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

Unlike the alpha and beta polypeptides of the core light-harvesting complex (LH1) of Rhodobacter (Rb.) sphaeroides, the alpha and beta polypeptides of the peripheral light-harvesting complex (LH2) of this organism will not fo rm a subunit complex by in vitro reconstitution with bacteriochlorophyll. G uided by prior experiments with the LH1 beta polypeptides of Rb. sphaeroide s and Rhodospirillum rubrum, which defined a set of interactions required t o stabilize the subunit complex, a series of mutations to the Rb. sphaeroid es LH2 beta polypeptide was prepared and studied to determine the minimal c hanges 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 t he conserved His residue which is known to be coordinated to bacteriochloro phyll); 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-ter minus from +4 to +10 was replaced with the amino acids found at the corresp onding positions in the LH1 beta polypeptide of Rb. sphaeroides. Only this last multiple mutant polypeptide formed subunit-type complexes in vitro. Th us, the importance of the C-terminal region, which encompasses conserved re sidues at positions +4, +6 and +7, is confirmed. Two mutants of the LH1 bet a polypeptide of Rb. sphaeroides were also constructed to further evaluate the interactions stabilizing the subunit complex and those necessary for ol igomerization of subunits to form LH1 complexes. In one of these mutants, T rp at position -10 was changed to Arg, as found in LH2 at this position, an d in the other His at position -18 was changed to Val. The results from the se mutants allow us to conclude that the residue at the -10 position is uni mportant in subunit formation or oligomerization, while the strictly conser ved His at -18 is not required for subunit formation but is very important in oligomerization of subunits to form LH1.