Light- and temperature-dependent dynamics of chromophore and protein structural changes in bathy phytochrome Agp2

Bacterial phytochromes are sensoric photoreceptors that transform light absorbed by the photosensor core module (PCM) to protein structural changes that eventually lead to the activation of the enzymatic output module. The underlying photoinduced reaction cascade in the PCM starts with the isomerization of the tetrapyrrole chromophore, followed by conformational relaxations, proton transfer steps, and a secondary structure transition of a peptide segment (tongue) that is essential for communicating the signal to the output module. In this work, we employed various static and time-resolved IR and resonance Raman spectroscopic techniques to study the structural and reaction dynamics of the Meta-F intermediate of both the PCM and the full-length (PCM and output module) variant of the bathy phytochrome Agp2 from Agrobacterium fabrum. In both cases, this intermediate represents a branching point of the phototransformation, since it opens an unproductive reaction channel back to the initial state and a productive pathway to the final active state, including the functional protein structural changes. It is shown that the functional quantum yield, i.e. the events of tongue refolding per absorbed photons, is lower by a factor of ca. two than the quantum yield of the primary photochemical process. However, the kinetic data derived from the spectroscopic experiments imply an increased formation of the final active state upon increasing photon flux or elevated temperature under photostationary conditions. Accordingly, the branching mechanism does not only account for the phytochrome's function as a light intensity sensor but may also modulate its temperature sensitivity.DFG, 221545957, SFB 1078: Protonation Dynamics in Protein FunctionTU Berlin, Open-Access-Mittel – 202

