Characterization of a folding intermediate of apoplastocyanin trapped by proline isomerization

Biochemistry. 1993 Nov 23;32(46):12299-310. doi: 10.1021/bi00097a005.

Abstract

The unfolding and refolding transitions of French bean apoplastocyanin (apo-Pc), a beta-sandwich protein, have been characterized. The apoprotein is stabilized by sodium sulfate and can be reversibly unfolded by guanidine hydrochloride (GuHCl). However, in contrast to holo-Pc, apo-Pc is unstable at low ionic strength, suggesting that the copper ion stabilizes the holoprotein. The equilibrium unfolding transition monitored by peptide circular dichroism (CD) and tyrosine fluorescence is described by a two-state model. The kinetics of the unfolding transition were monitored using a manual mixing technique and are consistent with a single two-state transition. In contrast, the kinetics of the refolding reaction measured by fluorescence and CD show two transitions with different rates. The relaxation time of the slower phase (800-1000 s) is almost independent of GuHCl concentration. The faster phase was observed only under strongly native conditions, and its relaxation time is GuHCl-dependent. Double-jump experiments and acceleration by cyclophilin demonstrate that both phases involve cis-trans isomerization of proline residues. The changes in fluorescence associated with the two phases are more than 150% of the total change expected from equilibrium experiments, indicating the presence of intermediate(s) with fluorescence greater than the unfolded state. Amide hydrogen-exchange experiments coupled with two-dimensional NMR spectroscopy demonstrate the formation of an intermediate in the very low refolding reaction in which amide protons in the beta-sheets are weakly protected from exchange. No CD evidence for nativelike beta-sheet formation was found for this intermediate. The NMR experiments suggest that the intermediate is compact with flexible beta-sheets and altered packing of the hydrophobic core. It has many of the characteristics of a molten globule. However, the 1H NMR spectrum of the intermediate exhibits a small number of shifted resonances that indicate the presence of specific tertiary interactions in a localized region. A mechanism for refolding of apoplastocyanin is proposed that includes two slow steps corresponding to trans-->cis isomerization of two prolines.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amides / chemistry
  • Apoproteins / chemistry*
  • Circular Dichroism
  • Fabaceae
  • Hydrogen / chemistry
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Plants, Medicinal
  • Plastocyanin / chemistry*
  • Proline / chemistry
  • Protein Denaturation
  • Protein Structure, Tertiary
  • Spectrometry, Fluorescence
  • Tyrosine / chemistry

Substances

  • Amides
  • Apoproteins
  • apoplastocyanin
  • Tyrosine
  • Hydrogen
  • Plastocyanin
  • Proline