pH-dependent stability and folding kinetics of a protein with an unusual alpha-beta topology: the C-terminal domain of the ribosomal protein L9.

Abstract:

:The folding kinetics and thermodynamics of the isolated C-terminal domain of the ribosomal protein L9 (CTL9) have been studied as a function of pH. CTL9 is an alpha-beta protein that contains a single beta-sheet with an unusual mixed parallel, anti-parallel topology. The folding is fully reversible and two-state over the entire pH range. Stopped-flow fluorescence and CD experiments yield the same folding rate, and the chevron plots have the characteristic V-shape expected for two-state folding. The values of DeltaG*(H2O) and the m value calculated from the kinetic experiments are in excellent agreement with the equilibrium measurements. The extrapolated initial amplitudes of both the stopped-flow fluorescence and CD measurements show that there is no detectable burst phase intermediate. The domain contains three histidine residues, two of which are largely buried in the native state. They do not participate in salt-bridges or take part in a hydrogen bonded network. NMR measurements reveal that the buried histidine residues have significantly perturbed pK(a) values in the native state. The equilibrium stability and the folding rate are found to be strongly dependent upon their ionization state. There is a linear relationship between the log of the folding rate and DeltaG* (H2O) . The protein is much more stable and folds noticeably faster at pH values above the native state pK(a) values. DeltaG*(H2O) of unfolding increases from 2.90 kcal mol(-1) at pH 5.0 to 6.40 kcal mol(-1) at pH 8.0 while the folding rate increases from 0.60 to 18.7 s(-1). Tanford linkage analysis revealed that the interactions involving the two histidine residues are largely developed in the transition state. The results are compared to other studies of the pH-dependence of folding.

journal_name

J Mol Biol

authors

Sato S,Raleigh DP

doi

10.1016/S0022-2836(02)00015-3

keywords:

subject

Has Abstract

pub_date

2002-04-26 00:00:00

pages

571-82

issue

2

eissn

0022-2836

issn

1089-8638

pii

S0022-2836(02)00015-3

journal_volume

318

pub_type

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