Zusammenfassung (Englisch)
Light-, Oxygen- and Votlage- (LOV) domains are blue-light activated photoreceptors that control various functions in plants and algae. They contain a non-covalently bound flavin mononucleotide (FMN) in their protein core. Upon blue-light illumination, the LOV domains undergo a reversible hotocycle. This photocycle is spectroscopically well understood, however the structural aspects of it are ...
Zusammenfassung (Englisch)
Light-, Oxygen- and Votlage- (LOV) domains are blue-light activated photoreceptors that control various functions in plants and algae. They contain a non-covalently bound flavin mononucleotide (FMN) in their protein core. Upon blue-light illumination, the LOV domains undergo a reversible hotocycle. This photocycle is spectroscopically well understood, however the structural aspects of it are still under debate. Currently available data for several LOV domains show that the lighttriggered activation induces a covalent bond formation between the C4a atom of FMN and the sulphur atom of a conserved cysteine. There is no large body of work, currently available, that shows the signal propagation beyond the immediate surrounding of FMN, as this is not easily achievable with the currently available techniques. X-ray crystallography can not completely describe the signal transduction pathway, probably due to the restraint imposed by the crystal lattice. NMR and CD techniques, on the other hand, did reveal the global changes induced with the LOV domain activation, such as dissociation and loss of helical structure of the
Jα helix. This ultimately leads to the activation of the linked effector domain or of the binding to the protein partner. However, the complete pathway within the LOV
domain still remains to be fully described.
A number of photoswitches have been developed that extend the currently available optogenetic tools. The LOV2 domain of A. sativa (AsLOV2) is often used as the photosensitive part of these constructs. One of the recent photoswitches that was
shown to control the motility and growth of living cells, was the LOV2-Rac1 fusion protein. This photoactivatable Rac1 (PA-Rac1) was shown to be reversibly activated
with localized blue light illuminations. The LOV domain in this construct serves as a photo-sensor, while the Rac1 domain controls downstream effectors.
Here we show the molecular mechanism of LOV domains from R. sphaeroides and C. reinhardtii as model-systems for the mechanism guiding the LOV-part of the PA-Rac1. In RsLOV we propose a fast mechanism, propagating from the FMNpocket, through the anti-parallel β-sheets, to the A’α helix, and with it, the Jα helix. The Jα helix forms a helix-turn-helix (HTH) motif, together with the Kα helix, so the signal is propagated from the FMN-binding pocket to the HTH-motif.
As the HTH-motif forms a dimerization surface, in RsLOV homodimers, blue-light activation also affects the association properties of RsLOV. In CrLOV we were able to show the mechanism and structural aspects guiding this LOV domain. Upon introducing a single point mutation, F41Y, the mechanism changes to an electron
transfer completely abolishing adduct formation. Furthermore, with additional muVVI Abstract tation of the cysteine to an alanine or a serine, the yield and protonation state of
the formed FMN radicals can be tuned. To further understand this electron transfer process occurring in CrLOV-F41Y mutant, we developed a theoretical method to
predict the initial donor and terminal acceptor residues in addition to predicting the complete electron transfer pathway, within a protein.
In this work, we also show the mechanism behind the Rac1 protein and its spliced variant, Rac1b, as they form the second unit of the PA-Rac1 construct. We show that upon activation of the Rac1 domain the switch 1 and switch 2 regions are
found in a closed conformation, as opposed to the Rac1b domain where the same regions form a released conformation, more open for downstream-partner binding.
Moreover, the 19-amino acid insertion in Rac1b is a key structural region that controls the signal propagation and opening of the binding site. We also show the mechanisms behind the protein complexes of Rac1 and Rac1b with p67phox and RhoGDI, and the induced changes in the interaction surfaces formed between these
domains.
Finally, using the knowledge of the model-systems, we bring the mechanisms together and show the signal transduction dynamics of the LOV2-Rac1b fusion construct. We identify the important aspects of the LOV2-Rac1b structure and the
structural effects that the 19-amino acid insertion cause. LOV2 domain sits on top of the 19-amino acid insertion which acts as a loading arm. This region extends and contracts thus releasing or binding the LOV2 and Rac1b units more tightly. The dynamical changes induced in the structure upon the activation of either of the two subunits that form this photoactivatable construct are also described. This includes
changes in the binding regions, interaction surface as well as the magnesium and calcium ions. While magnesium is important in nucleotide binding and recognition, our results suggest that the calcium ion plays no role in a calcium-deficient environment. The LOV2 domain of the fusion construct shows a mechanism similar to the ones seen in our model systems. The Jα helix partially looses its helical structure, showing breakage and unwinding of the geometry. We also show the complete unfolding of the small A’α helix. Furthermore, our results suggest coupling of the Gβ and Hβ sheets which corresponds to the early stages of the β-sheet tightening process, seen in FTIR experiments for isolated LOV domains. Finally, we propose that Rac1b is controlling several structural aspects, from unit binding to nucleotide
hydrolysis, while LOV2 has control over the partner binding as well as the release of the binding regions of Rac1b, responsible for the downstream effector binding.
Übersetzung der Zusammenfassung (Deutsch)
Light, Oxygen- und Voltage (LOV) Domänen sind Blaulicht aktivierte Photorezeptoren, die verschiedene Funktionen in Plazen und Algen kontrolieren. Bei Anregung mit blauem Licht folgt die LOV Domäne einem reversiblen Photozyklus. Der komplette Signalübertragungsweg der LOV Domänen ist immer noch nicht verstanden. In dieser Arbeit wird der Signalübertragungsweg von RsLOV, Rac1, Rac1b und deren ...
Übersetzung der Zusammenfassung (Deutsch)
Light, Oxygen- und Voltage (LOV) Domänen sind Blaulicht aktivierte Photorezeptoren, die verschiedene Funktionen in Plazen und Algen kontrolieren. Bei Anregung mit blauem Licht folgt die LOV Domäne einem reversiblen Photozyklus. Der komplette Signalübertragungsweg der LOV Domänen ist immer noch nicht verstanden. In dieser Arbeit wird der Signalübertragungsweg von RsLOV, Rac1, Rac1b und deren Konstrukte mit LOV2, p67phox und RhoGDI beschrieben. Desweiteren wird ein neuer Algorithmus des Elektronentransferswegs in Proteinen, sowie eine Änderung des Reaktionsmechanismuses in CrLOV, durch die Einführung von Single-Point-Mutationen beschrieben.
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