PMC:2724026 / 6789-16861 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/2724026","sourcedb":"PMC","sourceid":"2724026","source_url":"https://www.ncbi.nlm.nih.gov/pmc/2724026","text":"Results\n\nStudies of wild type FADD DD\nThe 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.\n\nChoice of mutations\nTo obtain Φ-values for core residues, and residues within elements of secondary structure, 64 mutations were made at 42 positions throughout the six helices: 23 of these were mutations of core residues, and 19 were residues where alanine to glycine “scanning” was performed, to report on the extent of secondary structure formation.23,24 Of the 23 core residues, most were non-disruptive replacements by alanine; however, in four cases this mutation was too destabilising and so non-conservative substitutions were made (Leu to Met and Trp to Phe). The residues to which the contacts are deleted and the number of heavy-atom side-chain contacts within 6 Å deleted on mutation are given in Table 1, which illustrates that truncation of core residues either reports mainly on contacts within one of the bundle cores or probes contacts in the central core. Thus, for example, in H4, R140 contacts only residues within B2, whereas L145 only makes long-range contacts with residues in H1 (in B1), through the central core.\n\nAnalysis 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.\n\nΦ-Value analysis\nΦ-Values obtained were either zero or fractional, with no Φ-value of 1 (Tables 2 and 3), indicating no part of the protein is as fully formed in the transition state as it is in the native state. To interpret a Φ-value analysis, it is customary to consider Φ-value patterns, rather than to try to interpret individual Φ-values. This allows one to determine which regions of the protein are fully unfolded, partially folded or fully folded in the transition state.28 The Φ-values obtained are thus generally classified into low, medium and high classes, with the boundaries chosen to reflect the overall Φ-value distribution (e.g. see Ref. 29): in our case, the cut-offs used are: low, Φ ≤ 0.15; medium, Φ = 0.16–0.5; high, Φ ≥ 0.51.\nWe will consider the structure of the transition state of FADD DD in terms of formation of the three structural cores, i.e. formation of the two three-helix bundles B1 and B2, and the formation of the central core (the four-helix motif). (A detailed analysis of each helix is given in the Supplementary Data). The Φ-values were mapped on the native structure, to allow the patterns of Φ-values to be interpreted.\n\nFormation of B1 (formed by H1, H5 and H6): (Fig. 3)\nFormation of the B1 core was probed by six mutations: H1, V103A; H5, V162A, L165A; and H6, V173A, L176A, V177A. The extent of helix formation was probed by six Ala to Gly mutations: H1, A98G, A102G; H5, A166G, A167G; and H6, A175G, A178G.\nAll three helices are partially folded but the B1 core can be considered as only very weakly structured. Only H5 and H6 make contact through the B1 core, through interactions between L165 at the C-terminus of H5 and V173 at the N-terminus of H6. H1 is only connected to the B1 core in the native state via V103, but the Φ-value of V103A is zero, suggesting that H1 does not contribute to the formation of the B1 core in the transition state (TS). (Note, however, that H1 and H5 do interact in the TS, through sidechain contacts on the central core side of the helices).\n\nFormation of B2 (formed by H2, H3 and H4): (Fig. 3)\nFormation of the B2 core was probed by six mutations: H2, W112A; H3, I126A, I129A, Y133A; and H4, R140A, S144A. The extent of helix formation was probed by 12 Ala to Gly mutations: H2, A113G, A114G, A117G; H3, A127G, A131G, A132G; and H4, A138G, A139G, A142G, A143G, A146G, A150G.\nHelix 4 is the most structured region of the entire protein, with high Ala to Gly Φ-values at the N-terminal end, becoming lower towards the C-terminal end. The two B2 core residues in this helix both have high Φ-values; W112 (the only hydrophobic residue from H2 which packs into this core) has a medium Φ-value. All the Φ-values of helix 3 are close to zero; this helix plays no role in the formation of B2 in the TS. Thus, we infer that the B2 core is loosely structured in the TS with H2 packing onto H4.