The primary structural photoresponse of phytochrome proteins captured by a femtosecond X-ray laser

Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The data reveal a twist of the D-ring, which leads to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signaling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the observed collective changes are important for the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light.Peer reviewe

Azido IR-probes : a novel way to study phytochromes

Fytokromit ovat kasveista, sienistä ja bakteereista löytyvien valoa aistivien proteiinien perhe, jotka toiminnallaan säätelevät näiden organismien kasvua ja kehitystä. Fytokromeilla on kaksi palautuvaa rakenteellista konformaatiota, punaista valoa absorboiva Pr ja kaukopunaista valoa absorboiva Pfr. Valosignaalin aiheuttama isomerisaatio proteiinin sisään hautautuneessa bilin-kromoforissa laukaisee konformaation muutoksen tilasta toiseen, mutta näiden suuren kokoluokan rakenteellisten muutosten havainnointi liuoksessa tai reaaliajassa on haastavaa. Tässä tutkimuksessa liitimme geneettisesti värähtelijäleiman, epäluonnollisen aminohapon p-atsidofenyylialaniinin (pAzF), yhdeksään keskeiseen paikkaan Deinococcus radiodurans bakteerifytokromissa (DrBphP). Leima on ihanteellinen hyödynnettäväksi Fourier-muunnosinfrapuna (FTIR) spektroskooppisessa analyysissa, sillä atsido-ryhmän (–N3) asymmetriset värähdykset sijaitsevat proteiinin IR-spektrin signaalittomalla taajuudella. Ympäröivän sähkökentän ja vetysitoutumisen herkästi aistivan leiman avulla voidaan siksi havaita paikallisia kemiallisia ja rakenteellisia muutoksia Pr- ja Pfr-tilojen välillä. Leiman taajuuden siirtymä paljasti selvästi havaittavia muutoksia neljän paikan lähiympäristössä. Odotettu muutos havainnollistettiin rakenteellisesti joustavassa hiuspinniulokkeessa sijaitsevassa Y472, missä leima siirtyi vahvoja vetysidoksia muodostavasta ympäristöstä eli liuottimesta Pr-tilassa heikkoja tai ei ollenkaan vetysidoksia muodostavaan ympäristöön, eli kohti proteiinia, Pfr:ssä. Samanlainen, melko radikaali muutos havaittiin lähellä isomerisoituvaa biliverdiinin D-rengasta paikassa Y176, minkä perusteella leiman vetysidospari voisi olla proteiinin sisäinen vesimolekyyli. Hiuspinniä kohti osoittavassa F203:ssa leima aisti ulokkeen uudelleen laskostumisen Pr- ja Pfr-tilojen välissä ja raportoi luultavasti hydrofobisesta ympäristöstä -heliksin vieressä Pfr:ssä. Kauempana biliverdiinistä ja hiuspinniulokkeesta sijaitsevassa R228 leiman ympäristössä havaittiin ainoastaan pienestä muutoksesta, jonka havaittujen taajuuksien ja paikan perusteella arvioitiin raportoivan jatkuvasta liuottimelle altistumisesta. FTIR- ja UV-Vis -spektroskopia paljastivat, että kaikissa yhdeksässä paikassa leima vaikutti fytokromin absorptio-ominaisuuksiin jonkin verran. Kuitenkin ainoastaan kolmessa paikassa leima häiritsi selvästi proteiinin laskostumista tai valomuutosominaisuuksia, mikä havaittiin pienenä puhdistussaantona tai poikkeavina UV-Vis absorptiospektreinä. Onnistuneesti liitettyjen leimojen todellinen vahvuus piilee niiden potentiaalissa paljastaa signaalin eteneminen reaaliajassa aikaerotteisen FTIR-spektroskopian avulla ja siten ratkaista valosignaloinnin mysteeri bakteerifytokromissa.Phytochromes are a family of photosensory proteins found in plants, fungi and bacteria, which function to support the growth and development of these organisms. Phytochromes populate two reversible structural conformations: red light absorbing Pr and far-red light absorbing Pfr. A photosignal causes the isomerization of the bilin chromophore buried inside the protein, which triggers the conversion from one state to the other, but site-specific observation of these large-scale changes in solution or in real time is challenging. In this study, a vibrational probe, an unnatural amino acid p-azidophenylalanine (pAzF), was genetically incorporated into nine key positions of the Deinococcus radiodurans bacteriophytochrome (DrBphP). This probe is ideal for Fourier-transform infrared (FTIR) spectroscopy analysis as the asymmetric vibrations of the azide group (–N3) locate in signal-free region of the protein IR spectrum. Furthermore, the probe is sensitive to its local electric environment, especially the H-bonding strength, and therefore allows the detection of site-specific structural changes between Pr and Pfr. In four positions, the frequency shift revealed clearly detectable changes in the local environment. The expected change in the structurally flexible hairpin extension was demonstrated in position Y472 where the probe moved from strongly H-bonding environment in Pr, i.e. solvent, to non- or weakly H-bonding environment in Pfr, i.e. toward the protein. Similar, quite extreme but opposite change in the local environment of the label was observed in Y176 near the biliverdin isomerizing D-ring, which suggested that the H-bonding partner of the label in Pfr is an intramolecular water molecule. In position F203 that points towards the hairpin extension, the label sensed the refolding of the extension between Pr and Pfr and reported probably on a hydrophobic environment next to the -helix in Pfr. Further from the D-ring and the hairpin, the label in R228 revealed a minor change, which, based on the observed frequencies and the position, was addressed to a continuous solvent exposure of the residue. FTIR and UV-Vis spectroscopy indicated that the label affected the DrBphP absorption properties to a certain degree in all nine positions, but only in three positions it severely impeded the protein folding or photoswitchability revealed by a low purification yield or aberrant UV-Vis absorption spectra. The true power of the successfully incorporated probes lies in their potential to reveal the signal propagation in real time by using time-resolved FTIR and, in this way, unveiling the mystery of the signal transduction in bacteriophytochromes

