Using fluorescence lifetime dequenching to estimate the average quinary stoichiometry of proteins in living cells

Abstract

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.

Special issue on fluorescence lifetime imaging (FLIM): from fundamentals to applications

“…They then monitor fluorescence lifetime dequenching during multiple cell divisions, and analyse the data based on a fluorescence lifetime self-quenching model. They find that at any given point in time, there are five or six weakly interacting partners in the immediate vicinity of any given protein in living HeLa cells [19].…”