11 research outputs found
Ubiquitin protease Ubp8 is necessary for S. cerevisiae respiration
No full textHealthy mitochondria are required in cell metabolism and deregulation of underlying mechanisms is often involved in human diseases and neurological disorders. Post-translational modifications of mitochondrial proteins regulate their function and activity, accordingly, impairment of ubiquitin proteasome system affects mitochondria homeostasis and organelle dynamics. In the present study we have investigated the role of the ubiquitin protease Ubp8 in S. cerevisiae respiration. We show that Ubp8 is necessary for respiration and its expression is upregulated in glycerol respiratory medium. In addition, we show that the respiratory defects in absence of Ubp8 are efficiently rescued by disruption of the E3 Ub-ligase Psh1, suggesting their epistatic link. Interestingly, we found also that Ubp8 is localized into mitochondria as single protein independently of SAGA complex assembly, thus suggesting an independent function from the nuclear one. We also show evidences on the importance of HAT Gcn5 in sustaining Ubp8 expression and affecting the amount of protein in mitochondria. Collectively, our results have investigated the role of Ubp8 in respiratory metabolism and highlight the role of ubiquitin related pathways in the mitochondrial functions of S. cerevisiae
From Mutation to Mechanism: an Approach to Understand Photoreceptors Function
No full textMicrobial rhodopsins and phytochromes are photoreceptors that use light to trigger biochemical processes. Upon light activation, they undergo a cyclic cascade of reactions leading to substrate transfer and/or conformational changes associated with the chromophore and protein. NmHR and UmRh1, two microbial rhodopsins, are a bacterial inward chloride pump and a fungal outward proton pump, respectively. While the role of NmHR from a marine bacterium is similar to that of the well-known archaeal inward chloride pump halorhodopsins (HsHR and NpHR), it shares a higher sequence homology with the bacterial outward sodium pump KR2. In addition, each rhodopsin contains a three-residue motif composed of amino acids involved in substrate transfer. The NTQ motif of NmHR is quite conserved in the sodium pump KR2 (NDQ), but is represented by a neutral TSA motif in archaebacterial halorhodopsins, suggesting a divergent evolution. A comprehensive mutational and spectroscopic analysis of residues known to be important for chloride transfer in archaeal halorhodopsins and for sodium transfer in KR2 revealed that chloride release and uptake in NmHR is different from that of archaeal halorhodopsins, but the architecture and residues involved in the process are more similar to KR2, despite the different charge and orientation of the transported ion.
UmRh1 is a rhodopsin found in the pathogenic fungus Ustilago maydis, which causes corn smut disease. This fungal rhodopsin shares a high sequence similarity with the well-known archaeal outward proton pump bacteriorhodopsin (HsBR), especially at residues known to be important for the proton pathway of the latter. The DTD motif of HsBR is conserved in UmRh1 and is represented by a DTE motif. However, extensive mutagenesis and spectroscopy revealed that the protonation dynamics of UmRh1 are slightly different from those of HsBR, especially in the proton uptake reaction. Furthermore, the pump activity of UmRh1 is enhanced in the presence of auxins such as IAA (indole-3-acetic acid). Using time-resolved spectroscopy on UmRh1 variants in the presence and absence of IAA, a potential role for auxin was elucidated. It is involved in the enhancement of the reprotonation mechanism and the proton release reaction associated with its pathogenic role.
