mass-sensitive-particle-tracking-to-characterize-membrane-associated

Mass-Sensitive Particle Tracking to Characterize Membrane-Associated Macromolecule Dynamics

Short-lived or transient interactions of macromolecules at and with lipid membranes, an interface where a multitude of essential biological reactions take ...

Fluorescent proteins (FPs) feature complex photophysical behavior that must be considered when studying the dynamics of fusion proteins in model systems and live cells. In this work, we characterize mNeonGreen (mNG), a recently introduced FP from the bilaterian Branchiostoma lanceolatum, in comparison to the well-known hydrozoan variants enhanced green fluorescent protein (EGFP) and Aequorea coerulescens GFP by steady-state spectroscopy and fluorescence correlation spectroscopy in solutions of different pH. Blind spectral unmixing of sets of absorption spectra reveals three interconverting electronic states of mNG: a nonfluorescent protonated state, a bright state showing bell-shaped pH dependence, and a similarly bright state dominating at high pH. The gradual population of the acidic form by external protonation is reflected by increased flickering at low pH in fluorescence correlation spectroscopy measurements, albeit with much slower flicker rates and lower amplitudes as compared to Aequorea GFPs. In addition, increased flickering of mNG indicates a second deprotonation step above pH 10 leading to a slight decrease in fluorescence. Thus, mNG is distinguished from Aequorea GFPs by a two-step protonation response with opposite effects that reflects a chemically distinct chromophore environment. Despite the more complex pH dependence, mNG represents a superior FP under a broad range of conditions.

Photophysical Behavior of mNeonGreen, an Evolutionarily Distant Green Fluorescent Protein.

In spite of their great importance in biology, methods providing access to spontaneous molecular interactions with and on biological membranes have been sparse. The recent advent of mass photometry to quantify mass distributions of unlabeled biomolecules landing on surfaces raised hopes that this approach could be transferred to membranes. Here, by introducing a new interferometric scattering (iSCAT) image processing and analysis strategy adapted to diffusing particles, we enable mass-sensitive particle tracking (MSPT) of single unlabeled biomolecules on a supported lipid bilayer. We applied this approach to the highly nonlinear reaction cycles underlying MinDE protein self-organization. MSPT allowed us to determine the stoichiometry and turnover of individual membrane-bound MinD/MinDE protein complexes and to quantify their size-dependent diffusion. This study demonstrates the potential of MSPT to enhance our quantitative understanding of membrane-associated biological systems.

Article	Year
Photophysical Behavior of mNeonGreen, an Evolutionarily Distant Green Fluorescent Protein. Biophysical journal\| PubMed ID: 29706225	2018
Mass-sensitive particle tracking to elucidate the membrane-associated MinDE reaction cycle. Nature methods\| PubMed ID: 34608318	2021

Frederik Steiert

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