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Crosstalk between the protein surface and hydrophobic core in a core-swapped fibronectin type III domain.

Billings KS, Best RB, Rutherford TJ, Clarke J - J. Mol. Biol. (2007)

Bottom Line: Remarkably, FNoTNc retains the structure of the parent proteins despite the extent of redesign, allowing us to gain insight into which components of each parent protein are responsible for different aspects of its behaviour.Naively, one would expect properties that appear to depend principally on the core to be similar to TNfn3, for example, the response to mutations, folding kinetics and side-chain dynamics, while properties apparently determined by differences in the surface and loops, such as backbone dynamics, would be more like FNfn10.For example, the anomalous response of FNfn10 to mutation is not solely a property of the core as we had previously suggested.

View Article: PubMed Central - PubMed

Affiliation: Cambridge University Chemical Laboratory, MRC Centre for Protein Engineering, Lensfield Road, Cambridge CB2 1EW, UK.

ABSTRACT
Two homologous fibronectin type III (fnIII) domains, FNfn10 (the 10th fnIII domain of human fibronectin) and TNfn3 (the third fnIII domain of human tenascin), have essentially the same backbone structure, although they share only approximately 24% sequence identity. While they share a similar folding mechanism with a common core of key residues in the folding transition state, they differ in many other physical properties. We use a chimeric protein, FNoTNc, to investigate the molecular basis for these differences. FNoTNc is a core-swapped protein, containing the "outside" (surface and loops) of FNfn10 and the hydrophobic core of TNfn3. Remarkably, FNoTNc retains the structure of the parent proteins despite the extent of redesign, allowing us to gain insight into which components of each parent protein are responsible for different aspects of its behaviour. Naively, one would expect properties that appear to depend principally on the core to be similar to TNfn3, for example, the response to mutations, folding kinetics and side-chain dynamics, while properties apparently determined by differences in the surface and loops, such as backbone dynamics, would be more like FNfn10. While this is broadly true, it is clear that there are also unexpected crosstalk effects between the core and the surface. For example, the anomalous response of FNfn10 to mutation is not solely a property of the core as we had previously suggested.

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Response of FNoTNc to mutation. Histograms of (a) central coremutations and (b) peripheral versus FNfn10 and TNfn3.Note that mutation of residues Pro5, Pro25 and Ser85 in FNoTNc causes littleloss of stability (as in FNfn10), whereas mutation of Phe92 (and Ile8, data notshown) results in a loss in stability close to that seen in TNfn3. (c) Backboneribbon representation of FNoTNc created using MacPyMOL. Peripheral core residuesP5, P25 and S85, which show little loss in stability in FNoTNc are coloured red,residues I8 and F92 are coloured cyan and other core residues are coloured blue.Data for FNfn10 and TNfn3 are taken from Ref. 19. Note that the ΔΔGD–Nfor I20A in FNfn10 was incorrectly reported in the original work.19 This has been remeasured for this study.
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fig2: Response of FNoTNc to mutation. Histograms of (a) central coremutations and (b) peripheral versus FNfn10 and TNfn3.Note that mutation of residues Pro5, Pro25 and Ser85 in FNoTNc causes littleloss of stability (as in FNfn10), whereas mutation of Phe92 (and Ile8, data notshown) results in a loss in stability close to that seen in TNfn3. (c) Backboneribbon representation of FNoTNc created using MacPyMOL. Peripheral core residuesP5, P25 and S85, which show little loss in stability in FNoTNc are coloured red,residues I8 and F92 are coloured cyan and other core residues are coloured blue.Data for FNfn10 and TNfn3 are taken from Ref. 19. Note that the ΔΔGD–Nfor I20A in FNfn10 was incorrectly reported in the original work.19 This has been remeasured for this study.

Mentions: A number of core residues were mutated in FNoTNc to investigate theresponse of the protein to mutation. These were positions that hadpreviously been investigated in the parent proteins FNfn10 and TNfn3.The thermodynamic stability of the mutant proteins was determined bychemical denaturation in guanidinium chloride (GdmCl). The mutations canclearly be divided into two categories. A few peripheral mutations(mutations in the A and G strands and the B–C loop) have little effecton stability as was previously observed in FNfn10 (Fig. 2a),whereas most other mutations were more typically destabilising. In thelatter case, the ΔΔGD–N wassimilar to (but in general slightly lower than) what had been observedpreviously in TNfn3 (Fig. 2b).The residues in each category are mapped onto a backbone ribbonrepresentation of FNoTNc in Fig.2c. ΔΔGD–N valuesfor all mutations are compared to those of FNfn10 and TNfn3 inSupplementary Table1.