Vibrationsspektroskopische Charakterisierung bakterieller Phytochrome

Phytochromes are light-sensitive proteins that contain an open tetrapyrrole as a chromophore. Found in various organisms, including plants, bacteria, cyanobacteria, and fungi, these photoreceptors regulate a wide range of physiological responses to light. Depending on the prevalent wavelength of incident light, phytochromes generally switch between two states, Pr (red-absorbing) and Pfr (far-red-absorbing). Interconversion occurs by illumination or through thermal reactions in the dark and comprises a number of intermediate states. The present study focused on protonation processes that take place during the photocycle, particularly the Pfr-to-Pr transformation. Correspondingly, a bathy phytochrome Agp2 from Agrobacterium fabrum was chosen as the main object for vibrational spectroscopic characterization. Agp2 is widely known for its complex pH-dependent behavior. Additional data were obtained from classical (i.e., with thermally stable Pr state) bacteriophytochromes from Stigmatella aurantiaca. All phytochromes studied here contain biliverdin IXα as a chromophore. In the first part of this thesis, the importance of five conserved amino acid residues in the chromophore-binding pocket of Agp2 was addressed. In total, eight different Agp2-PCM variants containing a single or a double amino acid substitution were characterized. The impact of each substitution on Pfr-to-Pr photoconversion and thermal reversion to Pfr was assessed under different pH conditions. Two essential residues were determined, Tyr165 and Arg211. Their substitution resulted in a Meta-F-like photoconversion product, which lacked the Pr-typical features: keto-enol tautomerization, secondary structure change in the conserved tongue region, and deprotonation of the propionic side chain of biliverdin ring C (propC). The other variants with substituted His278, Val244, or Phe192 were capable of forming a Pr state. However, they showed an increased pKa of propC deprotonation, which correlated with incomplete tongue restructuring at pH 7.8. The mild effects of His278 substitution in Agp2 were surprising in view of its close interaction with the chromophore, which is almost invariant among phytochromes. To further investigate the function of this histidine, phytochromes from S. aurantiaca were utilized. SaBphP2 is a typical representative of classical phytochromes, while its counterpart SaBphP1 naturally lacks the histidine, having a threonine instead. They were compared with each other as well as the respective His-Thr substitution variants. The Pr-to-Pfr photoconversion (including intermediate states) remained intact in all four SaBphPs despite an apparently lower Pfr yield in the Thr-containing variants. With regard to an earlier publication on this subject, as well as the data from Agp2, a purely regulatory role of the conserved histidine was proposed. The correlation between propC deprotonation and the extent of tongue restructuring, which was observed in Agp2, led to the suggestion that local electric field changes are essential for the formation of the Pr state. To check this hypothesis, the artificial amino acid p cyanophenylalanine (CNF) was introduced in Agp2 at two positions. FTIR spectroscopy was employed to monitor the vibrational Stark effect during each step of the photocycle. For the Agp2-Y165CNF-variant, the results were difficult to analyze due to the involvement of the CN group in hydrogen-bonding interactions. The data from Agp2-F192CNF, however, could be quantitatively analyzed. A significant change in the local electrostatic field was detected upon Meta-F-to-Pr transition, thus coinciding with propC deprotonation, whereas in the preceding reaction steps the electric field remained unchanged. The data obtained in this study allow a deeper and more detailed understanding of the mechanisms involved in the photocycle of Agp2, which have been proposed in previous publications. Also, this work provides experimental proof for the previously hypothesized relationship between the changes of the propC protonation state, secondary structure of the tongue, and local electrostatics. This study further documents the first application of vibrational Stark effect in phytochrome.Phytochrome sind lichtempfindliche Proteine, die ein offenes Tetrapyrrol als Chromophor enthalten. Diese Photorezeptoren kommen in verschiedenen Organismen vor, darunter Pflanzen, Bakterien, Cyanobakterien und Pilze. Sie regulieren eine Vielzahl physiologischer Reaktionen auf Licht. Abhängig von der vorherrschenden Wellenlänge des einfallenden Lichts wechseln Phytochrome im Allgemeinen zwischen zwei Zuständen, Pr (rot absorbierend) und Pfr (dunkelrot absorbierend). Die wechselseitige Umwandlung erfolgt durch Licht oder thermische Reaktionen im Dunkeln und umfasst eine Reihe von Zwischenzuständen. Die vorliegende Studie konzentrierte sich auf Protonierungsprozesse, die während des Photozyklus, insbesondere de Pfr-zu-Pr-Umwandlung, stattfinden. Entsprechend wurde ein Bathyphytochrom Agp2 aus Agrobacterium fabrum als Hauptobjekt für die schwingungsspektroskopische Charakterisierung ausgewählt. Agp2 ist bekannt für sein komplexes pH-abhängiges Verhalten. Zusätzliche Daten wurden von klassischen (d. h. mit thermisch stabilem Pr-Zustand) Bakteriophytochromen von Stigmatella aurantiaca erhalten. Alle untersuchten Phytochrome enthalten Biliverdin IXα als Chromophor. Im ersten Teil dieser Arbeit wurde die Bedeutung von fünf konservierten Aminosäureresten in der Chromophor-Bindungstasche von Agp2 untersucht. Insgesamt wurden acht verschiedene Agp2-PCM-Varianten charakterisiert, die eine einfache oder eine doppelte Aminosäuresubstitution enthielten. Der Einfluss jeder Substitution auf die Photokonversion von Pfr zu Pr und die thermische Reversion zu Pfr wurde unter verschiedenen pH-Bedingungen bewertet. Es wurden mit Tyr165 und Arg211 zwei essentielle Aminosäuren identifiziert. Ihre Substitution führte zu einem Meta-F-ähnlichen Photokonversionsprodukt, dem die Pr-typischen Merkmale fehlten, nämlich Keto-Enol-Tautomerisierung, Sekundärstrukturänderung in der konservierten tongue Region und Deprotonierung der Propionseitenkette des Biliverdin Rings C (propC). Die anderen Varianten mit substituiertem His278, Val244 oder Phe192 konnten einen Pr-Zustand bilden. Sie zeigten jedoch einen erhöhten pKa der propC-Deprotonierung, der mit einer unvollständigen Umstrukturierung der tongue bei pH 7.8 korrelierte. Die milden Effekte der His278-Substitution in Agp2 waren überraschend angesichts der engen Wechselwirkung mit dem Chromophor, wie sie bei nahezu allen Phytochromen vorherrscht. Um die Funktion dieses Histidins weiter zu untersuchen, wurden Phytochrome aus S. aurantiaca verwendet. SaBphP2 ist ein typischer Vertreter klassischer Phytochrome, während sein Gegenstück SaBphP1 von Natur aus statt dem Histidin ein Threonin enthält. Sie wurden miteinander sowie mit den jeweiligen His-Thr-Substitutionsvarianten verglichen. Die Photokonversion von Pr zu Pfr (einschließlich Intermediate) blieb in allen vier SaBphP Varianten intakt, trotz der offensichtlich geringeren Pfr-Ausbeute in den Thr-haltigen Varianten. Unter Berücksichtigung einer früheren Veröffentlichung zu diesem Thema sowie der Daten von Agp2 wurde eine rein regulatorische Rolle des konservierten Histidins vorgeschlagen. Die Korrelation zwischen der Deprotonierung von propC und dem Ausmaß der Umstrukturierung des tongue, die bei Agp2 beobachtet wurde, führte zu der Hypothese, dass elektrostatische Felder an der Bildung des Pr-Zustands beteiligt sind. Um dies zu untersuchen, wurde die artifizielle Aminosäure p-Cyanophenylalanin (CNF) an zwei Positionen in Agp2 eingeführt. FTIR-Spektroskopie wurde eingesetzt, um den Schwingungs-Stark-Effekt während des Photozyklus zu untersuchen. Eine Variante, Agp2-Y165CNF, lieferte aufgrund der Beteiligung der CN-Gruppe an Wasserstoffbrücken Daten, die nur schwer auswertbar waren. Im Gegensatz dazu konnten die Daten von Agp2-F192CNF quantitativ analysiert werden. Beim Übergang von Meta-F zu Pr wurde eine signifikante Änderung des lokalen elektrostatischen Feldes festgestellt, die gleichzeitig mit der propC-Deprotonierung erfolgt, während bei den vorgelagerten Reaktionen das lokale elektrische Feld nahezu unverändert blieb. Die im Verlauf dieser Studie erhaltenen Daten ermöglichen ein tieferes und detaillierteres Verständnis der Mechanismen, die am Photozyklus von Agp2 beteiligt sind und in früheren Veröffentlichungen vorgeschlagen wurden. Diese Arbeit liefert auch experimentelle Beweise für den zuvor vermuteten Zusammenhang zwischen den Änderungen des propC Protonierungszustandes, der Struktur der tongue und der lokalen Elektrostatik und dokumentiert zudem die erste Anwendung des Schwingungs-Stark-Effekts in Phytochromen

