Two members of our group, David Bína and Tomáš Polívka participated in a project that resulted in a publication in Nature. The paper, published on 31 August, describes the first complete structure of a cyanobacterial antenna, the phycobilisome. In contrast to expectations, two different conformations of phycobilisomes were identified and characterized. Further, the structure obtained by cryo-electron microscopy allowed us to find and visualize the binding sites of the orange carotenoid protein (OCP), which regulates the energy flow by quenching the antenna under high light conditions. Surprisingly, each phycobilisome can bind four OCPs arranged in two dimers, but the binding is possible only in one of the two phycobilisome conformations. Our task in this project was to model the flow of energy throughout the phycobilisomes with and without OCP. The model clearly showed that OCPs can efficiently quench phycobilisomes by energy transfer from the closest bilins to the carotenoid canthaxanthin bound to OCP.
We are not only presenting our works during scientific conferences, but we also like to participate in events that are aimed at a wider audience and kids. During June the group members became part of the two public events. On the 14th of June, Tomáš Polívka presented a public lecture at Science Café CB titled “What can happen in a trillionth of a second (and how to find out)?” He has shown how we can follow some chemical reactions in real-time and what can happen after light absorption by a molecule. The evening was finalized by open discussion that all participants could enjoy. On the 25 of June, we participated in the MakerDay organized by Objevarium in Ceske Budejovice. Our lab members, Valentyna Kuznetsova and Emrah Özcan, showed some fancy optical tricks to (not only) kids. In collaboration with the Mobile Laboratory, BC AV ČR we have enjoyed the day that exceeded our expectations on public interest in scientific activities.
Our Ph.D. student Ivana Šímová spent three weeks in May at the Technical University of Munich in the group of Prof. J. Hauer as a part of her doctoral internship. Being eager to play with some fancy optics, she spent most of the time together with another Ph.D. student Erika Keil by aligning and optimising a transient absorption setup that uses the achromatic second harmonic generation as a tool to obtain very short and spectrally broad pulses. This complicated task also included a very enjoyable evening session as you can see in the photos below. :)
In our latest paper, we focus mainly on the so-called S* state of carotenoids, routinely detected via the ultrafast spectroscopy for almost 30 years. We present a brief review of this still mysterious state, with the special emphasis on a model based on the vibrational energy relaxation approach (VERA) that aims to explain S* state origin. VERA proved itself to be able to explain the main characteristics of relaxation dynamics after one-photon excitation; nevertheless, the lineshapes after two-photon excitation are still beyond the current model of VERA. In the end of the article, we outline possible future directions in terms of theoretical and experimental methods needed to better describe energy dissipation effects in carotenoids including the first solvation shell.
Following the previous study, we shed the light on light-harvesting complex of G. phototropica again. This member of Gemmatimonadota phylum obtained its photosynthetic genes by a horizontal gene transfer from other bacterial species. Such event allowed G. phototropica to evolve and optimize its own original structure of light-harvesting units, which makes it a very appealing object to study. In a new paper just published in Science Advances, the structure of its light-harvesting complex has been resolved at 2.4 Å by cryo-EM and with the help of ultrafast spectroscopy, the energy-transfer network within the whole photosynthetic unit has been described. It turns out that the elegant double-ring organization of the complex not only provides a very efficient system of energy flow but also demonstrates the endless possibilities of evolution.
Some years back we have identified quenching mechanism in high-light induced proteins (Hlips) from cyanobacteria. The fruitful collaboration with the research group at the Institute of Microbiology of the Czech Academy of Sciences continues. In a new paper just published in Nature Communications, we show that the same quenching mechanism, energy transfer from excited chlorophyll-a to the lowest carotenoid excited state, is also active in two chlorophyll-binding proteins from plants. The Light-harvesting Like (LIL3) protein and Early-Light-Induced Protein (ELIP) bind zeaxanthin, which efficiently quenches excited chlorophyll via energy transfer mechanism. While LIL3 is very efficient quencher, ELIP in its native does not quench, but modification of its N-terminus induces quenching, pointing to the critical role of pigment protein interactions in quenching induction.
