Biological proteins are understood in terms of five structural levels-primary, secondary, tertiary, quaternary and quinary. The quinary structure is defined as the set of macromolecular interactions that are transient in vivo. This includes non-covalent protein-protein interactions occurring within the crowded intracellular environment. For much of twentieth century science, the canonical approach to studying biological proteins involved test tube environments. These uncrowded in vitro studies inadvertently failed to replicate and observe the quinary structures present within the original cells. Consequently, contemporary literature surrounding the fifth level of protein organisation is lacking. In particular, there is a lack of literature on the size of transient clusters within living cells. In an attempt to reconcile this gap in knowledge, we propose a quantitative method for estimating the average quinary stoichiometry in living cells. The method is based on lifetime self-quenching of fluorescently-labelled proteins in living cells. Close approach of two or more proteins in a quinary complex will result in self-quenching of the fluorescence lifetime from the fluorescent labels. Our method utilises the random mixing of proteins during cell division to mix fluorescently labelled with unlabelled proteins. Such mixing reduces the probability of adjacency between labelled proteins and, hence, decreases the probability of fluorescence lifetime quenching from labels. By monitoring fluorescence lifetime dequenching during multiple cell divisions, we can determine the average quinary structure in living proliferating cells. We demonstrate this method with a case study on cultured HeLa cells. The average quinary stoichiometry was found to be between five and six. That is, at any given point in time, there are five or six weakly interacting partners in the immediate neighbourhood of any given protein.

中文翻译：

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使用荧光寿命猝灭来估计活细胞中蛋白质的平均五元化学计量。

可以从五个结构水平（一级，二级，三级，四级和五级）理解生物蛋白质。醌结构被定义为体内瞬时的大分子相互作用的集合。这包括在拥挤的细胞内环境中发生的非共价蛋白-蛋白相互作用。在二十世纪的大部分科学中，研究生物蛋白质的规范方法涉及试管环境。这些未经拥挤的体外研究无意间未能复制并观察到原始细胞内存在的五元结构。因此，缺乏围绕蛋白质组织的第五水平的当代文献。特别是，缺乏关于活细胞内瞬时簇的大小的文献。为了调和知识上的差距，我们提出了一种定量方法，用于估计活细胞中的平均五元化学计量。该方法基于活细胞中荧光标记蛋白的终生自我猝灭。二元络合物中两种或多种蛋白质的紧密接近将导致荧光标记的荧光寿命自动猝灭。我们的方法利用细胞分裂过程中蛋白质的随机混合将荧光标记的蛋白质与未标记的蛋白质混合。这种混合降低了标记蛋白之间邻接的可能性，因此降低了荧光寿命从标记中猝灭的可能性。通过监测多个细胞分裂过程中荧光寿命的猝灭，我们可以确定活体增殖细胞中的平均五元结构。我们通过对培养的HeLa细胞进行案例研究证明了该方法。发现平均五元化学计量在5至6之间。也就是说，在任何给定的时间点，在任何给定蛋白质的直接邻域中都有五到六个弱相互作用的伴侣。