Tripping the light fantastic : signal transduction pathways in a bacterial phytochrome

Fytokromit ovat punaista ja kaukopunaista valoa aistivia reseptoreja kasveissa, bakteereissa, levissä ja sienissä. Ne säätelevät eliöiden kasvua ja kehitystä ympäröivien valo-olosuhteiden mukaisesti kääntämällä muutoin solulle näkymättömän valosignaalin biokemialliseen muotoon ja auttavat siten eliötä sopeutumaan ympäristöönsä. Valoaktivoituminen alkaa proteiinin sisälle hautautuneesta kromoforista, jossa tapahtuu absorption seurauksena rakenteellinen muutos. Tästä seuraa rakennemuutoksia proteiinin muissa osissa, mitkä lopulta säätelevät biokemiallista aktiivisuutta. Tätä ominaisuutta voidaan hyödyntää myös optogeneettisissä sovelluksissa solun toimintojen keinotekoiseen säätelyyn valon avulla. Tässä väitöskirjatyössä syvennyttiin valoaktivoituvana fosfataasina toimivan Deinococcus radiodurans -bakteerin fytokromin toimintaan sekä signaalinvälitykseen. Muiden fytokromien tavoin se koostuu sensori- ja toiminnallisesta yksiköstä. Sensori rakentuu kromoforin sitovasta sekä fytokromi-spesifisestä osasta (PHY), joka yhdistää sensorin biokemiallisesti toiminnalliseen osaan. Lisäksi PHY-osa sisältää tämän työn keskiössä olevan erikoisen ulokkeen, joka kurottautuu lähelle kromoforia ja uudelleen laskostuu valosyklin myötä. Tässä työssä kehitettiin UV–vis spektroskopiaa hyödyntävä menetelmä PHY-ulokkeen dynaamisuuden määrittämiseen. Ulokkeen dynaamisuuteen vaikutti sen kytkeytyminen kromoforiin, ja kokopitkässä systeemissä toiminnallinen osa stabiloi sitä. Liikehdinnän osoitettiin kytkeytyvän lisääntyneeseen toiminnallisen osan aktiivisuuteen johtaen epäspesifiseen signalointiin perustilassa. Toisaalta ilman uloketta systeemin biokemiallista toimintaa ei voitu aktivoida valolla, luultavasti koska ilman sitä signaali ei etene kromoforin sitovasta osasta eteenpäin. Ulokkeen uudelleenlaskostumista valosyklin aikana tutkittiin aikaerotteisella värähdysspektroskopialla yhdistettynä paikkaspesifiseen reportterimolekyyliin. Uloke oli aktiivinen kaikissa valosyklin välitiloissa ja päätti muutoksensa yhtäaikaisesti kromoforin kanssa. Ulokkeen dynaamisten ominaisuuksien kuvaus ja niiden yhdistäminen biokemiallisen aktiivisuuden muutoksiin tässä työssä osoittivat, että uloke on yksi avainkomponenteissa fytokromin signaalinvälityksessä.Phytochromes are red and far-red light sensing photoreceptors found in plants, bacteria, algae and fungi. They translate light cues into biochemical signalling cascades and thus allow organisms to adapt to the environment. They control growth and reproduction of nearly all vegetation, and have enormous potential for optogenetic applications to control desired cellular events by light. Light absorption causes isomerization of a bilin chromophore, which triggers structural rearrangements of the protein, ultimately controlling the biochemical activity of the system. In this thesis, spectroscopic, structural and biochemical data were combined to understand the function of a bacterial phytochrome from Deinococcus radiodurans. The system consists of a conserved photosensory module (PSM), which has a chromophore binding domain (CBD) and a phytochrome specific (PHY) domain. The PHY domain structurally and functionally connects the chormophore in CBD to the biologically