RECONSTITUTION OF CORE LIGHT-HARVESTING COMPLEXES OF PHOTOSYNTHETIC BACTERIA USING CHEMICALLY SYNTHESIZED POLYPEPTIDES - 2 - DETERMINATION OF STRUCTURAL FEATURES THAT STABILIZE COMPLEX-FORMATION AND THEIR IMPLICATIONS FOR THE STRUCTURE OF THE SUBUNIT COMPLEX

Chemically synthesized polypeptides have been utilized with a reconsti tution assay to determine the role of specific amino acid side chains in stabilizing the core light-harvesting complex (LH1) of photosynthet ic bacteria and its subunit complex. In the preceding paper [Meadows, K. A., Parkes-Loach, P. S., Kehoe, J. W., and Leach, P. A. (1998) Bioc hemistry 37, 3411-3417], it was demonstrated that 31-residue polypepti des (compared to 48 and 54 amino acids in the native polypeptides) hav ing the same sequence as the core region of the beta-polypeptide of Rh odobacter sphaeroides (sph beta 31) or Rhodospirillum rubrum (rr beta 31) could form subunit-type complexes. However, neither polypeptide in teracted with the native alpha-polypeptides to form a native LH1 compl ex. In this paper, it is demonstrated that larger segments of the nati ve Rb. sphaeroides beta-polypeptide possess native behavior in LH1 for mation. Polypeptides were synthesized that were six (sph beta 37) and ten amino acids (sph beta 41) longer than sph beta 31. Although sph be ta 37 exhibited behavior nearly identical to that of sph beta 31, sph beta 41 behaved more like the native polypeptide. In the case of rr be ta 31, a polypeptide with four additional amino acids toward the C ter minus was synthesized (rr beta 35). Because LH1-forming behavior was n ot recovered with this longer polypeptide, one or more of the three re maining amino acids at the C-terminal end of the native beta-polypepti de seem to play an important role in LH1 stabilization in Rs. rubrum. Three analogues of the core region of the Rb. sphaeroides beta-polypep tide were synthesized, in each of which one highly conserved amino aci d was changed. Evidence was obtained that the penultimate amino acid, a Trp residue, is especially important for subunit formation. When it was changed to Phe, the lambda(Max) of the subunit shifted from 823 to 811 nm and the association constant decreased about 500-fold. Changin g each of two other amino acids had smaller effects on subunit formati on. Changing Trp to Phe at the location six amino acid residues toward the C terminus from the His coordinated to Bch1 resulted in an approx imately 10-fold decrease in the association constant for subunit forma tion but did not affect the formation of a LH1-type complex compared t o sph beta 31. Finally, changing Arg to Leu at the location seven amin o acid residues toward the C terminus from the His coordinated to Bch1 decreased the association constant for subunit formation by about 30- fold. In this case, no LH1-type complex could be formed. On the basis of these results, in comparison with the crystal structure of the LH2 beta-polypeptide of Rhodospirillum molischianum, two possible structur es for the subunit complex are suggested.