Experimental Assessment of the Electronic and Geometrical Structure of a Near-Infrared Absorbing and Highly Fluorescent Microbial Rhodopsin

The recently discovered Neorhodopsin (NeoR) exhibits absorption and emission maxima in the near-infrared spectral region, which together with the high fluorescence quantum yield makes it an attractive retinal protein for optogenetic applications. The unique optical properties can be rationalized by a theoretical model that predicts a high charge transfer character in the electronic ground state (S0) which is otherwise typical of the excited state S1 in canonical retinal proteins. The present study sets out to assess the electronic structure of the NeoR chromophore by resonance Raman (RR) spectroscopy since frequencies and relative intensities of RR bands are controlled by the ground and excited state's properties. The RR spectra of NeoR differ dramatically from those of canonical rhodopsins but can be reliably reproduced by the calculations carried out within two different structural models. The remarkable agreement between the experimental and calculated spectra confirms the consistency and robustness of the theoretical approach.</p

Parameterization of a single H-bond in Orange Carotenoid Protein by atomic mutation reveals principles of evolutionary design of complex chemical photosystems

Introduction: Dissecting the intricate networks of covalent and non-covalent interactions that stabilize complex protein structures is notoriously difficult and requires subtle atomic-level exchanges to precisely affect local chemical functionality. The function of the Orange Carotenoid Protein (OCP), a light-driven photoswitch involved in cyanobacterial photoprotection, depends strongly on two H-bonds between the 4-ketolated xanthophyll cofactor and two highly conserved residues in the C-terminal domain (Trp288 and Tyr201).Method: By orthogonal translation, we replaced Trp288 in Synechocystis OCP with 3-benzothienyl-L-alanine (BTA), thereby exchanging the imino nitrogen for a sulphur atom.Results: Although the high-resolution (1.8 Å) crystal structure of the fully photoactive OCP-W288_BTA protein showed perfect isomorphism to the native structure, the spectroscopic and kinetic properties changed distinctly. We accurately parameterized the effects of the absence of a single H-bond on the spectroscopic and thermodynamic properties of OCP photoconversion and reveal general principles underlying the design of photoreceptors by natural evolution.Discussion: Such “molecular surgery” is superior over trial-and-error methods in hypothesis-driven research of complex chemical systems

