Minimal requirements for in vitro reconstitution of the structural subunit of light-harvesting complexes of photosynthetic bacteria

Unlike the α and β polypeptides of the core light-harvesting complex (LH1) of Rhodobacter (Rb.) sphaeroides, the α and β polypeptides of the peripheral light-harvesting complex (LH2) of this organism will not form a subunit complex by in vitro reconstitution with bacteriochlorophyll. Guided by prior experiments with the LH1 β polypeptides of Rb. sphaeroides and Rhodospirillum rubrum, which defined a set of interactions required to stabilize the subunit complex, a series of mutations to the Rb. sphaeroides LH2 β polypeptide was prepared and studied to determine the minimal changes necessary to enable it to form a subunit-type complex. Three mutants were prepared: Arg at position -10 was changed to Asn (numbering is from the conserved His residue which is known to be coordinated to bacteriochlorophyll); Arg at position -10 and Thr at position +7 were changed to Asn and Arg, respectively; and Arg at position -10 was changed to Trp and the C-terminus from +4 to +10 was replaced with the amino acids found at the corresponding positions in the LH1 β polypeptide of Rb. sphaeroides. Only this last multiple mutant polypeptide formed subunit-type complexes in vitro. Thus, the importance of the C-terminal region, which encompasses conserved residues at positions +4, +6 and +7, is confirmed. Two mutants of the LH1 β polypeptide of Rb. sphaeroides were also constructed to further evaluate the interactions stabilizing the subunit complex and those necessary for oligomerization of subunits to form LH1 complexes. In one of these mutants, Trp at position -10 was changed to Arg, as found in LH2 at this position, and in the other His at position -18 was changed to Val. The results from these mutants allow us to conclude that the residue at the -10 position is unimportant in subunit formation or oligomerization, while the strictly conserved His at -18 is not required for subunit formation but is very important in oligomerization of subunits to form LH1.[PUBLICATION ABSTRACT]