Confocal Spectroscopy to Study Dimerization, Oligomerization and Aggregation of Proteins: A Practical Guide

Abstract

Protein self-association is a key feature that can modulate the physiological role of proteins or lead to deleterious effects when uncontrolled. Protein oligomerization is a simple way to modify the activity of a protein, as the modulation of binding interfaces allows for self-activation or inhibition, or variation in the selectivity of binding partners. As such, dimerization and higher order oligomerization is a common feature in signaling proteins, for example, and more than 70% of enzymes have the potential to self-associate. On the other hand, protein aggregation can overcome the regulatory mechanisms of the cell and can have disastrous physiological effects. This is the case in a number of neurodegenerative diseases, where proteins, due to mutation or dysregulation later in life, start polymerizing and often fibrillate, leading to the creation of protein inclusion bodies in cells. Dimerization, well-defined oligomerization and random aggregation are often difficult to differentiate and characterize experimentally. Single molecule “counting” methods are particularly well suited to the study of self-oligomerization as they allow observation and quantification of behaviors in heterogeneous conditions. However, the extreme dilution of samples often causes weak complexes to dissociate, and rare events can be overlooked. Here, we discuss a straightforward alternative where the principles of single molecule detection are used at higher protein concentrations to quantify oligomers and aggregates in a background of monomers. We propose a practical guide for the use of confocal spectroscopy to quantify protein oligomerization status and also discuss about its use in monitoring changes in protein aggregation in drug screening assays.

Fluorescence biosensor for real-time interaction dynamics of host proteins with HIV-1 capsid tubes

“…Protein concentrations were estimated using single molecule spectroscopy with recombinant eGFP as a standard. 25 The cell-free expression mixture was diluted with wash buffer (supplemented with 2 mM Trolox, 2.5 mM protocatechuic acid and 0.25 U/mL protocatechuate-3,4-dioxygenase) to a final concentration of 370 nM GFP-tagged protein. This solution (40 µL) was drawn into the flow channel (100 µL/min) and subsequently washed out with imaging buffer (500 µL at 100 µL/min) while recording the CA K158C-AF647 channel and the GFP channel by time-lapse TIRF imaging.…”

Varicella zoster virus encodes a viral decoy RHIM to inhibit cell death

“…Single molecule fluorescence confocal spectroscopy was used to confirm interactions at a single molecule level. This method uses lasers focused on a very small confocal volume (250×250×800 nm) to detect fluorescent signals from two different proteins, labelled with two different fluorophores (60) (Fig 8A). Coincidence of fluorescent signals in mixtures of proteins at low concentration indicates the formation of hybrid complexes containing both protein partners.…”

The RHIM within the M45 protein from murine cytomegalovirus forms heteromeric amyloid fibrils with RIPK1 and RIPK3

“…Fluorescence co-localisation experiments were applied to detect RHIM-based hetero-oligomers against a background of monomeric proteins and the potential competing formation of homo-oligomers 36,37 . In these experiments, multiple different RHIM-containing fusion proteins were mixed together and maintained in monomeric form in 8 M urea-containing buffer.…”