TY  - JOUR
A1  - Schubert, Jonathan
A1  - Schulze, Andrea
A1  - Prodromou, Chrisostomos
A1  - Neuweiler, Hannes
T1  - Two-colour single-molecule photoinduced electron transfer fluorescence imaging microscopy of chaperone dynamics
JF  - Nature Communications
N2  - Many proteins are molecular machines, whose function is dependent on multiple conformational changes that are initiated and tightly controlled through biochemical stimuli. Their mechanistic understanding calls for spectroscopy that can probe simultaneously such structural coordinates. Here we present two-colour fluorescence microscopy in combination with photoinduced electron transfer (PET) probes as a method that simultaneously detects two structural coordinates in single protein molecules, one colour per coordinate. This contrasts with the commonly applied resonance energy transfer (FRET) technique that requires two colours per coordinate. We demonstrate the technique by directly and simultaneously observing three critical structural changes within the Hsp90 molecular chaperone machinery. Our results reveal synchronicity of conformational motions at remote sites during ATPase-driven closure of the Hsp90 molecular clamp, providing evidence for a cooperativity mechanism in the chaperone’s catalytic cycle. Single-molecule PET fluorescence microscopy opens up avenues in the multi-dimensional exploration of protein dynamics and allosteric mechanisms.
KW  - chaperones
KW  - fluorescence spectroscopy
KW  - molecular conformation
KW  - single-molecule biophysics
KW  - total internal reflection microscopy
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-265754
VL  - 12
ER  - 
TY  - JOUR
A1  - Merz, Viktor
A1  - Merz, Julia
A1  - Kirchner, Maximilian
A1  - Lenhart, Julian
A1  - Marder, Todd B.
A1  - Krueger, Anke
T1  - Pyrene-Based "Turn-Off" Probe with Broad Detection Range for Cu\(^{2+}\), Pb\(^{2+}\) and Hg\(^{2+}\) Ions
JF  - Chemistry—A European Journal
N2  - Detection of metals in different environments with high selectivity and specificity is one of the prerequisites of the fight against environmental pollution with these elements. Pyrenes are well suited for the fluorescence sensing in different media. The applied sensing principle typically relies on the formation of intra- and intermolecular excimers, which is however limiting the sensitivity range due to masking of e. g. quenching effects by the excimer emission. Herein we report a highly selective, structurally rigid chemical sensor based on the monomer fluorescence of pyrene moieties bearing triazole groups. This sensor can quantitatively detect Cu\(^{2+}\), Pb\(^{2+}\) and Hg\(^{2+}\) in organic solvents over a broad concentrations range, even in the presence of ubiquitous ions such as Na\(^{+}\), K\(^{+}\), Ca\(^{2+}\) and Mg\(^{2+}\). The strongly emissive sensor's fluorescence with a long lifetime of 165 ns is quenched by a 1 : 1 complex formation upon addition of metal ions in acetonitrile. Upon addition of a tenfold excess of the metal ion to the sensor, agglomerates with a diameter of about 3 nm are formed. Due to complex interactions in the system, conventional linear correlations are not observed for all concentrations. Therefore, a critical comparison between the conventional Job plot interpretation, the method of Benesi-Hildebrand, and a non-linear fit is presented. The reported system enables the specific and robust sensing of medically and environmentally relevant ions in the health-relevant nM range and could be used e. g. for the monitoring of the respective ions in waste streams.
KW  - probes
KW  - fluorescence spectroscopy
KW  - pyrene
KW  - heavy metals
KW  - luminescence
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-256803
VL  - 27
IS  - 31
ER  - 
TY  - JOUR
A1  - Rajab, Suhaila
A1  - Bismin, Leah
A1  - Schwarze, Simone
A1  - Pinggera, Alexandra
A1  - Greger, Ingo H.
A1  - Neuweiler, Hannes
T1  - Allosteric coupling of sub-millisecond clamshell motions in ionotropic glutamate receptor ligand-binding domains
JF  - Communications Biology
N2  - Ionotropic glutamate receptors (iGluRs) mediate signal transmission in the brain and are important drug targets. Structural studies show snapshots of iGluRs, which provide a mechanistic understanding of gating, yet the rapid motions driving the receptor machinery are largely elusive. Here we detect kinetics of conformational change of isolated clamshell-shaped ligand-binding domains (LBDs) from the three major iGluR sub-types, which initiate gating upon binding of agonists. We design fluorescence probes to measure domain motions through nanosecond fluorescence correlation spectroscopy. We observe a broad kinetic spectrum of LBD dynamics that underlie activation of iGluRs. Microsecond clamshell motions slow upon dimerization and freeze upon binding of full and partial agonists. We uncover allosteric coupling within NMDA LBD hetero-dimers, where binding of L-glutamate to the GluN2A LBD stalls clamshell motions of the glycine-binding GluN1 LBD. Our results reveal rapid LBD dynamics across iGluRs and suggest a mechanism of negative allosteric cooperativity in NMDA receptors.
KW  - fluorescence spectroscopy
KW  - kinetics
KW  - ligand-gated ion channels
KW  - molecular neuroscience
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-261678
VL  - 4
IS  - 1
ER  -