TY  - JOUR
A1  - Meiser, Elisabeth
A1  - Mohammadi, Reza
A1  - Vogel, Nicolas
A1  - Holcman, David
A1  - Fenz, Susanne F.
T1  - Experiments in micro-patterned model membranes support the narrow escape theory
JF  - Communications Physics
N2  - The narrow escape theory (NET) predicts the escape time distribution of Brownian particles confined to a domain with reflecting borders except for one small window. Applications include molecular activation events in cell biology and biophysics. Specifically, the mean first passage time τ can be analytically calculated from the size of the domain, the escape window, and the diffusion coefficient of the particles. In this study, we systematically tested the NET in a disc by variation of the escape opening. Our model system consisted of micro-patterned lipid bilayers. For the measurement of τ, we imaged diffusing fluorescently-labeled lipids using single-molecule fluorescence microscopy. We overcame the lifetime limitation of fluorescent probes by re-scaling the measured time with the fraction of escaped particles. Experiments were complemented by matching stochastic numerical simulations. To conclude, we confirmed the NET prediction in vitro and in silico for the disc geometry in the limit of small escape openings, and we provide a straightforward solution to determine τ from incomplete experimental traces.
KW  - membrane biophysics
KW  - single-molecule biophysics
Y1  - 2023
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-358121
VL  - 6
ER  - 
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  - Hartel, Andreas J.W.
A1  - Glogger, Marius
A1  - Jones, Nicola G.
A1  - Abuillan, Wasim
A1  - Batram, Christopher
A1  - Hermann, Anne
A1  - Fenz, Susanne F.
A1  - Tanaka, Motomu
A1  - Engstler, Markus
T1  - N-glycosylation enables high lateral mobility of GPI-anchored proteins at a molecular crowding threshold
JF  - Nature Communications
N2  - The protein density in biological membranes can be extraordinarily high, but the impact of molecular crowding on the diffusion of membrane proteins has not been studied systematically in a natural system. The diversity of the membrane proteome of most cells may preclude systematic studies. African trypanosomes, however, feature a uniform surface coat that is dominated by a single type of variant surface glycoprotein (VSG). Here we study the density-dependence of the diffusion of different glycosylphosphatidylinositol-anchored VSG-types on living cells and in artificial membranes. Our results suggest that a specific molecular crowding threshold (MCT) limits diffusion and hence affects protein function. Obstacles in the form of heterologous proteins compromise the diffusion coefficient and the MCT. The trypanosome VSG-coat operates very close to its MCT. Importantly, our experiments show that N-linked glycans act as molecular insulators that reduce retarding intermolecular interactions allowing membrane proteins to function correctly even when densely packed.
KW  - parasitology
KW  - cellular imaging
KW  - membrane biophysics
KW  - single-molecule biophysics
Y1  - 2016
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171368
VL  - 7
ER  -