PMC:2724026 / 9631-12553
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":"Analysis of mutant data\nThe change in stability upon mutation (ΔΔGD-N) was determined using equilibrium denaturation followed by changes in fluorescence. All equilibrium m-values were the same as WT within error, and so ΔΔGD-N was determined using a mean m-value (\u003cm\u003e) of 1.4 ± 0.21 kcal mol- 1 M- 1 as:25(1) ΔΔGD−N=\u003cm\u003e.δ[urea]50%where δ [urea]50% is the difference in the midpoint of denaturation between WT and mutant protein.\nFitting the kinetic data for FADD DD mutants proved to be complex. All kinetics were measured using intrinsic Trp fluorescence as a probe to allow data to be collected at a low concentration (≤ 1 μM) of protein As for WT, both refolding and unfolding data fit well to a single-exponential equation for all mutants. To avoid the possibility of complication from aggregation events observed in the WT protein, refolding data below 1.5 M urea were not used for fitting the chevron plots. An exception to this rule was made for highly destabilised mutants with a low [urea]50%, where the refolding arm was short (W112A, W148F, L161A and L165A). For S144A, all data below 2.5 M urea were omitted from chevron fitting, due to aggregation. All mutant chevron plots had linear folding arms with essentially the same slope (only three had refolding mkf values that were significantly different from the mean value of 1.7 M- 1).\nSome mutant chevron plots had linear unfolding limbs, as was seen in WT. However, many mutants exhibited some downward curvature in the unfolding arm of their chevron plot and for a few mutants this curvature was very significant (Fig. 2). (What curved chevron plots may mean is discussed later). It was therefore not possible to do any global fitting of the data. All chevrons were fit individually: first, the chevrons were fit to an equation with a quadratic term in the unfolding limb only (a “Hammond” fit,26 see Eq. (3)); second, each chevron plot was fit individually to an equation describing a linear dependence of both lnkf and lnku on the denaturant concentration (linear chevron fit, see Eq. (2)). To fit the kinetic data to the linear chevron fit, data points that were judged to be curving downwards were omitted from the fit. (All chevrons are shown in Supplementary Data Figs. S1 and S2, fit to a linear equation, identifying the points omitted.)\nMost importantly, all Φ-values in this work have been determined using refolding data only, to avoid any uncertainty that might arise from fits of the unfolding data. Also, to avoid a long extrapolation to 0 M denaturant27 and to eliminate any possible effects of aggregation, all Φ-values were calculated at 2 M urea. We show (Supplementary Data Fig. S 4) that the Φ-values determined using either linear or Hammond fits of the data are essentially identical. Thus, despite the complexity of the analysis, the Φ-values obtained are reproducible. The results of the kinetic analysis are shown in Tables 2 and 3.","divisions":[{"label":"title","span":{"begin":0,"end":23}},{"label":"p","span":{"begin":24,"end":428}},{"label":"label","span":{"begin":305,"end":308}},{"label":"p","span":{"begin":429,"end":1347}},{"label":"p","span":{"begin":1348,"end":2310}}],"tracks":[{"project":"2_test","denotations":[{"id":"19362094-2812029-62520519","span":{"begin":303,"end":305},"obj":"2812029"},{"id":"19362094-8356089-62520520","span":{"begin":1859,"end":1861},"obj":"8356089"},{"id":"19362094-16782128-62520521","span":{"begin":2531,"end":2533},"obj":"16782128"}],"attributes":[{"subj":"19362094-2812029-62520519","pred":"source","obj":"2_test"},{"subj":"19362094-8356089-62520520","pred":"source","obj":"2_test"},{"subj":"19362094-16782128-62520521","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#b693ec","default":true}]}]}}