Experimental Assessment of the Electronic and Geometrical Structure of a Near-Infrared Absorbing and Highly Fluorescent Microbial Rhodopsin

The recently discovered Neorhodopsin (NeoR) exhibits absorption and emission maxima in the near-infrared spectral region, which together with the high fluorescence quantum yield makes it an attractive retinal protein for optogenetic applications. The unique optical properties can be rationalized by a theoretical model that predicts a high charge transfer character in the electronic ground state (S0) which is otherwise typical of the excited state S1 in canonical retinal proteins. The present study sets out to assess the electronic structure of the NeoR chromophore by resonance Raman (RR) spectroscopy since frequencies and relative intensities of RR bands are controlled by the ground and excited state’s properties. The RR spectra of NeoR differ dramatically from those of canonical rhodopsins but can be reliably reproduced by the calculations carried out within two different structural models. The remarkable agreement between the experimental and calculated spectra confirms the consistency and robustness of the theoretical approach

Experimental Assessment of the Electronic and Geometrical Structure of a Near-Infrared Absorbing and Highly Fluorescent Microbial Rhodopsin

The recently discovered Neorhodopsin (NeoR) exhibits absorption and emission maxima in the near-infrared spectral region, which together with the high fluorescence quantum yield makes it an attractive retinal protein for optogenetic applications. The unique optical properties can be rationalized by a theoretical model that predicts a high charge transfer character in the electronic ground state (S0) which is otherwise typical of the excited state S1 in canonical retinal proteins. The present study sets out to assess the electronic structure of the NeoR chromophore by resonance Raman (RR) spectroscopy since frequencies and relative intensities of RR bands are controlled by the ground and excited state’s properties. The RR spectra of NeoR differ dramatically from those of canonical rhodopsins but can be reliably reproduced by the calculations carried out within two different structural models. The remarkable agreement between the experimental and calculated spectra confirms the consistency and robustness of the theoretical approach

Local electric field changes during thephotoconversion of the bathy phytochrome Agp2

Phytochromes switch between a physiologically inactive and active state via a light-induced reaction cascade, which is initiated by isomerization of the tetrapyrrole chromophore and leads to the functionally relevant secondary structure transition of a protein segment (tongue). Although details of the underlying cause–effect relationships are not known, electrostatic fields are likely to play a crucial role in coupling chromophores and protein structural changes. Here, we studied local electric field changes during the photoconversion of the dark state Pfr to the photoactivated state Pr of the bathy phytochrome Agp2. Substituting Tyr165 and Phe192 in the chromophore pocket by para-cyanophenylalanine (pCNF), we monitored the respective nitrile stretching modes in the various states of photoconversion (vibrational Stark effect). Resonance Raman and IR spectroscopic analyses revealed that both pCNF-substituted variants undergo the same photoinduced structural changes as wild-type Agp2. Based on a structural model for the Pfr state of F192pCNF, a molecular mechanical–quantum mechanical approach was employed to calculate the electric field at the nitrile group and the respective stretching frequency, in excellent agreement with the experiment. These calculations serve as a reference for determining the electric field changes in the photoinduced states of F192pCNF. Unlike F192pCNF, the nitrile group in Y165pCNF is strongly hydrogen bonded such that the theoretical approach is not applicable. However, in both variants, the largest changes of the nitrile stretching modes occur in the last step of the photoconversion, supporting the view that the proton-coupled restructuring of the tongue is accompanied by a change of the electric field.DFG, 390540038, EXC 2008: Unifying Systems in Catalysis "UniSysCat"DFG, 221545957, SFB 1078: Proteinfunktion durch Protonierungsdynami