Dr. Chunhong Yang

Current Research Topic

Light harvesting complex protein (LHCP) is the most abundant protein in the world. It forms with chlorophylls and xanthophylls the light harvesting complex (LHC) which enables plant's transferring solar radiation energy to chemical energy, which in turn forms base of almost all the substance in the world. The structure of LHC in the thylakoid membrane is the base of the fulfilment of its function. My research interest is that what is the roll of some amino acids in LHCP in stabilising the Light harvesting complex. The method to solve this question is to exchange some of the amino acids in the LHCP, which are proposed to be the binding sites of the Chlorophyll molecules, to reconstitute the complexes in vitro with wild type protein and mutant proteins, to compare characters (stability of the complexes, dissociation kinetics at elevated temperature and the stoichiometry of pigment molecules) of the reconstituted complexes of wild type and mutants and to see what is the effect of the mutation of the amino acid on the structure of LHC.
Nine mutants of LHCP were constructed with the point specific mutagenesis method based on PCR. Figure 1 shows the four amino acids in the LHCP being mutated and the replacing amino acids respectively. The five amino acids are located in all 4 a-helical domains, and are thought to be bind-sites of both chlorophyll a (H68 and Q197) and chlorophyll b (Q131 and H213). Each of these were exchanged with 2 amino acids (F and L replacing H, and E and S replacing Q). The exchange of the amino acid resulted in changing either of hydrophilic Aa to hydrophobic Aa (H to F or L) or of the electric charge of the molecule (Q to E or S).  Fig. of  LHC2
click on figure to enlarge
Green-Gelclick on figure to enlarge Fig 2 shows the isolation of the in vitro reconstituted complexes on a partially denaturing PAGE gel. The comparison of the green bands shows that mutation of the different amino acids changes the stability of the complexes differently. The exchange of Q131 has reduced dramatically the stability of the complex, which shows very weak bands in the green gel, while the exchange of H213 has little effect.

The following table shows the half-maximum time of the decay kinetics of fluorescence emission of chlorophyll a at elevated temperature (37°C). It shows that exchange of any of the four amino acids reduces the stability of the complexes at elevated temperature. All the mutants complexes show a shorter half-maximum time in the decay kinetics. Again the exchange of Q131 has the most effect on the half-maximum time and the exchange of H213 the miner effect.
Mutant T 1/2 (sec)
15h8 49,8
H213F 36,3
H213L 44,0
Q197E 16
Q197S 23,2
Q131E 11,9
Q131S 11,8
H68L 18,4
H68F 13,4
H681, E65Q 11,4

My research is now going on with the stoichiometry of the mutant, and to understand what is the role of some amino acids in stabilising the light harvesting complex.