active output module (OPM). Moreover, it includes a structurally flexible hairpin extension, "tongue", that extends to the vicinity of the chromophore and refolds during photoconversion. In the experimental part of the work, a method suitable to study dynamics of the tongue with UV–vis spectroscopy was established. With this method, combined with activity assays and other spectroscopic approaches, the dynamics of the tongue were shown to be dictated by the coupling with the CBD and stabilized in a complete system by the OPM. The tongue dynamics do not virtually affect the chromophore environment in the dark state, but were shown to be necessary for the thermal stability of the full-length system. The tongue is also crucial for controlling the biochemical activity of the system by light. Spectroscopic measurements suggested that in a tongueless systems, light signal becomes trapped within the CBD, which prohibits (de)activation of the OPM. During the photocycle, the tongue was shown to be in action in all intermediate states using site-selective labeling and timeresolved vibrational spectroscopy. The profound characterization of the tongue properties and dynamics with biophysical as well as biochemical approaches in this thesis revealed that the tongue is a key component in phytochrome allostery

The hairpin extension controls solvent access to the chromophore binding pocket in a bacterial phytochrome : a UV–vis absorption spectroscopy study

Solvent access to the protein interior plays an important role in the function of many proteins. Phytochromes contain a specific structural feature, a hairpin extension that appears to relay structural information from the chromophore to the rest of the protein. The extension interacts with amino acids near the chromophore, and hence shields the chromophore from the surrounding solvent. We envision that the detachment of the extension from the protein surface allows solvent exchange reactions in the vicinity of the chromophore. This can facilitate for example, proton transfer processes between solvent and the protein interior. To test this hypothesis, the kinetics of the protonation state of the biliverdin chromophore from Deinococcus radiodurans bacteriophytchrome, and thus, the pH of the surrounding solution, is determined. The observed absorbance changes are related to the solvent access of the chromophore binding pocket, gated by the hairpin extension. We therefore propose a model with an “open” (solvent-exposed, deprotonation-active on a (sub)second time-scale) state and a “closed” (solvent-gated, deprotonation inactive) state, where the hairpin fluctuates slowly between these conformations thereby controlling the deprotonation process of the chromophore on a minute time scale. When the connection between the hairpin and the biliverdin surroundings is destabilized by a point mutation, the amplitude of the deprotonation phase increases considerably. In the absence of the extension, the chromophore deprotonates essentially without any “gating”. Hence, we introduce a straightforward method to study the stability and fluctuation of the phytochrome hairpin in its photostationary state. This approach can be extended to other chromophore-protein systems where absorption changes reflect dynamic processes of the protein.peerReviewe

