PARENT SESSION
Posters P4Aa Chlorophyll and bilin based antenna systems. Abstracts (239-271)

Comformational changes of LHCB2 apoprotein and pigments during their reconstitution in vitro. Tingyun Kuang^*,1, Liangbi Li², Jing Leng³, ¹ 20 Nanxincun, Xiangshan, Haidian District, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China, China² 20 Nanxincun, Xiangshan, Haidian District, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China, China³ 20 Nanxincun, Xiangshan, Haidian District, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China, china

ABSTRACT- The most important steps in photosynthesis, including the absorption of light energy and transfer it to the reaction center, are achieved by light-harvesting antenna complexes. It is very likely that the arrangement of pigments within LHC II has been optimized with respect to antenna function. But questions how these pigments bind to the specific amino acid accurately, and how the apoprotein folds into its native structure, still remain unknown. The possibility to reconstitute LHC II from the apoprotein and pigments enables us to study the formation procedure in vitro. In this study, Lhcb2 gene, encodes one of the major light-harvesting complex II polypeptides, was cloned from pea and overexpressed in E.Coli. The lhcb2 apoprotein was purified from E.Coli and reconstituted with pigments in vitro, and the reconstituted complex exhibited similar electrophoresis behavior and spectroscopic properties as native one. To monitor the structure changes of Lhcb2 apoprotein and pigments binding to protein during the reconstitution, circular dichroism (CD) and fluorescence spectra were measured. According to the CD spectra in the far-UV region, LHCB2 apoprotein solubilized in reconstitution buffer containing LDS had a less ordered structure with low content of a-helix (about 17%) compared with native LHC II (about 45%). Upon the reconstitution procedure in vitro, the content of a-helix increased to 39.6%. The restoration of the fluorescence and CD spectra of pigment-protein complex in the visible region could be seen only upon freezing-thawing treatment. CD bands of Chl b at 645nm and 460nm appeared firstly, meanwhile the content of a-helix increased to 28%. Then other CD bands emerged subsequently after several cycles of freezing-thawing, and the fluorescence peak shifted from 674 nm to 681 nm. The second structure of protein finally folded into a way very resemble to the native one. It can be concluded from these data that during the reconstitution of light-harvesting complex, binding of Chl b molecules to outer sites was the first step of complex assembly, which led to the increase of a-helix. Further binding of other pigments appeared also to stabilize the protein structure and maintain the reconstituted complexes functional as well.

KEY WORDS: reconstitution, LHC II, Comformational changes