Carotenoids are important biomolecules that are ubiquitous in nature and find widespread application in medicine. In photosynthesis, they have a large role in light harvesting (LH) and photoprotection. They exert their LH function by donating their excited singlet state to nearby (bacterio)chlorophyll molecules. In photosynthetic bacteria, the efficiency of this energy transfer process can be as low as 30%. Here, we present evidence that an unusual pathway of excited state relaxation in carotenoids underlies this poor LH function, by which carotenoid triplet states are generated directly from carotenoid singlet states. This pathway, operative on a femtosecond and picosecond timescale, involves an intermediate state, which we identify as a new, hitherto uncharacterized carotenoid singlet excited state. In LH complex-bound carotenoids, this state is the precursor on the reaction pathway to the triplet state, whereas in extracted carotenoids in solution, this state returns to the singlet ground state without forming any triplets. We discuss the possible identity of this excited state and argue that fission of the singlet state into a pair of triplet states on individual carotenoid molecules constitutes the mechanism by which the triplets are generated. This is, to our knowledge, the first ever direct observation of a singlet-to-triplet conversion process on an ultrafast timescale in a photosynthetic antenna. C arotenoids (Cars) serve a variety of functions in biological systems. In photosynthesis, they act as light-harvesting (LH) pigments by absorbing sunlight in the blue and green parts of the solar spectrum and transferring the excited state energy to nearby (bacterio)chlorophylls (BChl) (1, 2). The BChl molecules subsequently transfer this energy to a photochemical energyconverting protein known as the reaction center (RC), where the excited state energy is fixed by means of a series of electron transfer reactions (3). During these energy-and electrontransfer processes, which may take up to hundreds of picoseconds, the singlet excited and charge-separated states of BChl are subject to intersystem crossing to the triplet state, which occurs on a timescale of several nanoseconds. Although produced with a small probability, these BChl triplet states are potentially harmful to the organism because they can promote molecular oxygen to its singlet excited state, which is a highly reactive and damaging species. Cars can efficiently accept and safely dissipate BChl triplet and singlet oxygen states, and this photoprotective quality is utilized by essentially all photosynthetic organisms (1, 2).The first singlet excited state of Cars, S 1 , carries gerade symmetry (with respect to inversion) as does the ground state, S 0 , and is therefore dipole-forbidden. The second excited singlet state, S 2 , carries ungerade symmetry and is dipole-allowed (1). The specific strong Car absorption in the blue and green regions of the visible part of the electromagnetic spectrum is caused by the transition to this second excited state,...

Spectral and kinetic information on energy transfer from carotenoids (Cars) to chlorophylls (Chls) within light-harvesting complex II (LHCII) and CP29 was obtained from femtosecond transient absorption study by using selective Car excitation (489 and 506 nm) and detecting the induced changes over a wide spectral interval (460-720 nm). By examining the evolution of entire spectral bands rather than looking at a few single traces, we were able to identify the species (pigments and/or electronic states) which participate in the energy flow, as well as the lifetimes and quantum yields of individual processes. Hence, it was found that the initially excited Car S 2 state decays very fast, with lifetimes of 70-90 fs in CP29 and 100 ( 20 fs in LHCII, via two competing channels: energy transfer to Chls (60-65%) and internal conversion to the lower, optically forbidden S 1 state (35-40%). In CP29, the energy acceptors are exclusively Chls a, while in LHCII, this is only valid for lutein and violaxanthin. In the latter case, neoxanthin transfers energy mostly to Chls b. In both complexes, ca. 15-20% of the initial Car excitations are transferred to Chls a via the S 1 level, with a time constant of around 1 ps, thus bringing the total Car-Chl transfer efficiency to ca. 80%. Given the yield of this process and the large difference between the transfer time and the intrinsic S 1 lifetime (∼20 ps), it seems that lutein is the only species active on this pathway. From the measured transfer rates, we estimated that a coupling of 280-330 cm -1 drives the transfer via the S 2 route, while a coupling value of around 100 cm -1 was estimated for the S 1 transfer. The Car S 2 state is coupled to both Q x and Q y states of the Chl through a Coulombic mechanism; from the available structural information, we estimated the dipole-dipole contribution to be 450-500 cm -1 . The S 1 state is coupled to the Chl a Q y transition via an exchange and/or a Coulombic mechanism.

Intrinsically disordered proteins (IDPs) have fluctuating heterogeneous conformations, which makes their structural characterization challenging. Although challenging, characterization of the conformational ensembles of IDPs is of great interest, since their conformational ensembles are the link between their sequences and functions. An accurate description of IDP conformational ensembles depends crucially on the amount and quality of the experimental data, how it is integrated, and if it supports a consistent structural picture. We used integrative modeling and validation to apply conformational restraints and assess agreement with the most common structural techniques for IDPs: Nuclear Magnetic Resonance (NMR) spectroscopy, Small-angle X-ray Scattering (SAXS), and single-molecule Förster Resonance Energy Transfer (smFRET). Agreement with such a diverse set of experimental data suggests that details of the generated ensembles can now be examined with a high degree of confidence. Using the disordered N-terminal region of the Sic1 protein as a test case, we examined relationships between average global polymeric descriptions and higher-moments of their distributions. To resolve apparent discrepancies between smFRET and SAXS inferences, we integrated SAXS data with NMR data and reserved the smFRET data for independent validation. Consistency with smFRET, which was not guaranteed a priori, indicates that, globally, the perturbative effects of NMR or smFRET labels on the Sic1 ensemble are minimal. Analysis of the ensembles revealed distinguishing features of Sic1, such as overall compactness and large end-to-end distance fluctuations, which are consistent with biophysical models of Sic1’s ultrasensitive binding to its partner Cdc4. Our results underscore the importance of integrative modeling and validation in generating and drawing conclusions from IDP conformational ensembles.

A spectral and functional assignment of the xanthophylls in monomeric and trimeric lightharvesting complex II of green plants has been obtained using HPLC analysis of the pigment composition, laser-flash induced triplet-minus-singlet, fluorescence excitation, and absorption spectra. It is shown that violaxanthin is not present in monomeric preparations, that it has most likely a red-most absorption maximum at 510 nm in the trimeric complex, and that it is involved in both light-harvesting and Chltriplet quenching. Two xanthophylls (per monomer) have an absorption maximum at 494 nm. These play a major role in both singlet and triplet transfer. These two are most probably the two xanthophylls resolved in the crystal structure, tentatively assigned to lutein, that are close to several chlorophyll molecules