Following the successful study of excited states of carotenoids after UV excitation, we have tuned our attention to carotenoids bound to proteins. To simplify the results, we have chosen proteins binding only one carotenoid and no further pigments, the orange carotenoid protein (OCP) and helical carotenoid protein (HCP). In this study, which has just been published in ChemPhotoChem journal, we have used 280 nm excitation to hit both protein (namely Tyr and Trp amino acids) and the carotenoid canthaxanthin, which also mildly absorbs at this wavelength. The results show that UV excitation enhances product formation in OCP in comparison with ‘standard’ excitation by blue-green light. In both OCP and HCP we have identified a canthaxanthin radical cation that is formed with about 5% efficiency exclusively after UV excitation.
After a year without working laser system we are back in full operation. The sad empty space on our optical table is again occupied by (now fully working) MaiTai, thus we spent most of the summer by putting all the pieces of our setup together, upgrading the beamlines and optimizing detection. At the end of July, we eventually run our first experiments after a long break.
Our new paper just published in the Journal of Chemical Physics Letters marks our long and winding road to a functional experimental setup for measurements of transient absorption spectra after two-photon excitation (2PE). After successful test of 2PE transient absorption experiment on carotenoids in solution we reported earlier, this new paper targets a more challenging sample – LHCII antenna from higher plants. By comparing the 2PE transient absorption spectra of LHCII and a mixture of the carotenoid lutein and chlorophyll-a in acetone, we were able to answer the important questions of carotenoid photophysics: Is the carotenoid selectively excited by 2PE or is there also significant contribution to 2PE from chlorophylls? If yes, how large fraction of 2PE photons is actually absorbed by carotenoids? The 2PE transient absorption experiment has proved to be an ideal tool to answer these questions. Our data show that only one third of 2PE photons is absorbed by carotenoids in LHCII, while the dominant contribution to 2PE signal originates from chlorophylls.
There is a number of papers addressing the carotenoid photophysics after excitation into their ‘color-determining’ excited state absorbing in the 400-550 nm spectral range. However, carotenoids also features spectral bands in the 250-330 nm range and whether excitation of these bands somehow changes the excited-state dynamics remains largely unknown. To add this piece of puzzle to the picture of carotenoid photophysics, we have chosen three keto-carotenoids, echinenone, canthaxanthin and rhodoxanthin, and followed their excited-state dynamics in the 400-1200 nm spectral region after excitation of UV absorption bands. The results are summarized in a paper just published in the ChemPhysChem journal. The key conclusion of our experiments is that the enigmatic S* signal is markedly enhanced after UV excitation, underlining its proposed relation to energy dissipation and storage of excess energy in molecular vibrations. We show that the S* signal is not associated with a single state; instead, contributions from both hot S1 state and non-equilibrated ground state forms the S* signal after UV excitation.
The group members are also involved in teaching and besides teaching university courses and labs, we often organize lab demonstrations for high school students. In the section For public, you can watch videos of simple do-it-yourself lab demonstrations using laser pointer as a light source.
Although our research primarily focuses on excited-state processes in carotenoids and photosynthetic light-harvesting proteins, time to time we also participate in other projects. Results of such excursion to another research area has been reported recently in Inorganic Chemistry journal. In collaboration with our colleagues from Institute of Inorganic Chemistry of the Czech Academy of Sciences, we studied properties of excited states of boron hydride clusters, compounds promising to serve as an active medium in lasers with emission in the blue spectral region. In a series of alkylated boron hydrides, the data measured in our lab revealed the excited-state absorption band, which in some compounds overlaps with the blue emission band, preventing potential laser action despite high fluorescence quantum yield.
Our group likes to work with ‘exotic’ photosynthetic antenna systems from various microorganisms, which contains unusual carotenoids. One such example is our new paper revealing energy transfer pathways in a membrane-bound antenna from the cryptophyte Rhodomonas salina. This antenna protein has few unusual features. First, it contains both chlorophyll-a and chlorophyll-c (therefore the name CAC antenna), which is combination characteristic for a number of algae, but CAC is antenna is one of a very few containing more chlorophyll-c than chlorophyll-a. Second, CAC binds the carotenoid alloxanthin, the only natural carotenoid containing two triple bonds. Both chlorophyll-c and alloxanthin transfers energy to chlorophyll-a, but efficiencies and time scales differ from ‘standard’ antenna such as LHCII, adding to the diversity of light-harvesting strategies in photosynthetic organisms.
