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. 2016:136:35-56.
doi: 10.1016/bs.mcb.2016.03.024. Epub 2016 Jun 14.

A FRET-based method for monitoring septin polymerization and binding of septin-associated proteins

E A Booth  1 , J Thorner  1
Affiliations

A FRET-based method for monitoring septin polymerization and binding of septin-associated proteins

E A Booth et al. Methods Cell Biol. 2016.

Abstract

Much about septin function has been inferred from in vivo studies using mainly genetic methods, and much of what we know about septin organization has been obtained through examination of static structures in vitro primarily by electron microscopy. Deeper mechanistic insight requires real-time analysis of the dynamics of the assembly of septin-based structures and how other proteins associate with them. We describe here a Förster resonance energy transfer (FRET)-based approach for measuring in vitro the rate and extent of filament formation from septin complexes, binding of other proteins to septin structures, and the apparent affinities of these interactions. FRET is particularly well suited for interrogating protein-protein interactions, especially on a rapid timescale; the spectral change provides an unambiguous indication of whether two elements within the system under study are associating and serves as a molecular-level "ruler" because it is very sensitive to the separation between the donor and acceptor fluorophores over biologically relevant distances (≤10nm). The necessary procedures involve generation of appropriate cysteine-less and single cysteine-containing septin variants, expression and purification of the heterooctameric complexes containing them, efficient labeling of the purified complexes with desired fluorophores, fluorimetric measurement of FRET, and appropriate safeguards and controls in data acquisition and analysis. Our methods can be used to interrogate the effects of buffer conditions, small molecules, and septin-binding proteins on septin filament assembly or stability; determine the effect of alternative septin subunits, mutational alterations, or posttranslational modifications on assembly; and, delineate the location of septin-binding proteins.

Keywords: Dye labeling; Filament formation; Fluorescence; Förster resonance energy transfer; Protein complexes; Protein engineering; Protein purification; Protein–protein interaction; Site-directed mutagenesis; Supramolecular organization.

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Figures

FIGURE 1
FIGURE 1. Preparation of fluorescently labeled septin heterooctamers
(A) Model of yeast septin heterooctamer. The star indicates the location of the solvent-accessible Cys in Cdc11(E294C) in an otherwise Cys-less heterooctamer. (B) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel profiles of the septin-containing fraction from three purification steps: immobilized metal ion affinity chromatography (IMAC) eluate; flow-through from chitin affinity chromatography; and peak fractions from size-exclusion chromatography (SEC). (C) Examples of Cdc11(E294C)-capped and otherwise Cys-less heterooctamers labeled with either AF555-maleimide (donor) or AF647-maleimide (acceptor) fluorophores, as indicated.
FIGURE 2
FIGURE 2. Analysis of Förster resonance energy transfer (FRET) fluorescence spectra using the principal component analysis (PCA) method
(A) Absorbance (solid line) and emission (dotted line) spectra for donor AF555 (orange (light gray in print versions)) and acceptor AF647 (blue (gray in print versions)) with the donor emission and acceptor excitation spectral overlap highlighted (green (dark gray in print versions)). (B) Stochastically, half of the junctions formed during end-on-end assembly of filaments from a 50:50 mixture of donor dye–labeled heterooctamers and acceptor dye– labeled heterooctamers should display FRET (arrowheads). (C) FRET spectra showing the decrease in the donor emission (left arrow) and increase in acceptor emission (right arrow) as an increasing concentration of acceptor-labeled heterooctamers was added to a fixed concentration (25 nM) of donor dye–labeled heterooctamers. (D) The FRET spectra shown in (C) corrected for the donor-only and the acceptor-only spectra, isolating the changes in donor and acceptor emission, demonstrating that these changes are indeed the principal components (inset) responsible for the FRET observed. (E) The data from the titration experiment in (C) replotted as a binding curve (black circles; error bars representing SEM), yielding a measured Kd = 24.28 ± 13.65 nM. Modeling assuming that association of the heterooctamers is completely stochastic predicts the accompanying curve for the FRET change (red (light gray in print versions) line).
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