title: Evaluation of structure-function relationships in the core light-harvesting complex of photosynthetic bacteria by reconstitution with mutant polypeptides.
abstract: Seven mutant LH1 polypeptides of Rhodobactor sphaeroides have been isolated, and their behaviors in in vitro reconstitution of LH1 and its subunit complex have been characterized. Two mutants were selected to address the increased stability of the subunit complex of Rb. sphaeroides compared with that of Rhodobacter capsulatus. We found that this difference can be largely ascribed to the existence of Tyr at position +4 in the beta-polypeptide (the numbering system used assigns position 0 to the His which provides the coordinating ligand to bacteriochlorophyll) of the former bacterium compared to Met in that position in the latter. The amount of energy involved in the increased interaction was 1.6 kcal/mol, which would be consistent with a hydrogen bond involving Tyr. Mutation of the His at position 0 to Asn allows an estimate of the binding energy for subunit formation contributed by coordination of the imidazole group of His to the Mg atom of bacteriochlorophyll of >4.5 kcal/mol per BChl. Finally, an evaluation of the role of amino acids in the C-terminal region of the alpha-polypeptide was begun. Reconstitution of a mutant alpha-polypeptide in which Trp at position +11 was changed to Phe resulted in optimal formation of an LH1-type complex whose lambda(max) was blue-shifted to 853 nm, the same as observed in the intact bacterium harboring this mutation. These results provide further confirmation that the environment of BChl in reconstituted LH1 complexes is the same as in vivo and support the assignment of this residue to a role in hydrogen bonding with the C3(1) carbonyl group of BChl. Two other mutants of the alpha-polypeptide in which 5 and 14 amino acids in the C-terminus were deleted were also examined. These were of interest because the latter mutant, unlike the former, resulted in a low level of expression of LH1 in intact cells. However, with both of these mutant polypeptides, reconstitution appeared identical to that of the native system. In the case of the mutant shortened by 14 amino acids, a small blue-shift in lambda(max) to 861 nm was observed, again reproducing the blue-shift exhibited by the intact cells. Thus, these results suggest that the lowered levels of in vivo expression observed in these two mutants are due to reduced incorporation of the alpha-polypeptide into the membrane or its increased degradation, rather than to decreased stabilization of the LH1 complex.
authors.affiliation: Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500, USA.
authors.forename: C M
authors.initials: CM
authors.lastname: Davis
authors.forename: P L
authors.initials: PL
authors.lastname: Bustamante
authors.forename: J B
authors.initials: JB
authors.lastname: Todd
authors.forename: P S
authors.initials: PS
authors.lastname: Parkes-Loach
authors.forename: P
authors.initials: P
authors.lastname: McGlynn
authors.forename: J D
authors.initials: JD
authors.lastname: Olsen
authors.forename: L
authors.initials: L
authors.lastname: McMaster
authors.forename: C N
authors.initials: CN
authors.lastname: Hunter
authors.forename: P A
authors.initials: PA
authors.lastname: Loach
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D002851:Chromatography, High Pressure Liquid; D045342:Light-Harvesting Protein Complexes; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000235:genetics; D012246:Rhodospirillum; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D045342:Light-Harvesting Protein Complexes; D045322:Photosynthetic Reaction Center Complex Proteins; C094744:light-harvesting complex 1, Rhodospirillum