Targeting protein-DNA interactions in prokaryotic systems

许多结构研究证实了sigma54与RNAP和DNA的结合。Sigma54结合来自转录起始位点的DNA -24和-12碱基对，启动DNA熔化，这是转录的重要步骤。大量的工作，包括DNA足迹，丙氨酸扫描突变研究，sigma54- rna -聚合酶在封闭构型下与DNA结合的低分辨率冷冻电子显微镜，以及高分辨率核磁共振结构，都表明sigma54与DNA的主要凹槽特异性和紧密结合。例如，Wemmer实验室使用核磁共振表明，来自Aquifex aeolicus的66个氨基酸长螺旋-转螺旋（HTH）基序与DNA结合。在这个c端HTH基序中，在377到386残基（ARRTVAKYRE）上有一个单独的α -螺旋，称为RpoN盒，负责结合DNA的主槽（PBD: 2O8K）。用Ala替代Arg378、Arg379、Tyr384和Arg385后，DNA结合明显降低。蛋白质核磁共振结构表明，该螺旋与启动子的-24区（5'-TGGCACG-3'）选择性相互作用。特别是，Arg378和Arg379定位于相互作用的-24元素，并与DNA非编码链上的Gua-25和Gua-26发生多重氢键和离子相互作用。这导致66-mer HTH基序和启动子区域之间的高亲和力相互作用（Kd = 114 nM）。尽管这些肽被建模为抑制转录，但有必要评估细胞活力，以确保下游的抗毒作用是可观察到的。采用标准肉汤微量稀释法，24对所有钉钉肽类似物的抑菌活性进行了评价。所有肽的最小抑制浓度（MIC）值为32微克/毫升或更高。由于与许多传统抗生素相比，该值很高，因此可以在低浓度下评估该肽，以研究其对革兰氏阴性菌毒力特性的影响。为了确定化合物是否对真核细胞具有细胞毒性，将WS1成纤维细胞暴露于每种化合物的连续稀释中。数据表明，这些化合物在10微米的浓度范围内对正常细胞没有毒性。为了鉴定合成肽的螺旋结构，将化合物溶解在水中进行了圆二色光谱分析。野生型sigma54肽是非结构化的，而钉接肽1-4都具有α -螺旋二级结构的特征谱。流式细胞术用于在大量细菌中获得每个钉接肽的有效性的高通量视角。通过利用其个体事件检测，确定了多肽的细胞摄取百分比。在大肠杆菌和铜绿假单胞菌的革兰氏阴性菌种中，所有四种肽类似物都能够渗透。与载体对照相比，野生型肽，即不存在短钉的序列，在PA01铜绿假单胞菌中渗透较少，在BW25113大肠杆菌中渗透水平不显著。对于每个肽，每个肽样品中至少有50%的细胞渗透。特别是，sigma54-2的外显率最高，而sigma54-4的外显率最低。为了确定细胞内的运输方式，叠氮化钠被用来灭活用于氢离子运输的atp酶。所有内部运输将通过非主动方法进行，例如被动或促进扩散。通过这两种类型的处理，可以确定钉接肽的摄取方式是通过主动还是被动运输。此外，在被动转运中，由于缺乏促进细胞进入所需的代谢手段，肽的进入应该比主动更快。大多数肽类似物在叠氮化钠处理后渗透性增加。这可能是一个指标，肽能够被细菌泵出一定程度。总的来说，该实验表明，与未装订的肽相比，装订肽以电荷无关的方式穿透细胞的能力更好。在共聚焦显微镜下进行了进一步的研究，以获得评估细胞外显率的低通量视觉方法。多肽与流式细胞术数据相关，其中一些细胞显示强荧光强度，而另一些细胞显示弱荧光强度。通过基于图像的渗透性检测方法，我们观察到大肠杆菌的双链DNA似乎聚集在远离膜的中心。在许多观察到的细胞中，许多细胞正在进行细胞分裂，但仍然大量摄取这些肽。在细胞膜中，由于缺乏绿色或红色膜染色的遮蔽，可以看到肽不能将自身整合到膜中。这些肽必须穿过细胞壁和细胞膜才能留在细菌的细胞质中。利用荧光各向异性结合荧光能量转移，对sigma54 RpoN与glnA -24位点的结合进行了深入研究。荧光各向异性是确定生物分子亲和力的有力工具，其中发射极化和分子旋转扩散依赖于分子结合。在本研究中，深入研究了FITC标记的sigma54钉接α -螺旋肽在不同浓度的双链-24 glnA序列下的各向异性变化，以确定肽与DNA之间的解离常数（kD）。接下来，我们使用荧光寿命成像显微镜（FLIM）装置来评估肽类似物与固相结合的30个碱基对的双链glnA DNA序列之间的特异性结合。福斯特共振能量转移（FRET）是一种广泛使用的技术，用于确定生物分子结合以及生物分子构象和结构变化的定量分析，其中FRET是在供体和受体荧光团（1-10 nm）接近时观察到的。选择FITC作为肽供体是因为它在488nm处激发，而选择Cy5作为受体是因为它缺乏488nm激发，在FITC的发射范围内吸收，在FITC的发射范围外发射。在这个实验装置中，Cy5标记的DNA（受体）被固定在金纳米颗粒（AuNP）薄膜上，后者的目的是增强单分子荧光事件。通过使用单分子事件，可以可视化DNA和肽之间的直接相互作用。然后将FITC标记的肽（供体）添加到AuNP-glnA偶联物中，仅在DNA与钉接肽的特异性结合时观察到FRET。当标记和未标记的FITC的最大值对齐时，DNA和肽之间的相互作用显示出由于在488nm处激发Cy5信号的存在而发生FRET。一种改良的经典运动试验被用来确定细菌通过软介质的能力。敲除MC4100菌株显示出一种表型，在这种表型中，细菌在远离接种点的地方不显著生长。在BW25113细胞中，所有肽相对于载体和野生型肽都表现出一定程度的抑制作用。从细菌生长直径的变化中可以看出，与敲除细胞系相比，肽不具有相同的运动抑制作用。由于肽降低了运动相关基因的表达，肽的整体能力确实显示了细菌鞭毛运动能力的降低。

A number of structural studies have confirmed the binding of sigma54 to RNAP and DNA. Sigma54 binds DNA -24 and -12 base pairs from the transcription start site to initiate DNA melting, an essential step in transcription. A substantial body of work, including DNA footprinting, alanine scanning mutation studies, low resolution cryoelectron microscopy of sigma54-RNA-polyermase bound to DNA in the closed configuration, and high resolution NMR structure has shown that sigma54 binds specifically and tightly to the major groove of DNA. For example the Wemmer lab used NMR to show that the 66 amino acid long helix-turn-helix (HTH) motif from in Aquifex aeolicus binds DNA. Within this C-terminal HTH motif, a single alpha-helix at residues 377 to 386 (ARRTVAKYRE), termed the RpoN box, is responsible for binding to the major groove of DNA (PBD: 2O8K). Replacement of Arg378, Arg379, Tyr384 and Arg385 with Ala decreased DNA binding substantially. The protein NMR structure shows that this helix interacts selectively with the -24 region (5'-TGGCACG-3') of the promoter. In particular, Arg378 and Arg379 are localized to the -24 element of the interaction and make multiple hydrogen bonding and ionic interactions with Gua-25 and Gua-26 on the non-coding strand of DNA based on significant line-broadening of a 15N-HSQC spectrum. This leads to a high affinity interaction between the 66-mer HTH motif and the promoter region (Kd = 114 nM) Although the peptides are modeled to inhibit transcription, it is necessary to assess cell viability to ensure that downstream antivirulence effects are observable. By using the standard broth microdilution method,24 the antimicrobial activity of all stapled peptide analogs was assessed. The minimum inhibitory concentration (MIC) value of all peptides is 32 microg/mL or higher. As this value is high compared to many conventional antibiotics, the peptide can be assessed at low concentrations to study its effects on virulence properties of Gram negative bacteria. To determine whether the compounds are cytotoxic to eukaryotic cells, WS1 fibroblasts were exposed to serial dilutions of each compound. The data demonstrate that the compounds are not toxic to normal cells over a concentration range peaking at 10 microM. In order to assess the helical structure of the synthesized peptides, circular dichroism spectroscopy was carried out by dissolving the compounds in water. While the wild type sigma54 peptide is unstructured, stapled peptides 1-4 all possess the hallmark spectrum of an alpha-helical secondary structure. Flow cytometry was used to gain a high-throughput perspective on the effectiveness of each stapled peptide in a large population of bacteria. By making use of its individual event detection, the percentage of cellular uptake of the peptides was determined. In both E. coli and P. aeruginosa species of Gram negative bacteria, all four peptide analogs are capable of penetration. Compared to the vehicle control, the wild type peptide, the sequence in which no staple is present, shows minor penetration in PA01 P. aeruginosa and insignificant levels of penetration in BW25113 E. coli. With each peptide, there is a penetration of at least 50% of cell in each peptide sample. In particular, sigma54-2 appears to have the highest penetrance whereas sigma54-4 has the least. To determine the mode of transport within the cell, sodium azide was used to inactivate ATPases used in hydrogen ion transport. All interior transport will be conducted through a non-active method such as passive or facilitated diffusion. With both types of treatments, it's possible to determine whether the mode of uptake of stapled peptides is through active or passive transport. Furthermore, with passive transport, the entry of the peptide should be faster than active due to the lack of metabolic means necessary to promote cell entry. The majority of peptide analogs increase in penetration upon sodium azide treatment. This may be an indicator that the peptide is able to be pumped out by bacteria to a degree. Overall, this assay demonstrates that stapled peptides are capable of penetrating cells better than their unstapled counterpart in a charge-independent manner. Further studies in confocal microscopy were conducted to gain a low-throughput visual method of assessing cell penetrance. The peptides correlate with the flow cytometry data in that some