The folding state of the lumenal loop determines the thermal stability of light-harvesting chlorophyll a/b protein.
The major light-harvesting protein of photosystem II (LHCIIb) is the most abundant chlorophyll-binding protein in the thylakoid membrane. It contains three membrane-spanning alpha helices; the first and third one closely interact with each other to form a super helix, and all three helices bind most of the pigment cofactors. The protein loop domains connecting the alpha helices also play an important role in stabilizing the LHCIIb structure. Single amino acid exchanges in either loop were found to be sufficient to significantly destabilize the complex assembled in vitro [Heinemann, B., and Paulsen, H. (1999) Biochemistry 38, 14088-14093. Mick, V., Eggert, K., Heinemann, B., Geister, S., and Paulsen, H (2004) Biochemistry 43, 5467-5473]. This work presents an analysis of such point mutations in the lumenal loop with regard to the extent and nature of their effect on LHCIIb stability to obtain detailed information on the contribution of this loop to stabilizing the complex. Most of the mutant proteins yielded pigment-protein complexes if their reconstitution and/or isolation was performed under mild conditions; however, the yields were significantly different. Several mutations in the vicinity of W97 in the N-proximal section of the loop gave low reconstitution yields even under very mild conditions. This confirms our earlier notion that W97 may be of particular relevance in stabilizing LHCIIb. The same amino acid exchanges accelerated thermal complex dissociation in the absence of lithium dodecyl sulfate (LDS) and raised the accessibility of the lumenal loop to protease; both effects were well correlated with the reduction in reconstitution yields. We conclude that a detachment of the lumenal loop is a possible first step in the dissociation of LHCIIb. Dramatically reduced complex yields in the presence but not in the absence of LDS were observed for some but not all mutants, particularly those near the C-proximal end of the loop. We conclude that complex stabilities in the absence and in the presence of LDS do not correlate and most likely are determined by different structural characteristics, at least in LHCIIb but maybe also in other membrane proteins.
Mick
Vera
V
Institut für Allgemeine Botanik, Johannes Gutenberg-Universität Mainz, D-55099 Mainz, Germany.
Geister
Sonja
S
Paulsen
Harald
H
D019943:Amino Acid Substitution / Q000235:genetics; D002734:Chlorophyll / Q000737:chemistry; D000077194:Chlorophyll A; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006868:Hydrolysis; D007700:Kinetics; D045342:Light-Harvesting Protein Complexes / Q000737:chemistry* / Q000235:genetics; D018532:Pisum sativum; D045332:Photosystem II Protein Complex / Q000737:chemistry* / Q000235:genetics; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D012967:Sodium Dodecyl Sulfate; D013696:Temperature; D014357:Trypsin / Q000737:chemistry
D045342:Light-Harvesting Protein Complexes; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; D002734:Chlorophyll; D012967:Sodium Dodecyl Sulfate; C037184:chlorophyll b; C028913:dodecyl sulfate; D014357:Trypsin; D000077194:Chlorophyll A