Conformational states of ribulosebisphosphate carboxylase and their interaction with chaperonin 60.
Conformational states of ribulosebisphosphate carboxylase (Rubisco) from Rhodospirillum rubrum were examined by far-UV circular dichroism (CD), tryptophan fluorescence, and 1-anilino-naphthalenesulfonate (ANS) binding. At pH 2 and low ionic strength (I = 0.01), Rubisco adopts an unfolded, monomeric conformation (UA1 state) as judged by far-UV CD and tryptophan fluorescence. As with other acid-unfolded proteins [Goto, Y., Calciano, L. J., & Fink, A. L. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 573-577], an intermediate conformation (A1 state) is observed at pH 2 and high ionic strength. The A1 state has an alpha-helical content equivalent to 64% of that present in the native dimer (N2 state). However, fluorescence measurements indicate that the tertiary structure of the A1 state is largely disordered. A site-directed mutant, K168E, which exists as a stable monomer [Mural, R. J., Soper, T. S., Larimer, F. W., & Hartman, F. C. (1990) J. Biol. Chem. 265, 6501-6505] was used to characterize the "native" monomer (N1 state). The far-UV CD spectra of the N1 and N2 states are almost identical, indicating a similar secondary structure content. However, the tertiary structure of the N1 state is less ordered than that of the N2 state. Nevertheless, when appropriately complemented in vitro, K168E forms an active heterodimer. Upon neutralization of acid-denatured Rubisco or dilution of guanidine hydrochloride-denatured Rubisco, unstable folding intermediates (I1 state) are rapidly formed. At concentrations at or below the "critical aggregation concentration" (CAC), the I1 state reverts spontaneously but slowly to the native states with high yield (greater than 65%). The CAC is temperature-dependent. At concentrations above the CAC, the I1 and the A1 states undergo irreversible aggregation. The commitment to aggregation is rapid [ef. Goldberg, M. E., Rudolph, R., & Jaenicke, R. (1991) Biochemistry 30, 2790-2797] and proceeds until the concentration of folding intermediate(s) has fallen to the CAC. In the presence of a molar excess of chaperonin 60 oligomers, the I1 state forms a stable binary complex. No stable binary complex between chaperonin 60 and the N1 state could be detected. Formation of the chaperonin 60-I1 binary complex arrests the spontaneous folding process. The I1 state becomes resistant to interaction with chaperonin 60 with kinetics indistinguishable from those associated with the appearance of the native states. In vitro complementation analysis indicated that the product of the chaperonin-facilitated process is monomeric.(ABSTRACT TRUNCATED AT 400 WORDS)
Central Research and Development Department, Dupont Company Experimental Station, Wilmington, Delaware 19880-0402.
S M
SM
van der Vies
P V
PV
Viitanen
A A
AA
Gatenby
G H
GH
Lorimer
R
R
Jaenicke
D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D018833:Chaperonins; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D012247:Rhodospirillum rubrum / Q000201:enzymology; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D014364:Tryptophan / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D011506:Proteins; C027132:1-anilino-8-naphthalenesulfonate; D014364:Tryptophan; D018833:Chaperonins; D012273:Ribulose-Bisphosphate Carboxylase