Drittes Physikalisches Institut - Biophysik

Fluorescence antibunching is a well-known technique for determining the number of independent emitters per molecule or molecular complex. It was rarely applied to autofluorescent proteins due to the necessity of collecting large numbers of fluorescence photons from a single molecule, which is usually impossible to achieve with rather photolabile autofluorescent proteins. Here, we measure fluorescence antibunching on molecules in solution, allowing us to accumulate data over a large number of molecules. We use that method for determining an average stoichiometry of molecular complexes. The proposed method is absolute in the sense that it does not need any calibration or referencing. We develop the necessary theoretical background and check the method on pure dye solutions and on molecular complexes with known stoichiometry.

For measurements of transient absorption a laser-raster technique has been found to be surprisingly sensitive. It utilizes spatial separation of excitation and probing by employing a fast-flowing jet stream. The time resolution is determined by the flow velocity and the focal diameters of the continuous excitation and probing laser beams. Changes in transmission can be detected in the spectral region

The conformational dynamics of proteins plays a key role in their complex physiological functions. Fluorescence resonance energy transfer (FRET) is a particular powerful tool for studying protein conformational dynamics, but requires efficient site-specific labeling with fluorescent reporter probes. We have employed different tris-NTA/fluorophore conjugates, which bind histidine-tagged proteins with high affinity, for sitespecific incorporation of FRET acceptors into proteins, which were covalently labeled with a donor fluorophore. We demonstrate versatile application of this approach for exploring the conformation of the type I interferon receptor ectodomains ifnar1-EC and ifnar2-EC. Substantial ligand-induced conformational changes of ifnar1-EC, but not ifnar2-EC, were observed by monitoring the fluorescence intensity and the fluorescence lifetime of the FRET donor. Time-resolved fluorescence correlation spectroscopy revealed a substantial conformational flexibility of ifnar1-EC and a ligand-induced tightening. Our results demonstrate that protein labeling with tris-NTA/fluorophores enables for efficient quantitative intramolecular FRET analysis.

of molecules in the sample. Current fluorescence lifetime technology is too slow by a factor of at least 1000 to detect this decay accurately within the millisecond timescale of a typical biochemical transient. Our system, based on a direct acquisition data collection approach, records the entire fluorescence decay with S/N > 100 from a single pulse form a 10 kHz microlaser. Using this approach, we can resolve individual fluorescence lifetime components comprising as little as 10% of a complex multi-exponential fluorescence decay. When used to monitor structural transitions by time resolved FRET, we are capable of isolating individual structural states within complex structural ensembles. Using this approach we were able to simultaneously monitor the binding of myosin to different classes of binding sites on actin filaments. This type of analysis is not possible with conventional fluorescence based stopped flow measurements.

The authors present a double frequency modulation technique to determine the¯uorescence power with an accuracy of at least 10 À5 . Quantitative analysis of the modulation transfer from the excitation to the¯uorescence power is used to derive the triplet state lifetime s DS and the rate constant for intersystem crossing k ED of¯uorescent dyes. For that purpose the correlation between the population of transient states and the decrease of¯uorescence power is measured. The experimental results of rhodamine 6G are presented. The in¯uence of molecular oxygen is also examined. The photophysical parameters can be determined with high accuracy even if absorption of excited states, photoproducts or other molecules is present. This sensitivity is sucient to determine the presence of a few hundred molecules in excited states. Ó

Using a highly sensitive spectrometer we measured the absorption of transient states of different dyes in liquid solution. Analysing the temporal and spectral dependency of the absorption we determined lifetimes as well as absorption spectra of transient states. The kinetic parameters are specifically influenced by interaction with certain bases. On investigating the acid-base equilibrium of the triplet state of different rhodamine dyes we found the deprotonated triplet state to be an efficient source of a long-lived transient species. We suggest a reaction scheme which explains the dependence of the observed triplet lifetime on the concentration of amine. The possible consequences for the photostability of the dye are discussed. q 1998 Elsevier Science B.V. 0009-2614r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.

During chemotaxis and phototaxis, sperm, algae, marine zooplankton, and other microswimmers move on helical paths or drifting circles by rhythmically bending cell protrusions called motile cilia or flagella. Sperm of marine invertebrates navigate in a chemoattractant gradient by adjusting the flagellar waveform and, thereby, the swimming path. The waveform is periodically modulated by Ca(2+) oscillations. How Ca(2+) signals elicit steering responses and shape the path is unknown. We unveil the signal transfer between the changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) and path curvature (κ). We show that κ is modulated by the time derivative d[Ca(2+)](i)/dt rather than the absolute [Ca(2+)](i). Furthermore, simulation of swimming paths using various Ca(2+) waveforms reproduces the wealth of swimming paths observed for sperm of marine invertebrates. We propose a cellular mechanism for a chemical differentiator that computes a time derivative. The cytoskeleton of cilia, th...

The type I interferon (IFN) receptor plays a key role in innate immunity against viral and bacterial infections. Here, we show by intramolecular Förster resonance energy transfer spectroscopy that ligand binding induces substantial conformational changes in the ectodomain of ifnar1 (ifnar1-EC). Binding of IFNα2 and IFNβ induce very similar conformations of ifnar1, which were confirmed by single-particle electron microscopy analysis of the ternary complexes formed by IFNα2 or IFNβ with the two receptor subunits ifnar1-EC and ifnar2-EC. Photo-induced electron-transfer-based fluorescence quenching and single-molecule fluorescence lifetime measurements revealed that the ligand-induced conformational change in the membranedistal domains of ifnar1-EC is propagated to its membrane-proximal domain, which is not involved in ligand recognition but is essential for signal activation. Temperature-dependent ligand binding studies as well as stopped-flow fluorescence experiments corroborated a multistep conformational change in ifnar1 upon ligand binding. Our results thus suggest that the relatively intricate architecture of the type I IFN receptor complex is designed to propagate the ligand binding event to and possibly even across the membrane by conformational changes.

The discovery of Förster resonance energy transfer (FRET) 1 has revolutionized our ability to measure inter-and intramolecular distances on the nanometre scale using fluorescence imaging. The phenomenon is based on electromagnetic-field-mediated energy transfer from an optically excited donor to an acceptor. We replace the acceptor molecule with a metallic film and use the measured energy transfer efficiency from donor molecules to metal surface plasmons 2 to accurately deduce the distance between the molecules and metal. Like FRET, this makes it possible to localize emitters with nanometre accuracy, but the distance range over which efficient energy transfer takes place is an order of magnitude larger than for conventional FRET. This creates a new way to localize fluorescent entities on a molecular scale, over a distance range of more than 100 nm. We demonstrate the power of this method by profiling the basal lipid membrane of living cells.

In recent years, new optical systems have been developed with the ability to collect light at very high angles of emission, exceeding the critical angle of total internal reflection. Prominent examples are solid-immersion lenses and paraboloid collectors. These systems achieve high efficiencies in fluorescence detection which is an important issue for sensitive applications in analytical chemistry and biochemical assays. The exclusive collection of supercritical angle fluorescence (SAF) allows for the detection of evanescent modes and thus to confine the detection volume within one wavelength to an interface. For conventional optical systems with high numerical aperture a precise wave-optical theory of imaging was developed by Richards and Wolf in the fifties of the last century. However, their theory is not directly applicable to non-imaging, strongly aberratic light collection systems systems that collect a significant part of light above the critical angle. Here, we extend the theory to describe the optical properties of such systems.