cells display strong fluorescence intensity and others display less. By using an image-based detection method for permeability, we observe that the double-stranded DNA of E. coli appears to aggregate in the center away from the membrane. In many of the cells observed, a number of the cells were undergoing cell division but still had significant uptake of these peptides. With the cell membranes, it is seen that the peptide does not integrate itself into the membrane due to a lack of green or masking of the red membrane stain. These peptides must cross both the cell wall and cell membrane to remain in the cytoplasm of bacteria. The binding of sigma54 RpoN with the -24 site of glnA was thoroughly examined through the use of fluorescence anisotropy coupled with fluorescence energy transfer. Fluorescence anisotropy is a powerful tool in determining biomolecular affinities where the emission polarization and the molecular rotational diffusion are dependent upon the molecular binding. In this study, the change in anisotropy of FITC tagged sigma54 stapled alpha-helical peptides at varying concentrations of the double stranded -24 glnA sequence were studied thoroughly to determine the dissociation constant (kD) between the peptide and the DNA. Next, we used the fluorescence lifetime imaging microscope (FLIM) setup in order to evaluate the specific binding between the peptide analogs and the double-stranded glnA DNA sequence of 30 base pairs in length bound to a solid phase. Foester resonance energy transfer (FRET) is a widely used technique in determining biomolecular binding along with the quantitative analysis of biomolecular conformational and structural changes where FRET is observed upon the close proximity of the donor and acceptor fluorophores (1-10 nm). FITC was selected as the peptide donor due to its excitation at 488 nm and Cy5 as the acceptor due to its lack of 488 nm excitation, its absorption within FITC's emission range, and its emission outside the FITC emission range. In this experimental setup, a Cy5 tagged DNA (acceptor) was immobilized on to a thin film of gold nanoparticles (AuNP), where the latter's purpose is to enhance the single molecular fluorescence events. By using single molecular events, it is possible to visualize the direct interaction between DNA and peptide. FITC tagged peptide (donor) was then added to the AuNP-glnA conjugates, with FRET only to be observed upon the specific binding of the DNA and the stapled peptide. As the maxima of both tagged and non-tagged FITC are aligned, the interaction between DNA and peptide displays a profile that shows FRET occurs due to the presence of a Cy5 signal from an excitation at 488 nm. A modified classical motility assay was used to determine the bacteria's ability to travel through soft media. Knockout MC4100 strain displays a phenotype in which the bacteria do not grow significantly away from the point of inoculation. In BW25113 cells, all peptides show a degree of inhibition relative to the vehicle and wild-type peptide. As demonstrated from the change in diameter of bacterial growth, it is shown that the peptides do not possess same motility inhibition compared to a knocked out cell line. As the peptide decreases the expression of motility-related genes, the overall ability of the peptides does demonstrate a decrease in flagellar mobility of the bacteria.