Quantification of biomolecular binding dynamics by Fluorescence Correlation Spectroscopy

Diffusion and molecular binding processes are indispensable for biological systems. A vital step towards the understanding of such dynamics and their interplay is a thorough quantification of all parameters involved. This work addresses the characterization of biomolecular diffusion and binding dynamics using fluorescence correlation spectroscopy (FCS). To quantify the reversible surface attachment of fluorescently labeled molecules, a novel method termed surface-integrated FCS (SI-FCS) is developed. Using this technique, the association and dissociation rates of receptor-ligand pairs can be determined over a wide range of time scales, ranging from hundreds of milliseconds to tens of seconds. The surface of interest is exposed to a widefield illumination and a highly sensitive electron-multiplying charge-coupled device (EMCCD) camera is used for detection, not only providing single-molecule sensitivity, but also enabling a parallel detection of the signal, which facilitates multiplexed SI-FCS measurements across the field of view. To validate this approach, we quantify the reversible hybridization of single-stranded deoxyribonucleic acid (DNA) using a standard total internal reflection fluorescence (TIRF) microscope. The nucleotide overlap was systematically varied to demonstrate the sensitivity of SI-FCS. Furthermore, this work extensively employs FCS in its more conventional form using a confocal microscope. The effect of refractive index mismatches on single-focus FCS measurements is thoroughly characterized and a regime in which unbiased experiments are possible is identified. Confocal FCS is used to monitor the filament formation of FtsZ proteins (filamenting temperature-sensitive mutant Z) and their breakage by the protein MipZ in vitro. Potential artifacts are identified and a novel model to analyze diffusing filaments in FCS experiments is derived, applied, and validated. These findings not only demonstrate that filament formation can be efficiently studied using confocal FCS, but also indicate that FtsZ from Caulobacter crescentus may intrinsically form small oligomers. Finally, this work characterizes the diffusion of biomolecules in lipid monolayers at the air-water interface using confocal FCS. A miniaturized fixed area-chamber, which requires only minute amounts of protein, is presented and validated. Using this design, monolayer experiments become accessible to studies where biomolecules can only be purified in small amounts. Moreover, the quantification of diffusion in monolayers using FCS is a major step towards the routine characterization of binding of biomolecules to lipid monolayers.Diffusion und molekulare Bindungsreaktionen sind elementare Prozesse in biologischen Systemen. Für das Verständnis solcher Dynamiken und deren Wechselwirkungen ist es letztlich unabdingbar die beteiligten Parameter exakt zu quantifizieren. Diesem Ziel folgend setzt sich diese Arbeit mit der Quantifizierung von Diffusions- und Bindungsdynamiken unter Nutzung der Fluoreszenzkorrelationsspektroskopie (FCS) auseinander. Um die Assoziations- und Dissoziationsraten von reversiblen Bindungsreaktionen an Oberflächen zu messen, wurde im Rahmen dieser Arbeit eine neuartige Methode namens "surface-integrated FCS" (SI-FCS) entwickelt. Mittels dieser Methode können Bindungsraten zwischen Rezeptoren und fluoreszierenden Liganden in Zeitbereichen von Millisekunden bis über einer Minute gemessen werden. Die zu untersuchende Oberfläche, an der die Bindungsreaktionen stattfinden, wird mit einer Weitfeldausleuchtung beschienen und die daraufhin emittierte Fluoreszenz von den Liganden wird mit einer sehr empfindlichen Kamera (electron-multiplying charge-coupled device) detektiert. Diese Flächendetektion verfügt nicht nur über ausreichende Empfindlichkeit um einzelne Moleküle zu detektieren, sondern ermöglicht auch die parallele Messung mehrerer Autokorrelationskurven im Sichtfeld. Zur Validierung dieses neuartigen Ansatzes wird die reversible Hybridisierung von Desoxyribonukleinsäuren (DNS) mit einem im Rahmen dieser Arbeit konstruierten totalreflexionsbasiertem Fluoreszenzmikroskop (TIRF Mikroskop) quantifiziert. Die Anzahl der hybridisierenden Basenpaare wird in dieser Studie systematisch variiert und drückt sich in klaren Änderungen der gemessenen Bindungsraten aus. Damit wird die Sensitivität der Methode unterstrichen. Darüber hinaus bedient sich diese Arbeit der konventionellen konfokalen FCS. Das Problem von Proben, die einen anderen Brechungsindex als den von Wasser aufweisen, wird intensiv im Kontext von FCS Messungen beleuchtet. Abschließend werden Messbedingungen aufgezeigt unter denen systematische Messfehler und Artefakte, die auf den Brechungsindex zurückzuführen sind, vermieden werden können. In einem Teil dieser Arbeit wird die konfokale FCS genutzt um die Polymerisation von FtsZ Proteinen (Filamenting Temperature-Sensitive Z), sowie deren Zerlegung durch das Protein MipZ, zu untersuchen. Potentielle Fehlerquellen solcher Messungen werden beleuchtet und ein neues Modell für die Analyse von konfokalen FCS Messungen an Filamenten wird hergeleitet. Die präsentierten Ergebnisse zeigen nicht nur, dass FCS eine geeignete Methode ist umWachstum und Zerfall von Filamenten im Allgemeinen zu charakterisieren, sondern liefern auch deutliche Hinweise, dass FtsZ aus dem Bakterium Caulobacter crescentus auch in Abwesenheit von Guanosintriphosphat (GTP) kurze Oligomere bildet. Letzteres ist insbesondere interessant, da typischerweise angenommen wird, dass FtsZ als monomeres Protein vorliegt und erst in Anwesenheit von GTP zu Filamenten polymerisiert. Abschließend quantifiziert diese Arbeit die Diffusion von Biomolekülen in Lipidmonoschichten an der Grenzfläche zwischen Luft und Wasser. Unter Verwendung der konfokalen FCS werden Messungen in Miniaturkammern durchgeführt und validiert. Mithilfe dieser Methode werden Messungen an Biomolekülen ermöglicht, die nur in sehr geringen Mengen aufgereinigt werden können. Die hier präsentierten Diffusionsmessungen stellen einen wichtigen Schritt hin zur FCS basierten Charakterisierung der Bindungskinetiken von Biomolekülen zu Lipidmonoschichten dar