Crosstalk between the protein surface and hydrophobic core in a core-swapped fibronectin type III domain.

Billings KS, Best RB, Rutherford TJ, Clarke J - J. Mol. Biol. (2007)

Response of FNoTNc to mutation. Histograms of (a) central coremutations and (b) peripheral versus FNfn10 and TNfn3.Note that mutation of residues Pro5, Pro25 and Ser85 in FNoTNc causes littleloss of stability (as in FNfn10), whereas mutation of Phe92 (and Ile8, data notshown) results in a loss in stability close to that seen in TNfn3. (c) Backboneribbon representation of FNoTNc created using MacPyMOL. Peripheral core residuesP5, P25 and S85, which show little loss in stability in FNoTNc are coloured red,residues I8 and F92 are coloured cyan and other core residues are coloured blue.Data for FNfn10 and TNfn3 are taken from Ref. 19. Note that the ΔΔGD–Nfor I20A in FNfn10 was incorrectly reported in the original work.19 This has been remeasured for this study.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2291452&req=5

fig2: Response of FNoTNc to mutation. Histograms of (a) central coremutations and (b) peripheral versus FNfn10 and TNfn3.Note that mutation of residues Pro5, Pro25 and Ser85 in FNoTNc causes littleloss of stability (as in FNfn10), whereas mutation of Phe92 (and Ile8, data notshown) results in a loss in stability close to that seen in TNfn3. (c) Backboneribbon representation of FNoTNc created using MacPyMOL. Peripheral core residuesP5, P25 and S85, which show little loss in stability in FNoTNc are coloured red,residues I8 and F92 are coloured cyan and other core residues are coloured blue.Data for FNfn10 and TNfn3 are taken from Ref. 19. Note that the ΔΔGD–Nfor I20A in FNfn10 was incorrectly reported in the original work.19 This has been remeasured for this study.
Mentions: A number of core residues were mutated in FNoTNc to investigate theresponse of the protein to mutation. These were positions that hadpreviously been investigated in the parent proteins FNfn10 and TNfn3.The thermodynamic stability of the mutant proteins was determined bychemical denaturation in guanidinium chloride (GdmCl). The mutations canclearly be divided into two categories. A few peripheral mutations(mutations in the A and G strands and the B–C loop) have little effecton stability as was previously observed in FNfn10 (Fig. 2a),whereas most other mutations were more typically destabilising. In thelatter case, the ΔΔGD–N wassimilar to (but in general slightly lower than) what had been observedpreviously in TNfn3 (Fig. 2b).The residues in each category are mapped onto a backbone ribbonrepresentation of FNoTNc in Fig.2c. ΔΔGD–N valuesfor all mutations are compared to those of FNfn10 and TNfn3 inSupplementary Table1.

Bottom Line: Remarkably, FNoTNc retains the structure of the parent proteins despite the extent of redesign, allowing us to gain insight into which components of each parent protein are responsible for different aspects of its behaviour.Naively, one would expect properties that appear to depend principally on the core to be similar to TNfn3, for example, the response to mutations, folding kinetics and side-chain dynamics, while properties apparently determined by differences in the surface and loops, such as backbone dynamics, would be more like FNfn10.For example, the anomalous response of FNfn10 to mutation is not solely a property of the core as we had previously suggested.

View Article: PubMed Central - PubMed

Affiliation: Cambridge University Chemical Laboratory, MRC Centre for Protein Engineering, Lensfield Road, Cambridge CB2 1EW, UK.

ABSTRACT
Two homologous fibronectin type III (fnIII) domains, FNfn10 (the 10th fnIII domain of human fibronectin) and TNfn3 (the third fnIII domain of human tenascin), have essentially the same backbone structure, although they share only approximately 24% sequence identity. While they share a similar folding mechanism with a common core of key residues in the folding transition state, they differ in many other physical properties. We use a chimeric protein, FNoTNc, to investigate the molecular basis for these differences. FNoTNc is a core-swapped protein, containing the "outside" (surface and loops) of FNfn10 and the hydrophobic core of TNfn3. Remarkably, FNoTNc retains the structure of the parent proteins despite the extent of redesign, allowing us to gain insight into which components of each parent protein are responsible for different aspects of its behaviour. Naively, one would expect properties that appear to depend principally on the core to be similar to TNfn3, for example, the response to mutations, folding kinetics and side-chain dynamics, while properties apparently determined by differences in the surface and loops, such as backbone dynamics, would be more like FNfn10. While this is broadly true, it is clear that there are also unexpected crosstalk effects between the core and the surface. For example, the anomalous response of FNfn10 to mutation is not solely a property of the core as we had previously suggested.

Show MeSH
Related in: MedlinePlus