Chiantia, S. | bioRxiv Science

bioRxiv2018

Structural determinants of the interactions between Influenza A Virus Matrix protein M1 and lipid membranes

Influenza A virus is a pathogen responsible for severe seasonal epidemics threatening human and animal populations every year. One of the ten proteins encoded by the viral genome, the matrix protein M1, is abundantly produced in infected cells and plays a structural role in determining the morphology of the virus. During assembly of new viral particles, M1 is recruited to the host cell membrane where it associates with lipids and other viral proteins. The structure of M1 is only partially known. In particular, structural details of M1 interactions with the cellular plasma membrane as well as M1-protein interactions and multimerization have not been clarified, yet.\n\nIn this work, we employed a set of complementary experimental and theoretical tools to tackle these issues. Using raster image correlation, surface plasmon resonance and circular dichroism spectroscopies, we quantified membrane association and oligomerization of full-length M1 and of different genetically engineered M1 constructs (i.e., N- and C-terminally truncated constructs and a mutant of the polybasic region, residues 95-105). Furthermore, we report novel information on structural changes in M1 occurring upon binding to membranes. Our experimental results are corroborated by an all-atom model of the full-length M1 protein bound to a negatively charged lipid bilayer.

biophysics↗

bioRxiv2018

Quantifying protein oligomerization in living cells: A systematic comparison of fluorescent proteins

Fluorescence fluctuation spectroscopy has become a popular toolbox for non-disruptive studies of molecular interactions and dynamics in living cells. The quantification of e.g. protein oligomerization and absolute concentrations in the native cellular environment is highly relevant for a detailed understanding of complex signaling pathways and biochemical reaction networks. A parameter of particular relevance in this context is the molecular brightness, which serves as a direct measure of oligomerization and can be easily extracted from temporal or spatial fluorescence fluctuations. However, fluorescent proteins (FPs) typically used in such studies suffer from complex photophysical transitions and limited maturation, potentially inducing non-fluorescent states, which strongly affect molecular brightness measurements. Although these processes have been occasionally reported, a comprehensive study addressing this issue is missing.\n\nHere, we investigate the suitability of commonly used FPs (i.e. mEGFP, mEYFP and mCherry), as well as novel red FPs (i.e. mCherry2, mRuby3, mCardinal, mScarlet and mScarlet-I) for the quantification of oligomerization based on the molecular brightness, as obtained by Fluorescence Correlation Spectroscopy (FCS) and Number&Brightness (N&B) measurements in living cells. For all FPs, we measured a lower than expected brightness of FP homo-dimers, allowing us to estimate, for each fluorescent label, the probability of fluorescence emission in a simple two-state model. By analyzing higher FP homo-oligomers and the Influenza A virus Hemagglutinin (HA) protein, we show that the oligomeric state of protein complexes can only be accurately quantified if this probability is taken into account. Further, we provide strong evidence that mCherry2, an mCherry variant, possesses a superior apparent fluorescence probability, presumably due to its fast maturation. We finally conclude that this property leads to an improved quantification in fluorescence cross-correlation spectroscopy measurements and propose to use mEGFP and mCherry2 as the novel standard pair for studying biomolecular hetero-interactions.

biophysics↗