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The mechanism of folding of Im7 reveals competition between functional and kinetic evolutionary constraints.

Friel CT, Smith DA, Vendruscolo M, Gsponer J, Radford SE - Nat. Struct. Mol. Biol. (2009)

Bottom Line: It is not clear whether this type of folding landscape results from insufficient evolutionary pressure to optimize folding efficiency, or arises from a conflict between functional and folding constraints.The results provide a comprehensive view of the folding process of this small protein.An analysis of the contributions of native and non-native interactions at different stages of folding reveals how the complexity of the folding landscape arises from concomitant evolutionary pressures for function and folding efficiency.

View Article: PubMed Central - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Mount Preston Street, Leeds LS2 9JT, UK.

ABSTRACT
Many proteins reach their native state through pathways involving the presence of folding intermediates. It is not clear whether this type of folding landscape results from insufficient evolutionary pressure to optimize folding efficiency, or arises from a conflict between functional and folding constraints. Here, using protein-engineering, ultra-rapid mixing and stopped-flow experiments combined with restrained molecular dynamics simulations, we characterize the transition state for the formation of the intermediate populated during the folding of the bacterial immunity protein, Im7, and the subsequent molecular steps leading to the native state. The results provide a comprehensive view of the folding process of this small protein. An analysis of the contributions of native and non-native interactions at different stages of folding reveals how the complexity of the folding landscape arises from concomitant evolutionary pressures for function and folding efficiency.

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Comparison of structural properties of TS1, I and TS2. (a) Representative members of the three ensembles. The segments forming the four helices in N are shown in red (I), green (II) magenta (III) and yellow (IV). (b) Diagram displaying the heterogeneity of different ensembles: the tube diameter is scaled by the average displacement to the mean structure. (c) Distribution of the radius of gyration of the entire polypeptide chain (red) and the core residues (15,18, 19, 22, 34, 38, 41, 44, 53, 54, 55, 68 and 72) (blue). The dashed lines indicate the radius of gyration of each set of residues calculated for N. (d) Distribution of the distances between the centres of mass of different helices: helices I-II, red; helices II-IV, green; helices I-IV, blue. The dashed lines indicate the distances calculated for N.
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Figure 5: Comparison of structural properties of TS1, I and TS2. (a) Representative members of the three ensembles. The segments forming the four helices in N are shown in red (I), green (II) magenta (III) and yellow (IV). (b) Diagram displaying the heterogeneity of different ensembles: the tube diameter is scaled by the average displacement to the mean structure. (c) Distribution of the radius of gyration of the entire polypeptide chain (red) and the core residues (15,18, 19, 22, 34, 38, 41, 44, 53, 54, 55, 68 and 72) (blue). The dashed lines indicate the radius of gyration of each set of residues calculated for N. (d) Distribution of the distances between the centres of mass of different helices: helices I-II, red; helices II-IV, green; helices I-IV, blue. The dashed lines indicate the distances calculated for N.

Mentions: Ensembles representative of TS1, I and TS2 determined by restrained MD simulations are shown in Fig. 4a,b. These ensembles are fully consistent with the experimental Φ-values. This result is expected for TS1 and TS2, since the Φ-values were used as a source of structural information, but is notable for the intermediate state, since in this case equilibrium hydrogen-exchange protection factors – but not Φ-values – were used as restraints (Supplementary Fig. S2)22. To assess the quality of the ensembles generated, Φ-values were back-calculated using FoldX28. In contrast to the native contact approximation used to restrain Φ-values during the structure calculations (see Methods) the free energy based back-calculation of Φ-values using FoldX is indifferent to whether contacts are native or non-native26. Importantly, as some experimental ΦTS1 values, especially those of L18A, L19A and L37A, depend critically on the reference state used for their determination (see above), a correct prediction of these ΦTS1 values relative to ΔΔGui and ΔΔGun computed over the ensembles generated provides a stringent test for the quality of the TS1 and I ensembles. Correlations of 0.79, 0.74, 0.73 between experimental and back-calculated Φ-values for TS1, I, TS2 (with respect to ΔΔGun), and 0.76 for TS1 (with respect to ΔΔGui), highlight the quality of all ensembles (Supplementary Fig. S2b). An additional validation of the structures results from the correct prediction of the experimentally determined βT values (Supplementary Methods online).


