA increase the local concentration of the HU protein by more than 4-fold. Moreover, BGB and BSA seem to suppress filament formation. On the other hand, PEG8000 is not a good crowding agent for concentrations above 9% (/), because it might interact with DNA, the protein, and/or surfaces. Together, these results reveal a complex interplay between the HU protein and the various crowding agents that should be taken into consideration when using crowding agents to mimic an in vivo system.
HU 是一种核质相关蛋白，在大多数真核生物中以高拷贝数（数万）表达。该蛋白被认为通过跨越基因组结合来组织和压缩 DNA。HU 的大多数研究都是在简单的体外系统中进行的，这些观察结果在多大程度上可以外推到活细胞中尚不清楚。在这项研究中，我们在接近生理条件的情况下研究 HU 的 DNA 结合特性。我们报告说，这些特性受到大分子拥挤和盐条件的影响。我们使用三种不同的拥挤剂（印迹级阻滞剂（BGB）、牛血清白蛋白（BSA）和聚乙二醇 8000（PEG8000））以及两种不同的 MgCl 条件来模拟细胞内环境。使用系绳粒子运动（TPM），我们表明 HU 蛋白的两种结合状态（压缩和延伸）之间的转变强烈受拥挤剂的影响。我们的观察表明，镁离子增强了 HU-DNA 的压缩并抑制了丝状形成，而 BGB 和 BSA 使 HU 蛋白的局部浓度增加了 4 倍以上。此外，BGB 和 BSA 似乎抑制了丝状形成。另一方面，PEG8000 对于浓度高于 9%（/）的不是一个好的拥挤剂，因为它可能与 DNA、蛋白质和/或表面相互作用。总之，这些结果揭示了 HU 蛋白与各种拥挤剂之间复杂的相互作用，在使用拥挤剂模拟体内系统时应考虑到这一点。