Another aspect that has intrigued scientists over the years is the link between light and chromophore isomerization in photoreceptors, which leads to conformational changes in the protein. How does the signal propagate in the early stages of the photocycle after photon absorption? By introducing the noncanonical amino acid p-cyano-phenylalanine in NmHR and the phytochrome Agp2 into positions of conserved tryptophan residues known to be involved in conformational changes around the chromophore, preliminary results have been obtained that shed light on the protein-chromophore interaction.Mikrobielle Rhodopsine und Phytochrome sind Photorezeptoren, die Licht nutzen, um biochemische Prozesse auszulösen. Nach der Aktivierung durch Licht durchlaufen sie eine zyklische Reaktionskaskade, die zu einem Substrattransfer und/oder Konformationsänderungen im Zusammenhang mit dem Chromophor und dem Protein führt. Die hier untersuchten zwei mikrobiellen Rhodopsine sind NmHR, eine bakterielle nach innen gerichtete Chloridpumpe, und UmRh1, eine nach außen gerichtete pilzliche Protonenpumpe. Während die Rolle des aus einem Meeresbakterium stammenden NmHR derjenigen der bekannten Halorhodopsine (HsHR und NpHR) aus Archaeen ähnelt, weist es eine größere Sequenzhomologie mit der bakteriellen Natrium-Auswärtspumpe KR2 auf. Darüber hinaus enthält jedes Rhodopsin ein aus drei Aminosäuren bestehtes Motiv auf, das am Substrattransfer beteiligt ist. Das NTQ-Motiv von NmHR ist in der Natriumpumpe KR2 (NDQ) weitgehend konserviert, wird aber in archaebakteriellen Halorhodopsinen durch ein neutrales TSA-Motiv repräsentiert, was auf eine divergente Evolution hindeutet. Eine umfassende Mutationsanalyse mit nachfolgender spektroskopischer Untersuchung von Aminosäureresten, die für den Chlorid-Transfer in archaealen Halorhodopsinen und für den Natrium-Transfer in KR2 bekannt sind, ergab, dass sich in NmHR die Chloridfreisetzung und Aufnahme von der in archaealen Halorhodopsinen unterscheidet, dass aber die Architektur und die an dem Prozess beteiligten Aminosäuren dem KR2 ähnlicher sind, trotz der unterschiedlichen Ladung und Ausrichtung des transportierten Ions.
UmRh1 ist ein Rhodopsin, welches in dem pathogenen Pilz Ustilago maydis vorkommt, der die Maisfleckenkrankheit verursacht. Dieses Pilzrhodopsin weist eine hohe Sequenzähnlichkeit mit der bekannten aus Archaeen stammenden Protonenpumpe Bakteriorhodopsin (HsBR) auf, vor allem an Stellen, die für den Protonenweg der letzteren wichtig sind. Das DTD-Motiv von HsBR ist in UmRh1 konserviert und wird durch ein DTE-Motiv dargestellt. Ausführliche Mutagenese und Spektroskopie zeigten jedoch, dass sich die Protonierungsdynamik von UmRh1 geringfügig von derjenigen von HsBR unterscheidet, insbesondere bei der Protonenaufnahmereaktion. Außerdem ist die Pumpaktivität von UmRh1 in Gegenwart von Auxinen wie IAA (Indol-3-Essigsäure) verstärkt. Durch zeitaufgelöste Spektroskopie an UmRh1-Varianten in Gegenwart und Abwesenheit von IAA wurde eine mögliche Rolle von Auxin aufgedeckt. Es ist wahrscheinlich an der Verstärkung des Reprotonierungsmechanismus und der Protonenfreisetzungsreaktion im Zusammenhang mit seiner pathogenen Rolle beteiligt.
Ein weiterer Aspekt, der die Wissenschaftler im Laufe der Jahre fasziniert hat, ist die Verbindung zwischen Licht und der Isomerisierung des Chromophors in den Photorezeptoren, die zu Konformationsänderungen des Proteins führt. Wie wird das Signal in den frühen Stadien des Photozyklus nach der Photonenabsorption weitergeleitet? Durch die Einführung der nicht-kanonischen Aminosäure p-Cyano-Phenylalanin in NmHR und in Phytochrom Agp2 in Positionen von konservierten Tryptophanresten, von denen bekannt ist, dass sie an Konformationsänderungen um das Chromophor herum beteiligt sind, wurden erste Ergebnisse erzielt, die Licht auf die Protein-Chromophor-Interaktion werfen
Nanosecond Transient IR Spectroscopy of Halorhodopsin in Living Cells
No full textThe ability to track minute changes of a single amino acid residue in a cellular environment is causing a paradigm shift in the attempt to fully understand the responses of biomolecules that are highly sensitive to their environment. Detecting early protein dynamics in living cells is crucial to understanding their mechanisms, such as those of photosynthetic proteins. Here, we elucidate the light response of the microbial chloride pump NmHR from the marine bacterium Nonlabens marinus, located in the membrane of living Escherichia coli cells, using nanosecond time-resolved UV/vis and IR absorption spectroscopy over the time range from nanoseconds to seconds. Transient structural changes of the retinal cofactor and the surrounding apoprotein are recorded using light-induced time-resolved UV/vis and IR difference spectroscopy. Of particular note, we have resolved the kinetics of the transient deprotonation of a single cysteine residue during the photocycle of NmHR out of the manifold of molecular vibrations of the cells. These findings are of high general relevance, given the successful development of optogenetic tools from photoreceptors to interfere with enzymatic and neuronal pathways in living organisms using light pulses as a noninvasive trigger