Do you see that empty on our optical table? Anybody who ever worked in a laser lab knows it is unusual to have such a large space without the typical forest of posts and holders of various optical components. Therefore, you may think something strange is going on in our lab. And you are of course correct. This empty space is where our MaiTai laser should be. We were unfortunate to have a serious MaiTai failure during the covid crisis so we had to send it for service to the Spectra Physics labs in Germany. Now the repaired MaiTai is back, but due to travelling restrictions, we are still waiting for a technician to install it. We hope to be ready for new experiments soon. Meanwhile we use this unexpected covid-related break to work on data measured just before the MaiTai failure and even to remove dust from some old data that each of us will certainly find in long-unopened folders in our computers.
We are happy to announce that our colleague Václav Šebelík has successfully completed his PhD work on 24th of September. We were happy to be able to attend his defense in person and strictly followed the recommendations for public events at that time. Though one of the opponents from Germany had to join us virtually due to the travel restrictions. The presentation continued with follow up discussion with the opponents, committee members, and other attendees. After the official part, we had celebrated the end of Vašek’s PhD journey in a slightly new manner. :)
Vašek has started his doctoral studies at the beginning of March 2016 and has quickly became a great fit to our lab. He has been a key person in developing the two-photon and z-scan experimental setups, a perfect partner for any fun or work-related projects in the lab, a “save my time” software developer, as well as our website developer.
Though our research focuses primarily on carotenoids, we have recently diverted our attention to closely related polyenes. Thanks to our collaboration with prof. Ron Christensen from Bowdoin College in Maine, and prof. Tae-Lim Choi from Seoul National University we had opportunity to study two extremely long polyenes having ‘synthetic’ conjugation length N~200. In our new paper published in PCCP, we reported on spectroscopic properties of these polyenes. The experiments were carried at ELI Beamlines laser facility to achieve sub-50 fs time resolution that is not available in our lab. The long polyenes have sub-picosecond lifetime of the S1 state, while the ‘enigmatic’ S* state lasts just slightly longer, 1.8 ps. Comparison of these new data with earlier studies of long polyenes/carotenoids showed that there is essentially no spectroscopic difference between linear conjugated systems having conjugation lengths 50 or 200. Thus, any linear conjugated system with N>50 can be considered as an ‘infinite’ polyene/carotenoid.
HCP’s (helical carotenoid proteins) is a new family of proteins closely related to OCP. Following recent structural characterization of one member of this family, in our recent paper we focused on detailed spectroscopic characterization of two HCP’s, HCP2 and HCP3 binding the carotenoid canthaxanthin. We used our new prism spectrometer to cover whole 400-1200 nm spectral region to capture dynamics of nearly all spectral features in transient absorption spectra. Our data show that both HCP’s exists in two ground state conformations; ‘blue’ conformation excited at 470 nm exhibits an extra band in the S* region, while the ‘red’ conformation excited at 570 nm gives a standard canthaxanthin transient absorption spectrum. For both conformations, the S1 lifetime of canthaxanthin is shorter than in solution, reflecting the twist of one of the terminal rings revealed by the X-ray structure. Despite detailed structural and spectroscopic characterization, the function of HCP’s in cyanobacteria remains unknown.
In a joint effort with the group of Alexander Ruban from the Queen Mary University in London, we have taken a slightly different approach to explore mechanism of non-photochemical quenching in LHCII. It is well-known fact that quenching can be induced by aggregation of LHCII complexes and, when working at low detergent conditions, it is nearly impossible to prevent this aggregation. In experiments described in a new paper in iScience, we have immobilized LHCII trimers in polyacrylamide gel in both quenched and unquenched conformation to prevent aggregation. Under these conditions, the aggregation-induced quenching is minimal and the observed quenching must be related to specific conformation of individual non-interacting LHCII trimers. Transient absorption experiments on LHCII in gel revealed a new carotenoid spectral band whose amplitude is related to quenching.
