Observing single F_oF₁-ATP synthase at work using an improved fluorescent protein mNeonGreen as FRET donor

Abstract

Adenosine triphosphate (ATP) is the universal chemical energy currency for cellular activities provided mainly by the membrane enzyme FoF1-ATP synthase in bacteria, chloroplasts and mitochondria. Synthesis of ATP is accompanied by subunit rotation within the enzyme. Over the past 15 years we have developed a variety of single-molecule FRET (smFRET) experiments to monitor catalytic action of individual bacterial enzymes in vitro. By specifically labeling rotating and static subunits within a single enzyme we were able to observe three-stepped rotation in the F1 motor, tenstepped rotation in the Fo motor and transient elastic deformation of the connected rotor subunits. However, the spatial and temporal resolution of motor activities measured by smFRET were limited by the photophysics of the FRET fluorophores. Here we evaluate the novel FRET donor mNeonGreen as a fusion to FoF1-ATP synthase and compare it to the previously used fluorophore EGFP. Topics of this manuscript are the biochemical purification procedures and the activity measurements of the fully functional mutant enzyme.

Towards monitoring conformational changes of the GPCR neurotensin receptor 1 by single-molecule FRET

“…The NTSR1-mNG purification is divided into four steps: Cell lysis and solubilization, nickel chelate affinity chromatography, neurotensin affinity chromatography and a final size exclusion chromatography step. In contrast to other membrane protein purifications such as purification of F O F 1 -ATP synthase [15, 30], cell lysis and membrane solubilization were done in one step. This strategy was rendered possible by the high efficiency of the subsequent purification step, i.e.…”

“…The construct ends with a decahistidine tag (H10) for purification. The gene encoding mNeonGreen (mNG) was amplified using the primer pair and with pACWU-BH1 as the template [15]. The deoxynucleotides corresponding to the mNG gene are underlined and those of restriction sites are in bold letters.…”

“…Here, we pursue the analysis of the conformational dynamics of monomeric liposome-reconstituted NTSR1 by smFRET. Towards this goal, we fused the brightest green fluorescent protein, mNeonGreen [14, 15], to the C terminus of NTSR1 and measured single-molecule anisotropy of the detergent-solubilized protein and of the receptor reconstituted in liposomes. Counting NTSR1 one-after-another revealed that most of the detergent-solubilized as well as the reconstituted NTSR1 is monomeric.…”

Fast ATP-dependent Subunit Rotation in Reconstituted FoF1-ATP Synthase Trapped in Solution

“…For smFRET recordings in the ABEL trap we prepared a specifically labeled enzyme. Briefly, we reassembled the purified F 1 part of F o F 1 -ATP synthase from E. coli with a cysteine mutation in the rotating ε-subunit at residue 56 (εH56C) 35b with the reconstituted F o part with a cysteine introduced at the C-terminus of the non-rotating a - subunit ( a CT) 38 . Cy3B-maleimide was attached to εH56C with a labeling efficiency of 74 % as the FRET donor and Alexa Fluor 647-maleimide was attached to a CT with a labeling efficiency of 32 % as the FRET acceptor.…”

Imaging cytochrome C oxidase and FoF1-ATP synthase in mitochondrial cristae of living human cells by FLIM and superresolution microscopy

“…Using the restriction enzymes HindIII (New England Biolabs) and KpnI (New England Biolabs) the -subunit of FoF1-ATP synthase variant 2 was tagged on the C terminus with mNeonGreen using a linker sequence Gly-Thr-Thr-Arg-Pro-Asp-Ile in between. The DNA sequence of the fluorescent protein mNeonGreen 13 was inserted after amplification of the mNeonGreen pACWUBH1 vector 14,15 , kindly provided by Dr. Gabriele Deckers-Hebestreit (University of Osnabrück), with restriction endonuclease EcoRV (New England Biolabs) and AvaI (New England Biolabs) in the expression vector pcDNA5/FRT (Thermo Fisher Scientific), which was designed for use with Flp-In TM system (Thermo Fisher Scientific). Correctness of sequences was confirmed by restriction analysis and by DNA sequencing.…”