Quantification of biomolecular binding dynamics by Fluorescence Correlation Spectroscopy

Diffusion and molecular binding processes are indispensable for biological systems. A vital step towards the understanding of such dynamics and their interplay is a thorough quantification of all parameters involved. This work addresses the characterization of biomolecular diffusion and binding dynamics using fluorescence correlation spectroscopy (FCS). To quantify the reversible surface attachment of fluorescently labeled molecules, a novel method termed surface-integrated FCS (SI-FCS) is developed. Using this technique, the association and dissociation rates of receptor-ligand pairs can be determined over a wide range of time scales, ranging from hundreds of milliseconds to tens of seconds. The surface of interest is exposed to a widefield illumination and a highly sensitive electron-multiplying charge-coupled device (EMCCD) camera is used for detection, not only providing single-molecule sensitivity, but also enabling a parallel detection of the signal, which facilitates multiplexed SI-FCS measurements across the field of view. To validate this approach, we quantify the reversible hybridization of single-stranded deoxyribonucleic acid (DNA) using a standard total internal reflection fluorescence (TIRF) microscope. The nucleotide overlap was systematically varied to demonstrate the sensitivity of SI-FCS. Furthermore, this work extensively employs FCS in its more conventional form using a confocal microscope. The effect of refractive index mismatches on single-focus FCS measurements is thoroughly characterized and a regime in which unbiased experiments are possible is identified. Confocal FCS is used to monitor the filament formation of FtsZ proteins (filamenting temperature-sensitive mutant Z) and their breakage by the protein MipZ in vitro. Potential artifacts are identified and a novel model to analyze diffusing filaments in FCS experiments is derived, applied, and validated. These findings not only demonstrate that filament formation can be efficiently studied using confocal FCS, but also indicate that FtsZ from Caulobacter crescentus may intrinsically form small oligomers. Finally, this work characterizes the diffusion of biomolecules in lipid monolayers at the air-water interface using confocal FCS. A miniaturized fixed area-chamber, which requires only minute amounts of protein, is presented and validated. Using this design, monolayer experiments become accessible to studies where biomolecules can only be purified in small amounts. Moreover, the quantification of diffusion in monolayers using FCS is a major step towards the routine characterization of binding of biomolecules to lipid monolayers.Diffusion und molekulare Bindungsreaktionen sind elementare Prozesse in biologischen Systemen. Für das Verständnis solcher Dynamiken und deren Wechselwirkungen ist es letztlich unabdingbar die beteiligten Parameter exakt zu quantifizieren. Diesem Ziel folgend setzt sich diese Arbeit mit der Quantifizierung von Diffusions- und Bindungsdynamiken unter Nutzung der Fluoreszenzkorrelationsspektroskopie (FCS) auseinander. Um die Assoziations- und Dissoziationsraten von reversiblen Bindungsreaktionen an Oberflächen zu messen, wurde im Rahmen dieser Arbeit eine neuartige Methode namens "surface-integrated FCS" (SI-FCS) entwickelt. Mittels dieser Methode können Bindungsraten zwischen Rezeptoren und fluoreszierenden Liganden in Zeitbereichen von Millisekunden bis über einer Minute gemessen werden. Die zu untersuchende Oberfläche, an der die Bindungsreaktionen stattfinden, wird mit einer Weitfeldausleuchtung beschienen und die daraufhin emittierte Fluoreszenz von den Liganden wird mit einer sehr empfindlichen Kamera (electron-multiplying charge-coupled device) detektiert. Diese Flächendetektion verfügt nicht nur über ausreichende Empfindlichkeit um einzelne Moleküle zu detektieren, sondern ermöglicht auch die parallele Messung mehrerer Autokorrelationskurven im Sichtfeld. Zur Validierung dieses neuartigen Ansatzes wird die reversible Hybridisierung von Desoxyribonukleinsäuren (DNS) mit einem im Rahmen dieser Arbeit konstruierten totalreflexionsbasiertem Fluoreszenzmikroskop (TIRF Mikroskop) quantifiziert. Die Anzahl der hybridisierenden Basenpaare wird in dieser Studie systematisch variiert und drückt sich in klaren Änderungen der gemessenen Bindungsraten aus. Damit wird die Sensitivität der Methode unterstrichen. Darüber hinaus bedient sich diese Arbeit der konventionellen konfokalen FCS. Das Problem von Proben, die einen anderen Brechungsindex als den von Wasser aufweisen, wird intensiv im Kontext von FCS Messungen beleuchtet. Abschließend werden Messbedingungen aufgezeigt unter denen systematische Messfehler und Artefakte, die auf den Brechungsindex zurückzuführen sind, vermieden werden können. In einem Teil dieser Arbeit wird die konfokale FCS genutzt um die Polymerisation von FtsZ Proteinen (Filamenting Temperature-Sensitive Z), sowie deren Zerlegung durch das Protein MipZ, zu untersuchen. Potentielle Fehlerquellen solcher Messungen werden beleuchtet und ein neues Modell für die Analyse von konfokalen FCS Messungen an Filamenten wird hergeleitet. Die präsentierten Ergebnisse zeigen nicht nur, dass FCS eine geeignete Methode ist umWachstum und Zerfall von Filamenten im Allgemeinen zu charakterisieren, sondern liefern auch deutliche Hinweise, dass FtsZ aus dem Bakterium Caulobacter crescentus auch in Abwesenheit von Guanosintriphosphat (GTP) kurze Oligomere bildet. Letzteres ist insbesondere interessant, da typischerweise angenommen wird, dass FtsZ als monomeres Protein vorliegt und erst in Anwesenheit von GTP zu Filamenten polymerisiert. Abschließend quantifiziert diese Arbeit die Diffusion von Biomolekülen in Lipidmonoschichten an der Grenzfläche zwischen Luft und Wasser. Unter Verwendung der konfokalen FCS werden Messungen in Miniaturkammern durchgeführt und validiert. Mithilfe dieser Methode werden Messungen an Biomolekülen ermöglicht, die nur in sehr geringen Mengen aufgereinigt werden können. Die hier präsentierten Diffusionsmessungen stellen einen wichtigen Schritt hin zur FCS basierten Charakterisierung der Bindungskinetiken von Biomolekülen zu Lipidmonoschichten dar