Following our recent discovery that non-conjugated acyloxy groups can essentially switch off the intramolecular charge transfer (ICT) state of fucoxanthin if it is at the opposite side of the molecule as the conjugated keto group, we have been searching for a keto-carotenoid, which would have the acyloxy group close to the conjugated keto group. Indeed, there exists a pair of keto-carotenoids, siphonaxanthin and siphonein, that have either no acyloxy group (siphonaxanthin) or an acyloxy group at the same side of the molecule as the keto-group (siphonein). In our new paper recently published in Photosynthesis Research we show that the “acyloxy switch” really works. Depending on the position of the acyloxy group in respect to the conjugated keto group, it can either switch off (acyloxy opposite to keto) or switch on (acyloxy at the same side as keto) the ICT state.
The coronavirus infection touches the work of all of us. Yet, thanks to modern technologies and young group members who know how to use them effectively we have organized a virtual group meeting. Obviously, the spirit of the group is not much affected by the current situation. We will continue this way of communicating the results until we can go back to our offices and labs and have standard group meetings.
A new paper from the group has just appeared online in Photochemical and Photobiological Sciences. Thanks to our colleagues from the University of Cagliari and from the Warsaw University of Life Sciences we got a carotenoid-binding protein that has not yet been studied by ultrafast spectroscopy. It comes from the bacterium Deinococcus radiodurans that is well-known for its ability to sustain high doses of UV radiation essentially without damage. The bacterial cell wall of the bacterium is coated by the S-layer, which contains highly ordered two-dimensional arrays of proteins, which form hexameric complexes binding the carotenoid deinoxanthin (S-layer Deinoxanthin Binding Complex – SDBC). The paper provides the first detailed spectroscopic characterization of this carotenoid, both in solution and bound to SDBC.
Our fresh Ph.D. student, Ivča Šímová, has just returned from the workshop “Optimization of light energy conversion in plants and microalgae”, organized under SE2B in Porto, Portugal. She has presented the first data from her Ph.D. project focused on energy transfer processes in unusual purple bacterial antenna. The recently described antenna system from Gemmatimonas phototrophica featuring antenna having two concentric rings of BChl-a, and so far unknown carotenoid is just such unusual antenna.
After publishing two papers on pump-dump-probe spectroscopy in 2018, we took a short break as the two main persons behind the pump-dump-probe setup (Robert and Valja) left the group. Now, when Valja is back after nearly two-year postdoc stay in the group of Janne Ihalainen in Jyvaskyla, we are reviving the PDP setup again with a hope to reveal new features of excited state dynamics of those fantastic orange molecules – carotenoids. In the new detection setup, the PDP experiment is coupled with the prism spectrometer so we can see the effect of dumping/re-pumping in much broader spectral window than two years ago.
The February issue of BBA-Bioenergetics published our latest contribution to research of OCP and related proteins. This time we join efforts with our colleagues from other labs to combine spectroscopy and structural analysis of two OCP forms, OCP1 and OCP2. From the spectroscopic point of view, the paper shows our first data measured with a prism spectrometer in the detection system allowing to measure transient absorption spectra in the 400-1200 nm spectral region. The data presented in the paper were collected using the setup of our friends at ELI Beamlines, but now we have our own detection with prism spectrometer to run the experiments – our own data will appear soon elsewhere! Of course, we need to give up some spectral resolution in such experimental arrangement, but to see all spectral and dynamical features in the whole 400-1200 nm spectrum is simply fantastic. In the figure bellow you can see an example of the super broadband white-light continuum from our lab generated in a sapphire plate as it is seen at the CCD detector after the prism spectrometer.
The laboratory of Optical Spectroscopy has been founded in 2005 at the Institute of Physical Biology based in Nové Hrady, University of South Bohemia in České Budějovice. December 2013 we have relocated the laboratory to the newly built university facility at the Faculty of Science, University of South Bohemia in České Budějovice. Since then, we had shared sextillions of femtoseconds of happy and exciting moments, and had lightened up many of the research ideas. Here is the short summary of the recent years in photos of social events...