Optogenetische, allosterische Regulierung der enzymatischen Aktivität am Beispiel der T7-RNA-Polymerase
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Date
2025
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Abstract
Biochemische Reaktionsnetze passen sich an Umweltbedingungen an, indem sie chemische und physikalische Reize wahrnehmen und regulierte Mechanismen zur Steuerung von Reaktionen verwenden. Zu den physikalischen Reizen zählen insbesondere Licht und Spannung. Natürliche Photorezeptoren können Licht bestimmter Wellenlängen absorbieren, was ihre Funktion in biologischen Systemen entscheidend macht.
Die Entdeckung dieser Photorezeptoren ermöglichte die Entwicklung verschiedener Strategien zur gezielten Steuerung der Enzymaktivität und zur Untersuchung ihrer Auswirkungen auf Reaktionskaskaden und zelluläre Prozesse. Eine vielversprechende Strategie besteht in der Fusion von Licht-, Sauerstoff- und Spannungsdomänen (LOV-Domänen) mit Effektorproteinen, um Enzyme allosterisch zu kontrollieren.
In einer vorangehenden Studie wurde in dieser Arbeitsgruppe bereits die LOV-Domäne des Saathafers (Avena sativa, AsLOV2) erfolgreich in eine Effektordomäne, die RNA-Polymerase des Bakteriophagen T7, unter Erhalt der Aktivität insertiert und die lichtinduzierte Inhibierung der Polymerase durch in vitro-Transkriptionsreaktionen (IVT) bestimmt.
In dieser Folgestudie wurden die Aktivitäten der zuvor erzeugten Varianten im kinetischen Detail untersucht (apparente Michaelis-Menten-Kinetik) und neue LOV-Polymerase-Fusionsvarianten auf der Grundlage weiterer Insertionskriterien generiert. Die Ergebnisse lieferten mechanistische Erkenntnisse darüber, wie eine LOV-Domänen-Insertion die Polymeraseaktivität in einer lichtabhängigen Weise beeinflussen kann: Alle bisher untersuchten aktiven und photoresponsiven Enzymvarianten wurden nach Bestrahlung mit blauem Licht bei 470 nm teilweise inhibiert, was durch spezifische Beeinflussung der Polymerase-Eingangs- oder Ausgangskanäle (Substrateingangs- oder Produktausgangskanäle oder beides) erklärt werden kann. Diese Beeinflussung konnte durch die Modellierung der Fusionsproteine mit AlphaFold abgeleitet werden. Darüber hinaus wurden die Inhibitionsarten aus Enzymkinetiken und Modellierungen abgeleitet und erste Regeln für die Insertion von allosterischen Domänen in Polymerasen der Polymerasefamilie I aufgestellt.
Ferner wurde die insertierte LOV-Domäne der vielversprechendsten Variante durch natürlich vorkommende, rigide Strukturmotive C-terminal verlängert und die Auswirkung der Verlängerung auf die Aktivität und lichtabhängige Inhibierung bestimmt. Zusätzlich wurden mit diesem Fusionsprotein erste in-cellulo-Untersuchungen zur lichtabhängigen Inhibierung in E. coli durchgeführt. Dabei konnte die in vitro beobachtete Inhibierung der Variante in den Zellen reproduziert werden.
Anschließend wurden die Initiations- und Elongationskomplexe des Wildtyp-Enzyms durch Proteinkristallisation und Röntgenbeugungsexperimente gelöst und die photoaktiven Varianten zur Strukturaufklärung in Kristallisationsexperimenten eingesetzt, wobei erste Kristalle einer LOV-T7-RNAP-Variante erhalten werden konnten.