Photo-Induced Depletion of Binding Sites in DNA-PAINT Microscopy

The limited photon budget of fluorescent dyes is the main limitation for localization precision in localization-based super-resolution microscopy. Points accumulation for imaging in nanoscale topography (PAINT)-based techniques use the reversible binding of fluorophores and can sample a single binding site multiple times, thus elegantly circumventing the photon budget limitation. With DNA-based PAINT (DNA-PAINT), resolutions down to a few nanometers have been reached on DNA-origami nanostructures. However, for long acquisition times, we find a photo-induced depletion of binding sites in DNA-PAINT microscopy that ultimately limits the quality of the rendered images. Here we systematically investigate the loss of binding sites in DNA-PAINT imaging and support the observations with measurements of DNA hybridization kinetics via surface-integrated fluorescence correlation spectroscopy (SI-FCS). We do not only show that the depletion of binding sites is clearly photo-induced, but also provide evidence that it is mainly caused by dye-induced generation of reactive oxygen species (ROS). We evaluate two possible strategies to reduce the depletion of binding sites: By addition of oxygen scavenging reagents, and by the positioning of the fluorescent dye at a larger distance from the binding site

Residence times of single membrane-targeted FtsZ molecules at the bilayer, dependent on nucleotide and free Mg²⁺.

<p>(A) Overlaid images of FtsZ-YFP-mts structures (yellow channel) incubated with GFP-Booster-Atto647N (nanobody) (single molecules: red channel) in the presence of GTP (a, b), GDP (c, d), and indicated free Mg²⁺ concentration. The protein concentration in all cases was around 0.2 μM. (B) Mean residence times of FtsZ-YFP-mts were calculated using an exponential fit of the cumulative residence time distribution. Mean residence times were measured for different GTP and Mg²⁺ conditions. Further details under Materials and methods and Results. GDP, guanosine diphosphate; GFP, green fluorescent protein; GTP, guanosine triphosphate; mts, membrane-targeting sequence; YFP, yellow fluorescent protein.</p

Free Mg²⁺ regulates protein surface concentration and thus self-organization of membrane-targeted GTP-FtsZ.

<p>Representative snapshots showing TIRFM images of FtsZ-YFP-mts (0.2 μM) polymers on the bilayer taken 2–3 min after the addition of 0.04 mM GTP in the presence of 1 mM and 5 mM free Mg²⁺ concentrations, respectively. Next to each image, the mean fluorescence intensity, proportional to the FtsZ-YFP-mts density on the membrane, is shown (average of 3 experiments). The protein network observed at 5 mM free Mg²⁺ correlates with a high FtsZ-YFP-mts density regime, at least 3-fold larger (approximately 1,500 A.U.) than required for ring formation (approximately 500 A.U.) The scale bar represents 5 μM. A.U., arbitrary units; GTP, guanosine triphosphate; mts, membrane-targeting sequence; TIRFM, total internal reflection fluorescence microscope; YFP, yellow fluorescent protein.</p

Nucleation and growth of FtsZ filaments into rings on SLBs.

<p>(A) Representative snapshots from a time-lapse experiment displaying different stages of ring formation. Images were taken every 10 s using TIRF illumination (YFP channel). Frames correspond to the times (min) indicated after the addition of GTP and Mg²⁺. (B) Polar clockwise growth of a single FtsZ from a nucleation point. Growth seems to occur stepwise and depend on the accessibility of small filaments nearby (panel 1 and 2). Lower local protein density (white arrow) correlates with a higher flexibility of the polymer (panels 7–12). Breakage occurs primarily in trailing regions (15). After about 3 min, a primitive ring made of 3 distinct short filaments is exhibited (17). (C) Directional filament gliding via treadmilling. Fragmentation or depolymerization destabilizes the trailing (“older”) edge as shown in the kymograph (d-labeled white line). Images in (B) and (C) were taken every 2 s, and the scale bar represents 500 nm. Further details are under “Results.” Movies reconstructed from the whole collection of images can be found in supporting information <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004845#pbio.2004845.s008" target="_blank">S2</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2004845#pbio.2004845.s009" target="_blank">S3</a> Movies. GTP, guanosine triphosphate; SLB, supported lipid bilayer; TIRF, total internal reflection fluorescence; YFP, yellow fluorescent protein.</p

(A) FtsZ-YFP-mts ring formation is dependent on GTP and protein surface concentration.