\n\nFormation of central core formed by H1, H2, H4 and H5 (Fig. 3)\nFormation of the central core was probed by nine mutations: H1, F101A, I104A; H2, L115M, L119M; H4, V141A, L145M, W148F; and H5, H160A, L161A. The extent of helix formation was probed by 13 Ala to Gly mutations: H1, A98G, A102G; H2, A113G, A114G, A117G; H4, A138G, A139G, A142G, A143G, A146G, A150G; and H5, A166G, S167G. (Note that in native FADD DD, H1 and H5 of the central core run parallel with each other and are packed orthogonally onto the parallel helix pair H2 and H4; Fig. 1).\nIn the TS, the central core is partly formed, principally through interaction of F101 and I104 in H1, which contact residues from all the other three helices; H5 also contributes significantly to core packing, via H160 and L161 at the N-terminal end. The central core residue in H2, L115, which packs onto residues in both H1 and H5 in the native state, has a medium Φ-value. Notably, although H4 is apparently well structured, the only core residue that contributes structure in the TS is V141 at the extreme N-terminus of H4. It has a high Φ-value, and appears to pin this end of H4 to H1 via an interaction with I104. In the native state, the central core of FADD DD is dominated at one end by W148F from H4, which has a Φ-value of zero. Thus, one end of the central core appears to be largely unstructured, and H4 is essentially attached only via contacts with H2 (via the B2 core).","divisions":[{"label":"title","span":{"begin":0,"end":7}},{"label":"sec","span":{"begin":9,"end":1801}},{"label":"title","span":{"begin":9,"end":37}},{"label":"p","span":{"begin":38,"end":1801}},{"label":"sec","span":{"begin":1803,"end":2840}},{"label":"title","span":{"begin":1803,"end":1822}},{"label":"p","span":{"begin":1823,"end":2840}},{"label":"sec","span":{"begin":2842,"end":5764}},{"label":"title","span":{"begin":2842,"end":2865}},{"label":"p","span":{"begin":2866,"end":3270}},{"label":"label","span":{"begin":3147,"end":3150}},{"label":"p","span":{"begin":3271,"end":4189}},{"label":"p","span":{"begin":4190,"end":5152}},{"label":"p","span":{"begin":5153,"end":5764}},{"label":"title","span":{"begin":5766,"end":5782}},{"label":"p","span":{"begin":5783,"end":6515}},{"label":"p","span":{"begin":6516,"end":6928}},{"label":"sec","span":{"begin":6930,"end":7790}},{"label":"title","span":{"begin":6930,"end":6981}},{"label":"p","span":{"begin":6982,"end":7220}},{"label":"p","span":{"begin":7221,"end":7790}},{"label":"sec","span":{"begin":7792,"end":8633}},{"label":"title","span":{"begin":7792,"end":7843}},{"label":"p","span":{"begin":7844,"end":8124}},{"label":"p","span":{"begin":8125,"end":8633}},{"label":"title","span":{"begin":8635,"end":8697}},{"label":"p","span":{"begin":8698,"end":9185}}],"tracks":[{"project":"2_test","denotations":[{"id":"19362094-9177173-62520517","span":{"begin":1284,"end":1286},"obj":"9177173"},{"id":"19362094-7756312-62520518","span":{"begin":2158,"end":2160},"obj":"7756312"},{"id":"19362094-17307875-62520518","span":{"begin":2158,"end":2160},"obj":"17307875"},{"id":"19362094-2812029-62520519","span":{"begin":3145,"end":3147},"obj":"2812029"},{"id":"19362094-8356089-62520520","span":{"begin":4701,"end":4703},"obj":"8356089"},{"id":"19362094-16782128-62520521","span":{"begin":5373,"end":5375},"obj":"16782128"},{"id":"19362094-1569556-62520522","span":{"begin":6246,"end":6248},"obj":"1569556"},{"id":"19362094-18538343-62520523","span":{"begin":6422,"end":6424},"obj":"18538343"}],"attributes":[{"subj":"19362094-9177173-62520517","pred":"source","obj":"2_test"},{"subj":"19362094-7756312-62520518","pred":"source","obj":"2_test"},{"subj":"19362094-17307875-62520518","pred":"source","obj":"2_test"},{"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"},{"subj":"19362094-1569556-62520522","pred":"source","obj":"2_test"},{"subj":"19362094-18538343-62520523","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#ecda93","default":true}]}]}}