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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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Related in: MedlinePlus

Dependence of the natural logarithm of the observed rate constants on the concentration of urea for selected Im7 variants. Solid lines show the best fit of the data to a three-state model with an on-pathway intermediate. The dashed line shows the best fit to the data for wild-type Im7 for comparison. (a) I7V, (b) L19A, (c) L38A, (d) I54V, (e) I72V, (f) A77G.
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Figure 3: Dependence of the natural logarithm of the observed rate constants on the concentration of urea for selected Im7 variants. Solid lines show the best fit of the data to a three-state model with an on-pathway intermediate. The dashed line shows the best fit to the data for wild-type Im7 for comparison. (a) I7V, (b) L19A, (c) L38A, (d) I54V, (e) I72V, (f) A77G.

Mentions: To obtain information about the extent of secondary structure formation in TS1, Ala13 and Ala77, which are solvent exposed in the native state and located in helices I and IV, respectively (Fig. 1a), were truncated to Gly and the folding and unfolding kinetics of the variants measured as described above. These substitutions have only a small effect on kui (kui = 1150s−1 and 1648s−1 for A13G and A77G, respectively, compared with 1574s−1 for the wild-type protein) (Fig. 2 and Supplementary Table 1 online), but decrease the stability of I and N (Supplementary Table 2 online), indicating that these residues are not well-ordered in TS1, but form helical structure in I and TS2. Accordingly, Φ-values calculated for TS1, I and TS2 are 0.29±0.07, 1.33±0.20 and 1.39±0.09, respectively, for A13G and −0.02±0.06, 0.82±0.22 and 0.81±0.10, respectively for A77G. The substitution V33A increases the helical propensity in the N-terminal region of helix II (Fig. 1a). While accurate Φ-values could not be determined for this substitution since ΔΔGun is small (Supplementary Table 1), this substitution also has little effect on kui. By contrast with I and TS2 which contain native-like helices I, II and IV14,17, helical structure is not detected in the vicinity of the sites investigated in TS1.


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)

Dependence of the natural logarithm of the observed rate constants on the concentration of urea for selected Im7 variants. Solid lines show the best fit of the data to a three-state model with an on-pathway intermediate. The dashed line shows the best fit to the data for wild-type Im7 for comparison. (a) I7V, (b) L19A, (c) L38A, (d) I54V, (e) I72V, (f) A77G.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2651959&req=5

Figure 3: Dependence of the natural logarithm of the observed rate constants on the concentration of urea for selected Im7 variants. Solid lines show the best fit of the data to a three-state model with an on-pathway intermediate. The dashed line shows the best fit to the data for wild-type Im7 for comparison. (a) I7V, (b) L19A, (c) L38A, (d) I54V, (e) I72V, (f) A77G.
Mentions: To obtain information about the extent of secondary structure formation in TS1, Ala13 and Ala77, which are solvent exposed in the native state and located in helices I and IV, respectively (Fig. 1a), were truncated to Gly and the folding and unfolding kinetics of the variants measured as described above. These substitutions have only a small effect on kui (kui = 1150s−1 and 1648s−1 for A13G and A77G, respectively, compared with 1574s−1 for the wild-type protein) (Fig. 2 and Supplementary Table 1 online), but decrease the stability of I and N (Supplementary Table 2 online), indicating that these residues are not well-ordered in TS1, but form helical structure in I and TS2. Accordingly, Φ-values calculated for TS1, I and TS2 are 0.29±0.07, 1.33±0.20 and 1.39±0.09, respectively, for A13G and −0.02±0.06, 0.82±0.22 and 0.81±0.10, respectively for A77G. The substitution V33A increases the helical propensity in the N-terminal region of helix II (Fig. 1a). While accurate Φ-values could not be determined for this substitution since ΔΔGun is small (Supplementary Table 1), this substitution also has little effect on kui. By contrast with I and TS2 which contain native-like helices I, II and IV14,17, helical structure is not detected in the vicinity of the sites investigated in TS1.

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
Related in: MedlinePlus