<p>At low protein concentration and no GTP and Mg2+, FtsZ transiently binds to the membrane without forming visible structures. With GTP, dynamic chiral rings are formed as a function of time. Once stable swirls are built, they exhibit a mean velocity of 34 nm/s and a turnover time of short fragments of 11.5 s. From the velocity and the turnover time, the average length of the protofilaments can be estimated to 390 nm. However, ring formation is only observed at intermediate protein density regimes. At high protein density, a parallel network of filaments (nematic phase) is observed. (B) To guarantee chirality, attachment needs to have a perpendicular component to both the ring curvature and filament polarity to have a preferential binding face. In this case, the mts interacts with the flat surface on opposite sides of the FtsZ filament, so curvature is also in the opposite direction. (C) We here suggest that an intrinsic helical FtsZ shape, characterized by a radius and a pitch, can alternatively explain previous FtsZ-induced inwards/outwards deformations in the following way: Due to the intrinsic pitch, the growing filament would either pull up (left) or push down (right) the surface. On the contrary, if the surface is not deformable (SLB), the filament would experience a strain, get destabilized, and eventually break upon growth. GDP, guanosine diphosphate; GTP, guanosine triphosphate; GUV, giant unilamellar vesicles; mts, membrane-targeting sequence; SLB, supported lipid bilayer; YFP, yellow fluorescent protein.</p

Steady-state treadmilling and chirality of FtsZ vortices: Dependence on GTPase activity and location of the mts.

<p>(A-C) Left panels: Representative snapshots of the rings formed upon addition of GTP (4 mM) and Mg²⁺ (5 mM) by (A) FtsZ-YFP-mts, whose mts is located at the C-terminus of FtsZ; (B) mts-H-FtsZ-YFP, with mts located at the N-terminus of FtsZ; and (C) FtsZ*[T108A]-YFP-mts, a variant of FtsZ-mts with diminished GTPase activity. Right panels: Kymograph analysis showing (A) a positive slope that corresponds to the apparent clockwise rotation time of the selected ring (red circle); (B) a negative slope that corresponds to an apparent counterclockwise rotation, indicating that the position of the mts determines the chirality of the apparent rotation; (C) no apparent slope corresponding to static rings, suggesting that the apparent rotation in (A) and (B) is mediated by GTP hydrolysis. (D) Velocity distributions for FtsZ-YFP-mts (red) and mts-H-FtsZ-YFP (blue) with mean rotational speed values of 34 nm/s and 25 nm/s, respectively. Further details under Materials and methods and Results. GTP, guanosine triphosphate; mts, membrane-targeting sequence; YFP, yellow fluorescent protein.</p

Dependence of FtsZ-YFP-mts vortex formation on protein surface concentration.

<p>(A) Time dependence of the average fluorescence intensity of FtsZ-YFP-mts on the bilayer upon 4 mM GTP and 5 mM Mg²⁺ addition, as measured by TIRFM, at 0.2 (blue line) and 0.5 (red line) μM protein concentration. The gray area marks the intensity when first closed rings are observed, which is approximately the same for both protein concentrations. After closed rings have formed, the further accumulation of protein at the surface is strongly concentration dependent. The dashed line represents the phase in which clearly discernible, locally stable dynamic vortices are observed. While at 0.2 μM, the system reaches this regime after an elapsed time of 45 min (time point 3); at 0.5 μM, it only takes approximately 20 min (time point 2). (B) Representative images of the experiment shown in panel (A). Frames were taken at elapsed times in minutes. Right: Ring size distributions at time points 2 and 3, indicated in panel (A), with average diameters of 0.94 +/− 0.16 μm, <i>N</i> = 140, and 0.98 +/− 0.14 μm, <i>N</i> = 128, respectively. Size distributions of rings are similar since both correspond to the same protein surface density (approximately 880 A.U.) Further details are under “Results.” A.U., arbitrary units; GTP, guanosine triphosphate; mts, membrane-targeting sequence; TIRFM, total internal reflection fluorescence microscope; YFP, yellow fluorescent protein.</p