The interconnecting hairpin extension "arm" : An essential allosteric element of phytochrome activity

In red-light sensing phytochromes, isomerization of the bilin chromophore triggers structural and dynamic changes across multiple domains, ultimately leading to control of the output module (OPM) activity. In between, a hairpin structure, "arm", extends from an interconnecting domain to the chromophore region. Here, by removing this protein segment in a bacteriophytochrome from Deinococcus radiodurans (DrBphP), we show that the arm is crucial for signal transduction. Crystallographic, spectroscopic, and biochemical data indicate that this variant maintains the properties of DrBphP in the resting state. Spectroscopic data also reveal that the armless systems maintain the ability to respond to light. However, there is no subsequent regulation of OPM activity without the arms. Thermal denaturation reveals that the arms stabilize the DrBphP structure. Our results underline the importance of the structurally flexible interconnecting hairpin extensions and describe their central role in the allosteric coupling of phytochromes.peerReviewe

The structural effect between the output module and chromophore-binding domain is a two-way street via the hairpin extension

Signal transduction typically starts with either ligand binding or cofactor activation, eventually affecting biological activities in the cell. In red light-sensing phytochromes, isomerization of the bilin chromophore results in regulation of the activity of diverse output modules. During this process, several structural elements and chemical events influence signal propagation. In our study, we have studied the full-length bacteriophytochrome from Deinococcus radiodurans as well as a previously generated optogenetic tool where the native histidine kinase output module has been replaced with an adenylate cyclase. We show that the composition of the output module influences the stability of the hairpin extension. The hairpin, often referred as the PHY tongue, is one of the central structural elements for signal transduction. It extends from a distinct domain establishing close contacts with the chromophore binding site. If the coupling between these interactions is disrupted, the dynamic range of the enzymatic regulation is reduced. Our study highlights the complex conformational properties of the hairpin extension as a bidirectional link between the chromophore-binding site and the output module, as well as functional properties of diverse output modules.peerReviewe

Site-by-site tracking of signal transduction in an azidophenylalanine-labeled bacteriophytochrome with step-scan FTIR spectroscopy

Kurttila M, Stucki-Buchli B, Rumfeldt J, et al. Site-by-site tracking of signal transduction in an azidophenylalanine-labeled bacteriophytochrome with step-scan FTIR spectroscopy. Physical Chemistry Chemical Physics. 2021;23:5615-5628

The primary structural photoresponse of phytochrome proteins captured by a femtosecond X-ray laser

Phytochrome proteins control the growth, reproduction, and photosynthesis of plants, fungi, and bacteria. Light is detected by a bilin cofactor, but it remains elusive how this leads to activation of the protein through structural changes. We present serial femtosecond X-ray crystallographic data of the chromophore-binding domains of a bacterial phytochrome at delay times of 1 ps and 10 ps after photoexcitation. The data reveal a twist of the D-ring, which leads to partial detachment of the chromophore from the protein. Unexpectedly, the conserved so-called pyrrole water is photodissociated from the chromophore, concomitant with movement of the A-ring and a key signaling aspartate. The changes are wired together by ultrafast backbone and water movements around the chromophore, channeling them into signal transduction towards the output domains. We suggest that the observed collective changes are important for the phytochrome photoresponse, explaining the earliest steps of how plants, fungi and bacteria sense red light