Biochemical reaction networks adapt to environmental conditions by sensing chemical and physical stimuli and using regulated mechanisms to control reactions. Physical stimuli include, in particular, light and voltage. Natural photoreceptors can absorb light of certain wavelengths, which is important for their function in biological systems. The discovery of these photoreceptors has enabled the development of various strategies to specifically control enzyme activity and to study their effects on reaction cascades and cellular processes. One promising strategy is the fusion of light, oxygen and voltage (LOV) domains with effector proteins to allosterically control enzymes. In a previous study, the LOV domain from oat (Avena sativa, AsLOV2) was successfully inserted into an effector domain, the RNA polymerase of bacteriophage T7, while maintaining its activity, and the light-induced inhibition of the polymerase was determined by in vitro transcription reactions (IVT). In this follow-up study, the activities of the previously generated variants were kinetically investigated in detail (apparent Michaelis-Menten kinetics) and new LOV polymerase fusion variants were designed based on additional insertion criteria. The results provided mechanistic insights into how LOV domain insertion can influence polymerase activity in a light-dependent manner: All previously studied active and photoresponsive enzyme variants were partially inhibited after irradiation with blue light at 470 nm, which can be explained by specific interactions with polymerase input or output tunnels (substrate entry or product exit tunnel or both). This influence could be derived by modeling the fusion proteins with AlphaFold. In addition, the types of inhibition were deduced from enzyme kinetics and modeling and first rules for the insertion of allosteric domains in polymerases of polymerase family I were established. Furthermore, the inserted LOV domain of the most promising variant was extended C-terminally by naturally available rigid structural motifs, and the effect of the extension on activity and light-dependent inhibition was determined. In addition, the first in cellulo studies on light-dependent inhibition in E. coli were carried out with this fusion protein. The inhibition of the variant observed in vitro could be reproduced in the cells. Subsequently, the initiation and elongation complexes of the wildtype enzyme were solved by protein crystallization and X-ray diffraction experiments, and the photoactive variants were used for structure elucidation in crystallization experiments, whereby the first crystals of a LOV-T7-RNAP variant were obtained.
Biochemical reaction networks adapt to environmental conditions by sensing chemical and physical stimuli and using regulated mechanisms to control reactions. Physical stimuli include, in particular, light and voltage. Natural photoreceptors can absorb light of certain wavelengths, which is important for their function in biological systems. The discovery of these photoreceptors has enabled the development of various strategies to specifically control enzyme activity and to study their effects on reaction cascades and cellular processes. One promising strategy is the fusion of light, oxygen and voltage (LOV) domains with effector proteins to allosterically control enzymes. In a previous study, the LOV domain from oat (Avena sativa, AsLOV2) was successfully inserted into an effector domain, the RNA polymerase of bacteriophage T7, while maintaining its activity, and the light-induced inhibition of the polymerase was determined by in vitro transcription reactions (IVT). In this follow-up study, the activities of the previously generated variants were kinetically investigated in detail (apparent Michaelis-Menten kinetics) and new LOV polymerase fusion variants were designed based on additional insertion criteria. The results provided mechanistic insights into how LOV domain insertion can influence polymerase activity in a light-dependent manner: All previously studied active and photoresponsive enzyme variants were partially inhibited after irradiation with blue light at 470 nm, which can be explained by specific interactions with polymerase input or output tunnels (substrate entry or product exit tunnel or both). This influence could be derived by modeling the fusion proteins with AlphaFold. In addition, the types of inhibition were deduced from enzyme kinetics and modeling and first rules for the insertion of allosteric domains in polymerases of polymerase family I were established. Furthermore, the inserted LOV domain of the most promising variant was extended C-terminally by naturally available rigid structural motifs, and the effect of the extension on activity and light-dependent inhibition was determined. In addition, the first in cellulo studies on light-dependent inhibition in E. coli were carried out with this fusion protein. The inhibition of the variant observed in vitro could be reproduced in the cells. Subsequently, the initiation and elongation complexes of the wildtype enzyme were solved by protein crystallization and X-ray diffraction experiments, and the photoactive variants were used for structure elucidation in crystallization experiments, whereby the first crystals of a LOV-T7-RNAP variant were obtained.
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Keywords
Optogenetik, T7-RNA-Polymerase, AsLOV2, Michaelis-Menten-Kinetik, Photochemie, Molekularbiologie, Proteinkristallographie
Subjects based on RSWK
Fotochemie, Optogenetik