PMC:2724026 / 6827-8590
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2724026","sourcedb":"PMC","sourceid":"2724026","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2724026","text":"The equilibrium stability of wild type (WT) FADD was determined at pH 7.0, 25 °C in urea with unfolding monitored by changes in both intrinsic fluorescence and elipticity at 222 nm. Fluorescence and circular dichroism (CD) data overlaid (data not shown), indicating folding to be a completely reversible, co-operative, two-state transition. Data from five experiments gave an average [D]50% (the concentration of denaturant at which half the molecules are unfolded) of 4.8 ± 0.1 M and a mean m-value of 1.4 ± 0.2 kcal mol− 1 M− 1; this gives a mean free energy of unfolding for WT of 6.7 ± 0.15 kcal mol− 1. Kinetic rate constants obtained by fluorescence are in agreement with those obtained by CD. WT FADD DD contains one proline residue, but kinetic data fit well to a single exponential at all concentrations of urea. The chevron plot for WT FADD DD fit well to Eq. (2) (see Materials and Methods) with a linear dependence of both lnkf and lnku (where kf is the folding rate constant and ku is the unfolding rate constant) on the concentration of urea (Fig. 2). No data were analysed below 1.5 M urea, because the refolding rate constants become dependent upon protein concentration. This behaviour has been ascribed to transient aggregation.22 Linear extrapolation of chevron data to 0 M urea gives a kfH2O of 960 ± 60 s- 1 and a kuH2O of 0.04 ± 0.01 s- 1; WT kinetic m-values give a β-Tanford (βT) value of 0.76 (see Materials and Methods), indicating that the transition state is compact. The free energy of unfolding derived from equilibrium and kinetic data was compared at 2 M urea (to avoid long extrapolation to 0 M urea); the WT kinetic2M stability is 3.5 ± 0.1 kcal mol- 1, compared with that from equilibrium2M experiments of 3.9 ± 0.2 kcal mol- 1.","tracks":[{"project":"2_test","denotations":[{"id":"19362094-9177173-62520517","span":{"begin":1246,"end":1248},"obj":"9177173"}],"attributes":[{"subj":"19362094-9177173-62520517","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ec93b4","default":true}]}]}}