Evaluation of Structure-Function Relationships in the Core Light-Harvesting Complex of Photosynthetic Bacteria by Reconstitution with Mutant Polypeptides
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- 作者：Christine M. Davis ; Peggy L. Bustamante ; John B. Todd ; Pamela S. Parkes-Loach ; Peter McGlynn ; John D. Olsen ; Liam McMaster ; C. Neil Hunter ; and Paul A. Loach
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：March 25, 1997
- 年：1997
- 卷：36
- 期：12
- 页码：3671 - 3679
- 全文大小：191K
- 年卷期：v.36,no.12(March 25, 1997)
- ISSN：1520-4995
文摘
Seven mutant LH1 polypeptides of Rhodobactorsphaeroides have been isolated, and theirbehaviors in in vitro reconstitution of LH1 andits subunit complex have been characterized. Twomutantswere selected to address the increased stability of the subunit complexof Rb. sphaeroides compared withthat of Rhodobacter capsulatus. We foundthat this difference can be largely ascribed to the existenceofTyr at position +4 in the 
-polypeptide (the numbering system usedassigns position 0 to the His whichprovides the coordinating ligand to bacteriochlorophyll) of the formerbacterium compared to Met in thatposition in the latter. The amount of energy involved in theincreased interaction was 1.6 kcal/mol,which would be consistent with a hydrogen bond involving Tyr.Mutation of the His at position 0 to Asnallows an estimate of the binding energy for subunit formationcontributed by coordination of the imidazolegroup of His to the Mg atom of bacteriochlorophyll of >4.5 kcal/molper BChl. Finally, an evaluationof the role of amino acids in the C-terminal region of the
-polypeptide was begun. Reconstitution of amutant -polypeptide in which Trp at position +11 was changed toPhe resulted in optimal formation ofan LH1-type complex whose 
max was blue-shifted to 853nm, the same as observed in the intact bacteriumharboring this mutation. These results provide furtherconfirmation that the environment of BChl inreconstituted LH1 complexes is the same as in vivo andsupport the assignment of this residue to a rolein hydrogen bonding with the C31 carbonyl group of BChl.Two other mutants of the -polypeptide inwhich 5 and 14 amino acids in the C-terminus were deleted were alsoexamined. These were of interestbecause the latter mutant, unlike the former, resulted in a low levelof expression of LH1 in intact cells.However, with both of these mutant polypeptides, reconstitutionappeared identical to that of the nativesystem. In the case of the mutant shortened by 14 amino acids, asmall blue-shift in max to 861 nm wasobserved, again reproducing the blue-shift exhibited by the intactcells. Thus, these results suggest thatthe lowered levels of in vivo expression observed in thesetwo mutants are due to reduced incorporationof the -polypeptide into the membrane or its increased degradation,rather than to decreased stabilizationof the LH1 complex.
-polypeptide (the numbering system usedassigns position 0 to the His whichprovides the coordinating ligand to bacteriochlorophyll) of the formerbacterium compared to Met in thatposition in the latter. The amount of energy involved in theincreased interaction was 1.6 kcal/mol,which would be consistent with a hydrogen bond involving Tyr.Mutation of the His at position 0 to Asnallows an estimate of the binding energy for subunit formationcontributed by coordination of the imidazolegroup of His to the Mg atom of bacteriochlorophyll of >4.5 kcal/molper BChl. Finally, an evaluationof the role of amino acids in the C-terminal region of the-polypeptide was begun. Reconstitution of amutant -polypeptide in which Trp at position +11 was changed toPhe resulted in optimal formation ofan LH1-type complex whose max was blue-shifted to 853nm, the same as observed in the intact bacteriumharboring this mutation. These results provide furtherconfirmation that the environment of BChl inreconstituted LH1 complexes is the same as in vivo andsupport the assignment of this residue to a rolein hydrogen bonding with the C31 carbonyl group of BChl.Two other mutants of the -polypeptide inwhich 5 and 14 amino acids in the C-terminus were deleted were alsoexamined. These were of interestbecause the latter mutant, unlike the former, resulted in a low levelof expression of LH1 in intact cells.However, with both of these mutant polypeptides, reconstitutionappeared identical to that of the nativesystem. In the case of the mutant shortened by 14 amino acids, asmall blue-shift in max to 861 nm wasobserved, again reproducing the blue-shift exhibited by the intactcells. Thus, these results suggest thatthe lowered levels of in vivo expression observed in thesetwo mutants are due to reduced incorporationof the -polypeptide into the membrane or its increased degradation,rather than to decreased stabilizationof the LH1 complex.