The Photoreaction of the Proton-Pumping Rhodopsin 1 From the Maize Pathogenic Basidiomycete Ustilago maydis
No full textMicrobial rhodopsins have recently been discovered in pathogenic fungi and have been postulated to be involved in signaling during the course of an infection. Here, we report on the spectroscopic characterization of a light-driven proton pump rhodopsin (UmRh1) from the smut pathogen Ustilago maydis, the causative agent of tumors in maize plants. Electrophysiology, time-resolved UV/Vis and vibrational spectroscopy indicate a pH-dependent photocycle. We also characterized the impact of the auxin hormone indole-3-acetic acid that was shown to influence the pump activity of UmRh1 on individual photocycle intermediates. A facile pumping activity test was established of UmRh1 expressed in Pichia pastoris cells, for probing proton pumping out of the living yeast cells during illumination. We show similarities and distinct differences to the well-known bacteriorhodopsin from archaea and discuss the putative role of UmRh1 in pathogenesis
Membrane Protein Activity Induces Specific Molecular Changes in Nanodiscs Monitored by FTIR Difference Spectroscopy
No full textIt is well known that lipids neighboring integral membrane proteins directly influence their function. The opposite effect is true as well, as membrane proteins undergo structural changes after activation and thus perturb the lipidic environment. Here, we studied the interaction between these molecular machines and the lipid bilayer by observing changes in the lipid vibrational bands via FTIR spectroscopy. Membrane proteins with different functionalities have been reconstituted into lipid nanodiscs: Microbial rhodopsins that act as light-activated ion pumps (the proton pumps NsXeR and UmRh1, and the chloride pump NmHR) or as sensors (NpSRII), as well as the electron-driven cytochrome c oxidase RsCcO. The effects of the structural changes on the surrounding lipid phase are compared to mechanically induced lateral tension exerted by the light-activatable lipid analogue AzoPC. With the help of isotopologues, we show that the ν(C = O) ester band of the glycerol backbone reports on changes in the lipids’ collective state induced by mechanical changes in the transmembrane proteins. The perturbation of the nanodisc lipids seems to involve their phase and/or packing state. (13)C-labeling of the scaffold protein shows that its structure also responds to the mechanical expansion of the lipid bilayer
A Detailed View on the (Re)isomerization Dynamics in Microbial Rhodopsins Using Complementary Near-UV and IR Readouts
No full textIsomerization is a key process in many (bio)chemical systems. In microbial rhodopsins, the photoinduced isomerization of the all-trans retinal to the 13-cis isomer initiates a cascade of structural changes of the protein. The interplay between these changes and the thermal relaxation of the isomerized retinal is one of the crucial determinants for rhodopsin functionality. It is therefore important to probe this dynamic interplay with chromophore specific markers that combine gapless temporal observation with spectral sensitivity. Here we utilize the near-UV and mid-IR fingerprint region in the framework of a systematic (time-resolved) spectroscopic study on H+- (HsBR, (G)PR), Na+- (KR2, ErNaR) and Cl−-(NmHR) pumps. We demonstrate that the near-UV region is an excellent probe for retinal configuration and—being sensitive to the electrostatic environment of retinal—even transient ion binding, which allows us to pinpoint protein specific mechanistic nuances and chromophore-charge interactions. The combination of the near-UV and mid-IR fingerprint region hence provides a spectroscopic analysis tool that allows a detailed, precise and temporally fully resolved description of retinal configurations during all stages of the photocycle
DataSheet1_The Photoreaction of the Proton-Pumping Rhodopsin 1 From the Maize Pathogenic Basidiomycete Ustilago maydis.docx
No full textMicrobial rhodopsins have recently been discovered in pathogenic fungi and have been postulated to be involved in signaling during the course of an infection. Here, we report on the spectroscopic characterization of a light-driven proton pump rhodopsin (UmRh1) from the smut pathogen Ustilago maydis, the causative agent of tumors in maize plants. Electrophysiology, time-resolved UV/Vis and vibrational spectroscopy indicate a pH-dependent photocycle. We also characterized the impact of the auxin hormone indole-3-acetic acid that was shown to influence the pump activity of UmRh1 on individual photocycle intermediates. A facile pumping activity test was established of UmRh1 expressed in Pichia pastoris cells, for probing proton pumping out of the living yeast cells during illumination. We show similarities and distinct differences to the well-known bacteriorhodopsin from archaea and discuss the putative role of UmRh1 in pathogenesis.</p
Hydrogen Bonding and Noncovalent Electric Field Effects in the Photoconversion of a Phytochrome
