Feedback mechanisms that dissipate excess photoexcitations in light-harvesting complexes (LHCs) are necessary to avoid detrimental oxidative stress in most photosynthetic eukaryotes. Here we demonstrate the unique ability of LHCSR, a stress-related LHC from the model organism Chlamydomonas reinhardtii, to sense pH variations, reversibly tuning its conformation from a light-harvesting state to a dissipative one. This conformational change is induced exclusively by the acidification of the environment, and the magnitude of quenching is correlated to the degree of acidification of the environment. We show that this ability to respond to different pH values is missing in the related major LHCII, despite high structural homology. Via mutagenesis and spectroscopic characterization, we show that LHCSR's uniqueness relies on its peculiar C-terminus subdomain, which acts as a sensor of the lumenal pH, able to tune the quenching level of the complex.

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The mannitol transporter EII(mtl) from Escherichia coli is responsible for the uptake of mannitol over the inner membrane and its concomitant phosphorylation. EII(mtl) is functional as a dimer and its membrane-embedded C domain, IIC(mtl), harbors one high affinity mannitol binding site. To characterize this domain in more detail the microenvironments of thirteen residue positions were explored by 5-fluorotryptophan (5-FTrp) fluorescence spectroscopy. Because of the simpler photophysics of 5-FTrp compared to Trp, one can distinguish between the two 5-FTrp probes present in dimeric IIC(mtl). At many labeled positions, the microenvironment of the 5-FTrps in the two protomers differs. Spectroscopic properties of three mutants labeled at positions 198, 251, and 260 show that two conserved motifs (Asn194-His195 and Gly254-Ile255-His256-Glu257) are located in well-structured parts of IIC(mtl). Mannitol binding has a large impact on the structure around position 198, while only minor changes are induced at positions 251 and 260. Phosphorylation of the cytoplasmic B domain of EII(mtl) is sensed by 5-FTrp at positions 30, 42, 251 and 260. We conclude that many parts of the IIC(mtl) structure are involved in the sugar translocation. The structure of EII(mtl), as investigated in this work, differs from the recently solved structure of a IIC protein transporting diacetylchitobiose, ChbC, and also belonging to the glucose superfamily of EII sugar transporters. In EII(mtl), the sugar binding site is more close to the periplasmic face and the structure of the 2 protomers in the dimer is different, while both protomers in the ChbC dimer are essentially the same.

The mannitol transporter from Escherichia coli, EII mtl , belongs to a class of membrane proteins coupling the transport of substrates with their chemical modification. EII mtl is functional as a homodimer, and it harbors one high affinity mannitol-binding site in the membrane-embedded C domain (IIC mtl remains the same. We conclude that during the transport cycle, the phosphorylated B domain has to move to the mannitol-binding site, located in the middle of the membrane, to phosphorylate mannitol.Membrane-embedded transport proteins can be divided in three different classes based on their energy-coupling mechanism (1): (i) primary transport systems, which comprise transporters that use chemical energy or light to actively transport a specific solute over the membrane; (ii) secondary transporters, which are transporters in which the uphill transport of a solute is coupled to the downhill transport of a chemical compound along a gradient; and (iii) group translocation systems, which couple active transport with the chemical modification of the solute. The Enzyme II sugar transporters of the phosphoenolpyruvate-dependent group translocation system (PTS) 2 (2, 3) are the only known transporters belonging to the third class. These transporters phosphorylate their sugar during transport over the membrane. The Escherichia coli PTS transporters specific for glucose, ␤-glucosides, mannitol, and mannose are the best characterized members (4 -9). Enzyme II proteins consist of two cytoplasmic domains, IIA and IIB, involved in phosphoryl group transfer, and a membrane-spanning IIC domain, showing the sugar specificity (10). In some Enzyme II proteins, like the mannose transporter, a second membrane-spanning domain, IID, is present. Depending on its sugar specificity, Enzyme II occurs as separate domains or as fused constructs between two or three domains (11). In EII mtl , the mannitol-specific transporting and phosphorylating enzyme from E. coli, all three domains IIA mtl (12), IIB mtl (13), and IIC mtl (membrane-embedded domain of EII mtl ) are covalently linked (Fig. 1). Mannitol becomes phosphorylated during transport and is released as mannitol-1-phosphate in the cytoplasm (7). The phosphate group originates from phosphoenolpyruvate (PEP) and is transferred to the various EII sugar translocators in the cytoplasmic membrane via two general PTS proteins, EI and HPr (Fig. 1). The phosphoryl transfer reactions from PEP, via EI, HPr, and the A domain to the B domain of Enzyme II are well understood because three-dimensional structures are available for these proteins combined with detailed pre-steady state kinetic data in the case of the glucose transporter, EII glc , from E. coli (14,15). This type of information is not available for the last phosphorylation step from the B domain to the sugar, bound at the C domain. Moreover, no information is available regarding which residues bind the sugar and which residues facilitate the transport of the sugar.For a better understanding of how Enzyme II proteins work, detailed str...

The trimeric light-harvesting complexes II (LHCII) of plants and green algae are pigment-protein complexes involved in light harvesting and photoprotection. Different conformational states have been proposed to be responsible for their different functions. At present, detergent-solubilized LHCII is used as a model for the "light-harvesting conformation", whereas the "quenched conformation" is mimicked by LHCII aggregates. However, none of these conditions seem to perfectly reproduce the properties of LHCII in vivo. In addition, several monomeric LHC complexes are not fully stable in detergent. There is thus a need to find conditions that allow analyzing LHCs in vitro in stable and, hopefully, more native-like conformations. Here, we report a study of LHCII, the major antenna complex of plants, in complex with amphipols. We have trapped trimeric LHCII and monomeric Lhcb1 with either polyanionic or non-ionic amphipols and studied the effect of these polymers on the properties of the complexes. We show that, as compared to detergent solutions, amphipols have a stabilizing effect on LHCII. We also show that the average fluorescence lifetime of LHCII trapped in an anionic amphipol is ~30% shorter than in α-dodecylmaltoside, due to the presence of a conformation with 230-ps lifetime that is not present in detergent solutions.