@article{RatHeibyBunzetal.2018, author = {Rat, Charlotte and Heiby, Julia C. and Bunz, Jessica P. and Neuweiler, Hannes}, title = {Two-step self-assembly of a spider silk molecular clamp}, series = {Nature Communications}, volume = {9}, journal = {Nature Communications}, doi = {10.1038/s41467-018-07227-5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-225016}, pages = {4779, 1-11}, year = {2018}, abstract = {Web spiders synthesize silk fibers of unique strength and extensibility through the controlled self-assembly of protein building blocks, so-called spidroins. The spidroin C-terminal domain is highly conserved and connects two polypeptide chains through formation of an all-helical, intertwined dimer. Here we use contact-induced fluorescence self-quenching and resonance energy transfer in combination with far-UV circular dichroism spectroscopy as three orthogonal structural probes to dissect the mechanism of folding and dimerization of a spidroin C-terminal domain from the major ampullate gland of the nursery web spider Euprosthenops australis. We show that helices forming the dimer core assemble very rapidly and fold on association. Subsequently, peripheral helices fold and dock slowly onto the preformed core. Lability of outer helices facilitates formation of a highly expanded, partially folded dimer. The high end-to-end distance of chain termini in the partially folded dimer suggests an extensibility module that contributes to elasticity of spider silk.}, language = {en} } @article{RajabBisminSchwarzeetal.2021, author = {Rajab, Suhaila and Bismin, Leah and Schwarze, Simone and Pinggera, Alexandra and Greger, Ingo H. and Neuweiler, Hannes}, title = {Allosteric coupling of sub-millisecond clamshell motions in ionotropic glutamate receptor ligand-binding domains}, series = {Communications Biology}, volume = {4}, journal = {Communications Biology}, number = {1}, doi = {10.1038/s42003-021-02605-0}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-261678}, year = {2021}, abstract = {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.}, language = {en} } @article{HeibyGoretzkiJohnsonetal.2019, author = {Heiby, Julia C. and Goretzki, Benedikt and Johnson, Christopher M. and Hellmich, Ute A. and Neuweiler, Hannes}, title = {Methionine in a protein hydrophobic core drives tight interactions required for assembly of spider silk}, series = {Nature Communications}, volume = {10}, journal = {Nature Communications}, doi = {10.1038/s41467-019-12365-5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-202539}, pages = {4378}, year = {2019}, abstract = {Web spiders connect silk proteins, so-called spidroins, into fibers of extraordinary toughness. The spidroin N-terminal domain (NTD) plays a pivotal role in this process: it polymerizes spidroins through a complex mechanism of dimerization. Here we analyze sequences of spidroin NTDs and find an unusually high content of the amino acid methionine. We simultaneously mutate all methionines present in the hydrophobic core of a spidroin NTD from a nursery web spider's dragline silk to leucine. The mutated NTD is strongly stabilized and folds at the theoretical speed limit. The structure of the mutant is preserved, yet its ability to dimerize is substantially impaired. We find that side chains of core methionines serve to mobilize the fold, which can thereby access various conformations and adapt the association interface for tight binding. Methionine in a hydrophobic core equips a protein with the capacity to dynamically change shape and thus to optimize its function.}, language = {en} } @article{SchubertSchulzeProdromouetal.2021, author = {Schubert, Jonathan and Schulze, Andrea and Prodromou, Chrisostomos and Neuweiler, Hannes}, title = {Two-colour single-molecule photoinduced electron transfer fluorescence imaging microscopy of chaperone dynamics}, series = {Nature Communications}, volume = {12}, journal = {Nature Communications}, doi = {10.1038/s41467-021-27286-5}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-265754}, year = {2021}, abstract = {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.}, language = {en} } @article{HeibyRajabRatetal.2017, author = {Heiby, Julia C. and Rajab, Suhaila and Rat, Charlotte and Johnson, Christopher M. and Neuweiler, Hannes}, title = {Conservation of folding and association within a family of spidroin N-terminal domains}, series = {Scientific Reports}, volume = {7}, journal = {Scientific Reports}, doi = {10.1038/s41598-017-16881-6}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-159272}, pages = {16789}, year = {2017}, abstract = {Web spiders synthesize silk fibres, nature's toughest biomaterial, through the controlled assembly of fibroin proteins, so-called spidroins. The highly conserved spidroin N-terminal domain (NTD) is a pH-driven self-assembly device that connects spidroins to super-molecules in fibres. The degree to which forces of self-assembly is conserved across spider glands and species is currently unknown because quantitative measures are missing. Here, we report the comparative investigation of spidroin NTDs originating from the major ampullate glands of the spider species Euprosthenops australis, Nephila clavipes, Latrodectus hesperus, and Latrodectus geometricus. We characterized equilibrium thermodynamics and kinetics of folding and self-association using dynamic light scattering, stopped-flow fluorescence and circular dichroism spectroscopy in combination with thermal and chemical denaturation experiments. We found cooperative two-state folding on a sub-millisecond time scale through a late transition state of all four domains. Stability was compromised by repulsive electrostatic forces originating from clustering of point charges on the NTD surface required for function. pH-driven dimerization proceeded with characteristic fast kinetics yielding high affinities. Results showed that energetics and kinetics of NTD self-assembly are highly conserved across spider species despite the different silk mechanical properties and web geometries they produce.}, language = {en} }