Intracellular pH modulates quinary structure

Abstract

NMR spectroscopy can provide information about proteins in living cells. pH is an important characteristic of the intracellular environment because it modulates key protein properties such as net charge and stability. Here, we show that pH modulates quinary interactions, the weak, ubiquitous interactions between proteins and other cellular macromolecules. We use the K10H variant of the B domain of protein G (GB1, 6.2 kDa) as a pH reporter in Escherichia coli cells. By controlling the intracellular pH, we show that quinary interactions influence the quality of in-cell 15 N-1 H HSQC NMR spectra. At low pH, the quality is degraded because the increase in attractive interactions between E. coli proteins and GB1 slows GB1 tumbling and broadens its crosspeaks. The results demonstrate the importance of quinary interactions for furthering our understanding of protein chemistry in living cells.

Transient protein-protein interactions perturb E. coli metabolome and cause gene dosage toxicity

“…During the same time, the term ‘quinary structure’ (the fifth level of protein structural organization) was coined by McConkey (McConkey, 1982) to describe transient protein-protein interactions. These interactions are not only low in thermodynamic stability (K D  > 1 μM), but also have a low kinetic barrier (Wirth and Gruebele, 2013), thereby making them amenable to modulation by cellular variations like metabolite concentrations, pH (Cohen et al, 2015; Cohen and Pielak, 2016), crowding, etc. Till date, the multi-enzyme complex metabolon remains to be the best example of a quinary structure.…”

Simple NMR methods for evaluating higher order structures of monoclonal antibody therapeutics with quinary structure

“…The quinary interaction of trans -type has been demonstrated to stabilize protein structure in crowded solutions, i.e. , in cells, using H-D exchange NMR [38,39]. Here the cis -type of quinary structure attributes has been derived from dilution-injection-SEC-FPLC curves (Fig.…”

A cell is more than the sum of its (dilute) parts: A brief history of quinary structure

“…To explore the role of charge‐charge interactions in quinary structure, we quantified GB1 stability in E. coli cells at different pH values. Using the K10H variant of GB1 to report on intracellular pH and a buffer to control the intracellular pH, we showed that the quality of the in‐cell HSQC spectrum degrades at lower pH values . We used the quenched lysate method to quantify GB1 stability at different pH values to show that lowering the pH destabilizes the protein to a greater extent in cells than in buffer .…”