Probing DNA - Transcription Factor Interactions Using Single-Molecule Fluorescence Detection in Nanofluidic Devices

Single-molecule fluorescence detection offers powerful ways to study biomolecules and their complex interactions. Here, nanofluidic devices and camera-based, single-molecule Förster resonance energy transfer (smFRET) detection are combined to study the interactions between plant transcription factors of the auxin response factor (ARF) family and DNA oligonucleotides that contain target DNA response elements. In particular, it is shown that the binding of the unlabeled ARF DNA binding domain (ARF-DBD) to donor and acceptor labeled DNA oligonucleotides can be detected by changes in the FRET efficiency and changes in the diffusion coefficient of the DNA. In addition, this data on fluorescently labeled ARF-DBDs suggest that, at nanomolar concentrations, ARF-DBDs are exclusively present as monomers. In general, the fluidic framework of freely diffusing molecules minimizes potential surface-induced artifacts, enables high-throughput measurements, and proved to be instrumental in shedding more light on the interactions between ARF-DBDs monomers and between ARF-DBDs and their DNA response element.</p

Iron assimilation and utilization in anaerobic ammonium oxidizing bacteria

International audienceThe most abundant transition metal in biological systems is iron. It is incorporated into protein cofactors and serves either catalytic, redox or regulatory purposes. Anaerobic ammonium oxidizing (anammox) bacteria rely heavily on iron-containing proteins – especially cytochromes – for their energy conservation, which occurs within a unique organelle, the anammoxosome. Both their anaerobic lifestyle and the presence of an additional cellular compartment challenge our understanding of iron processing. Here, we combine existing concepts of iron uptake, utilization and metabolism, and cellular fate with genomic and still limited biochemical and physiological data on anammox bacteria to propose pathways these bacteria may employ

SMA-SH: Modified Styrene–Maleic Acid Copolymer for Functionalization of Lipid Nanodiscs

Challenges in purification and subsequent functionalization of membrane proteins often complicate their biochemical and biophysical characterization. Purification of membrane proteins generally involves replacing the lipids surrounding the protein with detergent molecules, which can affect protein structure and function. Recently, it was shown that styrene–maleic acid copolymers (SMA) can dissolve integral membrane proteins from biological membranes into nanosized discs. Within these nanoparticles, proteins are embedded in a patch of their native lipid bilayer that is stabilized in solution by the amphipathic polymer that wraps the disc like a bracelet. This approach for detergent-free purification of membrane proteins has the potential to greatly simplify purification but does not facilitate conjugation of functional compounds to the membrane proteins. Often, such functionalization involves laborious preparation of protein variants and optimization of labeling procedures to ensure only minimal perturbation of the protein. Here, we present a strategy that circumvents several of these complications through modifying SMA by grafting the polymer with cysteamine. The reaction results in SMA that has solvent-exposed sulfhydrils (SMA-SH) and allows tuning of the coverage with SH groups. Size exclusion chromatography, dynamic light scattering, and transmission electron microscopy demonstrate that SMA-SH dissolves lipid bilayer membranes into lipid nanodiscs, just like SMA. In addition, we demonstrate that, just like SMA, SMA-SH solubilizes proteoliposomes into protein-loaded nanodiscs. We covalently modify SMA-SH-lipid nanodiscs using thiol-reactive derivatives of Alexa Fluor 488 and biotin. Thus, SMA-SH promises to simultaneously tackle challenges in purification and functionalization of membrane proteins

Upon adding denaturant, apoflavodoxin’s donor emission spectrum and acceptor excitation spectrum shift to the red.

<p>(a) Normalized emission spectra of ‘donor-only’ apoflavodoxin. The inset zooms in on the fluorescence emission maximum, which shifts from 518 to 521 nm upon adding 6.9 M GuHCl. (b) Normalized excitation spectrum of acceptor of d69-a178. GuHCl concentrations are 0.1 M (solid line), 1.7 M (dotted line) and 6.9 M (dashed line), respectively.</p

Covalent attachment of dye-labels to enable FRET-based probing of folding of various regions of apoflavodoxin.

<p>In all protein variants, donor label (i.e., A488) is attached to residue 69. (a) Positions of dye labels within the topology of flavodoxin. A488 is represented by a bright green star and acceptor label (i.e., A568) by a pink star. (b) Cartoon representation of d69-a1, showing in blue the backbone that intervenes residues 1 and 69. (c) d69-a131, with the backbone intervening residues 69 and 131 in green. (d) d69-a178, with the backbone intervening residues 69 and 178 in orange. A488 is shown in bright green, and A568 in pink. Cartoons are generated with PyMOL (Schrödinger, LLC, Palo Alto, Ca, USA) using the crystal structure of <i>A. vinelandii</i> flavodoxin (pdb ID 1YOB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045746#pone.0045746-Alagaratnam1" target="_blank">[56]</a>) and the molecular structures of A488 and A568, as provided by Invitrogen.</p

The denaturant-dependence of fluorescence signals of doubly dye-labeled apoflavodoxin reveals properties of apoflavodoxin’s folding intermediate.

<p>Shown are fluorescence data of ‘donor-only’ protein (open circles), d69-a1 (blue circles), d69-a131 (green diamonds) and of d69-a178 (orange squares). Protein concentration is 62.5 nM. (a) Fluorescence emission intensity of tryptophan at 330 nm with excitation at 280 nm. (b) Fluorescence emission intensity of donor (A488) at 515 nm with excitation at 450 nm. (c) Fluorescence emission intensity of acceptor (A568) at 630 nm with excitation at 580 nm. (d) Sensitized fluorescence emission intensity of acceptor at 630 nm with excitation at 450 nm. (e) Apparent FRET efficiency (<i>E_app</i>), calculated by using data of panels (b) and (d) and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045746#pone.0045746.e003" target="_blank">equation 3</a>. Standard deviations of the fluorescence signals shown vary between 1 to 3% of the measured signal intensities.</p

Denaturant-dependencies of the reorientation rates of dye labels attached to apoflavodoxins.

<p>Shown are the <i>D</i>_⊥ data of A488 of ‘donor-only’ protein (open circles), and of A568 of d69-a1 (blue circles), d69-a131 (green diamonds) and d69-a178 (orange squares), respectively.</p