The mechanism of folding of Im7 reveals competition between functional and kinetic evolutionary constraints.

Friel CT, Smith DA, Vendruscolo M, Gsponer J, Radford SE - Nat. Struct. Mol. Biol. (2009)

Comparison of structural properties of TS1, I and TS2. (a) Representative members of the three ensembles. The segments forming the four helices in N are shown in red (I), green (II) magenta (III) and yellow (IV). (b) Diagram displaying the heterogeneity of different ensembles: the tube diameter is scaled by the average displacement to the mean structure. (c) Distribution of the radius of gyration of the entire polypeptide chain (red) and the core residues (15,18, 19, 22, 34, 38, 41, 44, 53, 54, 55, 68 and 72) (blue). The dashed lines indicate the radius of gyration of each set of residues calculated for N. (d) Distribution of the distances between the centres of mass of different helices: helices I-II, red; helices II-IV, green; helices I-IV, blue. The dashed lines indicate the distances calculated for N.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2651959&req=5

Figure 5: Comparison of structural properties of TS1, I and TS2. (a) Representative members of the three ensembles. The segments forming the four helices in N are shown in red (I), green (II) magenta (III) and yellow (IV). (b) Diagram displaying the heterogeneity of different ensembles: the tube diameter is scaled by the average displacement to the mean structure. (c) Distribution of the radius of gyration of the entire polypeptide chain (red) and the core residues (15,18, 19, 22, 34, 38, 41, 44, 53, 54, 55, 68 and 72) (blue). The dashed lines indicate the radius of gyration of each set of residues calculated for N. (d) Distribution of the distances between the centres of mass of different helices: helices I-II, red; helices II-IV, green; helices I-IV, blue. The dashed lines indicate the distances calculated for N.
Mentions: Ensembles representative of TS1, I and TS2 determined by restrained MD simulations are shown in Fig. 4a,b. These ensembles are fully consistent with the experimental Φ-values. This result is expected for TS1 and TS2, since the Φ-values were used as a source of structural information, but is notable for the intermediate state, since in this case equilibrium hydrogen-exchange protection factors – but not Φ-values – were used as restraints (Supplementary Fig. S2)22. To assess the quality of the ensembles generated, Φ-values were back-calculated using FoldX28. In contrast to the native contact approximation used to restrain Φ-values during the structure calculations (see Methods) the free energy based back-calculation of Φ-values using FoldX is indifferent to whether contacts are native or non-native26. Importantly, as some experimental ΦTS1 values, especially those of L18A, L19A and L37A, depend critically on the reference state used for their determination (see above), a correct prediction of these ΦTS1 values relative to ΔΔGui and ΔΔGun computed over the ensembles generated provides a stringent test for the quality of the TS1 and I ensembles. Correlations of 0.79, 0.74, 0.73 between experimental and back-calculated Φ-values for TS1, I, TS2 (with respect to ΔΔGun), and 0.76 for TS1 (with respect to ΔΔGui), highlight the quality of all ensembles (Supplementary Fig. S2b). An additional validation of the structures results from the correct prediction of the experimentally determined βT values (Supplementary Methods online).

Bottom Line: It is not clear whether this type of folding landscape results from insufficient evolutionary pressure to optimize folding efficiency, or arises from a conflict between functional and folding constraints.The results provide a comprehensive view of the folding process of this small protein.An analysis of the contributions of native and non-native interactions at different stages of folding reveals how the complexity of the folding landscape arises from concomitant evolutionary pressures for function and folding efficiency.

View Article: PubMed Central - PubMed

Affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Mount Preston Street, Leeds LS2 9JT, UK.

ABSTRACT
Many proteins reach their native state through pathways involving the presence of folding intermediates. It is not clear whether this type of folding landscape results from insufficient evolutionary pressure to optimize folding efficiency, or arises from a conflict between functional and folding constraints. Here, using protein-engineering, ultra-rapid mixing and stopped-flow experiments combined with restrained molecular dynamics simulations, we characterize the transition state for the formation of the intermediate populated during the folding of the bacterial immunity protein, Im7, and the subsequent molecular steps leading to the native state. The results provide a comprehensive view of the folding process of this small protein. An analysis of the contributions of native and non-native interactions at different stages of folding reveals how the complexity of the folding landscape arises from concomitant evolutionary pressures for function and folding efficiency.

Show MeSH