No full textA profound understanding of protein structure and mechanism requires dedicated experimental and theoretical tools to elucidate electrostatic and hydrogen bonding interactions in proteins. In this work, we employed an approach to disentangle noncovalent and hydrogen-bonding electric field changes during the reaction cascade of a multidomain protein, i.e., the phytochrome Agp2. The approach exploits the spectroscopic properties of nitrile probes commonly used as reporter groups of the vibrational Stark effect. These probes were introduced into the protein through site-specific incorporation of noncanonical amino acids resulting in four variants with different positions and orientations of the nitrile groups. All substitutions left structures and the reaction mechanism unchanged. Structural models of the dark states (Pfr) were used to evaluate the total electric field at the nitrile label and its transition dipole moment. These quantities served as an internal standard to calculate the respective properties of the photoinduced products (Lumi-F, Meta-F, and Pr) based on the relative intensities of the nitrile stretching bands. In most cases, the spectral analysis revealed two substates with a nitrile in a hydrogen-bonded or hydrophobic environment. Using frequencies and intensities, we managed to extract the noncovalent contribution of the electric field from the individual substates. This analysis resulted in profiles of the noncovalent and hydrogen-bond-related electric fields during the photoinduced reaction cascade of Agp2. These profiles, which vary significantly among the four variants due to the different positions and orientations of the nitrile probes, were discussed in the context of the molecular events along the Pfr → Pr reaction cascade
Hydrogen Bonding and Noncovalent Electric Field Effects in the Photoconversion of a Phytochrome
No full textA profound understanding of protein structure and mechanism requires dedicated experimental and theoretical tools to elucidate electrostatic and hydrogen bonding interactions in proteins. In this work, we employed an approach to disentangle noncovalent and hydrogen-bonding electric field changes during the reaction cascade of a multidomain protein, i.e., the phytochrome Agp2. The approach exploits the spectroscopic properties of nitrile probes commonly used as reporter groups of the vibrational Stark effect. These probes were introduced into the protein through site-specific incorporation of noncanonical amino acids resulting in four variants with different positions and orientations of the nitrile groups. All substitutions left structures and the reaction mechanism unchanged. Structural models of the dark states (Pfr) were used to evaluate the total electric field at the nitrile label and its transition dipole moment. These quantities served as an internal standard to calculate the respective properties of the photoinduced products (Lumi-F, Meta-F, and Pr) based on the relative intensities of the nitrile stretching bands. In most cases, the spectral analysis revealed two substates with a nitrile in a hydrogen-bonded or hydrophobic environment. Using frequencies and intensities, we managed to extract the noncovalent contribution of the electric field from the individual substates. This analysis resulted in profiles of the noncovalent and hydrogen-bond-related electric fields during the photoinduced reaction cascade of Agp2. These profiles, which vary significantly among the four variants due to the different positions and orientations of the nitrile probes, were discussed in the context of the molecular events along the Pfr → Pr reaction cascade.DFG, 221545957, SFB 1078: Proteinfunktion durch ProtonierungsdynamikDFG, 390540038, EXC 2008: Unifying Systems in Catalysis "UniSysCat"TU Berlin, Open-Access-Mittel – 202
DataSheet1_Membrane Protein Activity Induces Specific Molecular Changes in Nanodiscs Monitored by FTIR Difference Spectroscopy.docx
No full textIt is well known that lipids neighboring integral membrane proteins directly influence their function. The opposite effect is true as well, as membrane proteins undergo structural changes after activation and thus perturb the lipidic environment. Here, we studied the interaction between these molecular machines and the lipid bilayer by observing changes in the lipid vibrational bands via FTIR spectroscopy. Membrane proteins with different functionalities have been reconstituted into lipid nanodiscs: Microbial rhodopsins that act as light-activated ion pumps (the proton pumps NsXeR and UmRh1, and the chloride pump NmHR) or as sensors (NpSRII), as well as the electron-driven cytochrome c oxidase RsCcO. The effects of the structural changes on the surrounding lipid phase are compared to mechanically induced lateral tension exerted by the light-activatable lipid analogue AzoPC. With the help of isotopologues, we show that the ν(C = O) ester band of the glycerol backbone reports on changes in the lipids’ collective state induced by mechanical changes in the transmembrane proteins. The perturbation of the nanodisc lipids seems to involve their phase and/or packing state. 13C-labeling of the scaffold protein shows that its structure also responds to the mechanical expansion of the lipid bilayer.</p
