High-Pressure Spectroscopy Study of Chromophore-Binding Hydrogen Bonds in Light-Harvesting Complexes of Photosynthetic Bacteria
Date
2013-06-28
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Abstract
Fotosünteesivate purpurbakterite valgust koguvad antennid (LH1 ja LH2) on sobivad mudelsüsteemid membraansete valkude ehitusliku stabiilsuse uurimiseks. Käesolevas töös on uuritud kõrge rõhu all vesiniksidemete (H-side) tugevust, mis seovad uuritud valkudes bakterklorofülle (BChl). BChl-d uuritud valkudes (LH1-s 32-56 ja LH2-s 27) moodustavad ringe ja ahelaid, ja nende ülesandeks on koguda valguse energia ning suunata see bakteri elutegevuseks. H-sidemed, mis seovad BChl, moodustuvad valgu aminohappe külgahela positiivse laenguga vesiniku ja negatiivse laenguga BChl hapniku vahel, on suhteliselt nõrgad sidemed, kuid neil on väga suur roll elufunktsioone hoidvate valkude funktsioonide ja struktuuri stabiilsuse säilitamisel. Käesolevas töös on kasutatud BChl-le, kui sensoreid, mis kajastavad enda seisundit valgu sisemuses H-sidemete kaudu ning selle jälgimiseks on rakendatud nende valgust neelavate omaduste muutusi.
Uurimises on leitud, et kõrge hüdrostaatilise rõhu all oma loomulikus membraanses ümbruses paiknevad antennid on äärmiselt stabiilsed (taluvad kuni 30000 atm rõhku). Kuid detergenti viiduna (mis on kunstlik membraan) ilmutavad need antennid oma neeldumisspektri tavapärases punanihkumises rõhu kasvamise käigus nihkumist spektri sinisesse ossa, mis toimub LH1 puhul 10000 atm juures ja LH2 puhul 4000 atm juures, ja sellega kaasneb ka spektririba laienemine. Toodud efektid viitavad BChl liikumisvabaduse kasvule valgus, mis viitab BChl ja valgu vaheliste H-sidemete katkemisele. Rõhu vähendamisel sininihe ja laienemine on pöörduvad. Katsetulemuste alusel hinnatud BChl-e paigal hoidvate H-sidemete katkemise energia ületab 10-20 korda neid tavapäraselt ümbritsevast termilisest energiast. See tagab nende valkude märkimisväärse stabiilsuse nende elukeskkonnas. Lisaks on tuvastatud võimalik H-sidemete kooperatiivne katkemine ning valgus sisalduva karotenoidi ja valgu kontsentratsiooni ning glütserooli stabiliseeriv mõju valgu ehituse stabiliseerimisel.
The light-harvesting antenna complexes from purple photosynthetic bacteria are convenient model systems to examine the poorly understood role of hydrogen bonds as stabilizing factors in membrane protein complexes. The non-covalently bound arrays of bacteriochlorophyll chromophores within native and genetically modified variants of light-harvesting complexes were used to monitor local changes in the chromophore binding sites induced by externally applied hydrostatic pressure. A unique combination of optical spectroscopy with genetic and noninvasive physical (high-pressure) engineering applied in this work provides the first demonstration and quantification of the rupture of multiple hydrogen bonds in the bacteriochlorophyll binding pockets of the LH1 and LH2 membrane chromoproteins. While the membrane-bound complexes demonstrate very high resilience to pressures reaching 3 GPa, the detergent-isolated complexes reveal characteristic discontinuities of the absorption band shifts and broadenings around 1.1 GPa and 0.5 GPa in the case of the wild type LH1 and LH2 complexes, respectively, which evidence reversible and cooperative breakage of H-bonds. Genetic manipulations leading to exchange of native carotenoids, partial loss of chromophores, and/or H-bonds that bind the chromophores to the surrounding protein scaffold were found to significantly destabilize the membrane chromoproteins under high pressure. Co-solvents such as glycerol as well as high protein concentration, on the other hand, were able to stabilize not only detergent-isolated, which was known previously, but also the membrane-embedded chromoproteins. The energy required to break the H-bonds in wild type LH1 and LH2 complexes is 10–20 times greater than the average thermal energy at physiological temperatures, which secures their great stability under functional conditions. This study thereby provides important insights into design principles of natural photosynthetic complexes.
The light-harvesting antenna complexes from purple photosynthetic bacteria are convenient model systems to examine the poorly understood role of hydrogen bonds as stabilizing factors in membrane protein complexes. The non-covalently bound arrays of bacteriochlorophyll chromophores within native and genetically modified variants of light-harvesting complexes were used to monitor local changes in the chromophore binding sites induced by externally applied hydrostatic pressure. A unique combination of optical spectroscopy with genetic and noninvasive physical (high-pressure) engineering applied in this work provides the first demonstration and quantification of the rupture of multiple hydrogen bonds in the bacteriochlorophyll binding pockets of the LH1 and LH2 membrane chromoproteins. While the membrane-bound complexes demonstrate very high resilience to pressures reaching 3 GPa, the detergent-isolated complexes reveal characteristic discontinuities of the absorption band shifts and broadenings around 1.1 GPa and 0.5 GPa in the case of the wild type LH1 and LH2 complexes, respectively, which evidence reversible and cooperative breakage of H-bonds. Genetic manipulations leading to exchange of native carotenoids, partial loss of chromophores, and/or H-bonds that bind the chromophores to the surrounding protein scaffold were found to significantly destabilize the membrane chromoproteins under high pressure. Co-solvents such as glycerol as well as high protein concentration, on the other hand, were able to stabilize not only detergent-isolated, which was known previously, but also the membrane-embedded chromoproteins. The energy required to break the H-bonds in wild type LH1 and LH2 complexes is 10–20 times greater than the average thermal energy at physiological temperatures, which secures their great stability under functional conditions. This study thereby provides important insights into design principles of natural photosynthetic complexes.
Description
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Keywords
fototroofsed bakterid, kromofoorid, membraanivalgud, vesinikside, spektroskoopia, phototrophic microbes, chromophores, membrane proteins, hydrogen bond, spectroscopy