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
Jw. Kehoe et al., 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, Biochemistry, 37(10), 1998, pp. 3418-3428
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.