pH jump studies of the folding of the multidomain ribosomal protein L9: the structural organization of the N-terminal domain does not affect the anomalously slow folding of the C-terminal domain

Biochemistry. 2000 Apr 25;39(16):4955-62. doi: 10.1021/bi992608u.

Abstract

The folding kinetics of the multidomain ribosomal protein L9 were studied using pH jump stopped-flow fluorescence and circular dichroism (CD) in conjunction with guanidine hydrochloride (GdnHCl) jump stopped-flow CD experiments. Equilibrium CD and 1D (1)H NMR measurements demonstrated that the C-terminal domain unfolds below pH 4 while the N-terminal domain remains fully folded. Thus, the N-terminal domain remains folded during the pH jump experiments. The folding rate constant of the C-terminal domain was determined to be 3.5 s(-1) by pH jump experiments conducted in the absence of denaturant using stopped-flow CD and fluorescence. CD-detected GdnHCl jump measurements showed that the N- and C-terminal domains fold independently each by an apparent two-state mechanism. The folding rate constant for the N-terminal domain and the C-terminal domain in the absence of denaturant were calculated to be 760 and 4. 7 s(-1), respectively. The good agreement between the pH jump and the denaturant concentration jump experiments shows that the folding rate of the C-terminal domain is the same whether or not the N-terminal domain is folded. This result suggests that the slow folding of the C-terminal domain is not a consequence of unfavorable interactions with the rest of the protein chain during refolding. This is an interesting result since contact order analysis predicts that the folding rate of the C-terminal domain should be noticeably faster. The folding rate of the isolated N-terminal domain was also measured by stopped-flow CD and was found to be the same as the rate for the domain in the intact protein.

Publication types

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

MeSH terms

  • Acids / pharmacology
  • Circular Dichroism
  • Deuterium Oxide / metabolism
  • Fluorescence
  • Geobacillus stearothermophilus / chemistry*
  • Guanidine / pharmacology
  • Hydrogen-Ion Concentration
  • Kinetics
  • Magnetic Resonance Spectroscopy
  • Models, Molecular
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Protein Denaturation / drug effects
  • Protein Folding*
  • Protein Structure, Tertiary
  • Ribosomal Proteins / chemistry*
  • Ribosomal Proteins / genetics
  • Ribosomal Proteins / metabolism*
  • Sequence Deletion / genetics
  • Thermodynamics

Substances

  • Acids
  • Peptide Fragments
  • Ribosomal Proteins
  • ribosomal protein L9
  • Deuterium Oxide
  • Guanidine