Anthrax lethal factor (LF) is a critical component of the anthrax toxin, and functions intracellularly as a zinc-dependent endopeptidase targeting proteins involved in maintaining critical host signaling pathways. To reach the cytoplasm, LF requires to be unfolded and guided through the narrow protective antigen pore in a pH-dependent process. The current study sought to address the question as to whether LF is capable of retaining its metal ion when exposed to a low-pH environment (similar to that found in late endosomes) and an unfolding stress (induced by urea). Using a combination of tryptophan fluorescence spectroscopy and chelation studies, we show that a decrease in the pH value (from 7.0 to 5.0) leads to a pronounced shift in the onset of structural alterations in LF to lower urea concentrations. More importantly, the enzyme was found to retain its Zn²⁺ ion beyond the unfolding transitions monitored by Trp fluorescence, a finding indicative of tight metal binding to LF in a non-native state. In addition, an analysis of red-edge excitation shift (REES) spectra suggests the protein to maintain residual structure (a feature necessary for metal binding) even at very high denaturant concentrations. Furthermore, studies using the chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) revealed LF's Zn²⁺ ion to become accessible to complexation at urea concentrations in between those required to cause structural changes and metal dissociation. This phenomenon likely originates from the conversion of a PAR-inaccessible (closed) to a PAR-accessible (open) state of LF at intermediate denaturant concentrations.

炭疽致死因子（LF）是炭疽毒素的重要组成部分，在细胞内起着锌依赖性内肽酶靶向蛋白的作用，参与维持关键宿主信号通路。为了到达细胞质，需要在依赖于pH的过程中将LF展开并引导通过狭窄的保护性抗原孔。当前的研究试图解决以下问题：LF在暴露于低pH环境（类似于晚期内体中发现的环境）和展开应力（由尿素引起）时是否能够保留其金属离子。结合使用色氨酸荧光光谱法和螯合研究，我们发现pH值的降低（从7.0到5.0）导致LF的结构改变开始时尿素浓度明显降低。更重要的是，在Trp荧光监测下，超过²⁺离子的未折叠转变表明，紧密的金属以非天然状态结合到LF。此外，对红边激发位移（REES）光谱的分析表明，即使在非常高的变性剂浓度下，该蛋白质也能保持残留结构（金属结合所必需的特征）。此外，使用发色螯合剂4-（2-吡啶基偶氮）间苯二酚（PAR）进行的研究显示，在尿素浓度介于LF的Zn ²⁺离子之间，络合可使其络合，从而引起结构变化和金属解离。此现象可能是由于在中间变性剂浓度下，LF的PAR不可访问（封闭）状态转换为LF的PAR可访问（开放）状态引起的。