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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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Side-chain dynamics. The methylS2 values of (a) FNoTNc (b) FNfn10 and(c) TNfn3 are shown projected onto the protein structure. The three structuresare in approximately the same orientation with the C-terminus at the top of themolecule. S2 ranges from zero to unity,with higher values of S2 indicatinggreater conformational restriction. FNoTNc has a cluster of highly mobileresidues in the core as does TNfn3. Figures created using MacPyMOL. (d) Acomparison of S2 values of identicalside chains in FNoTNc and TNfn3. (A similar comparison has not been shown forFNfn10 and FNoTNc since very few core residues have the same identity and so theside-chain dynamics per residue are not directly comparable.) Data for FNfn10and TNfn3 were taken from Ref. 22.
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fig6: Side-chain dynamics. The methylS2 values of (a) FNoTNc (b) FNfn10 and(c) TNfn3 are shown projected onto the protein structure. The three structuresare in approximately the same orientation with the C-terminus at the top of themolecule. S2 ranges from zero to unity,with higher values of S2 indicatinggreater conformational restriction. FNoTNc has a cluster of highly mobileresidues in the core as does TNfn3. Figures created using MacPyMOL. (d) Acomparison of S2 values of identicalside chains in FNoTNc and TNfn3. (A similar comparison has not been shown forFNfn10 and FNoTNc since very few core residues have the same identity and so theside-chain dynamics per residue are not directly comparable.) Data for FNfn10and TNfn3 were taken from Ref. 22.

Mentions: The relaxation of the operator terms IzCzDz, IzCzDy and IzCz was measured to give deuterium relaxation time constantsT1(D) andT2(D)31 (Supplementary Table5). Order parameters,S2, were determined for eachmethyl group using the standard model-free formalism as previouslydescribed22 (Supplementary Table6). The dynamics data for those methyl groups that hadoverlapping peaks were treated with caution, as the contribution fromthe overlapped peaks cannot be separated. Nevertheless, we have someconfidence in these results due to the agreement of the order parametersfor residues that have both overlapped and well-resolved methyl groups.S2 ranges from zero tounity, with higher values of S2indicating greater conformational restriction. The methylS2 values are shownprojected onto the protein structure and compared to the parent proteinsin Fig. 6. Within the core of FNoTNc is a cluster of deeplyburied residues, which have unusually low order parameters, as haspreviously been observed in TNfn3.


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)

Side-chain dynamics. The methylS2 values of (a) FNoTNc (b) FNfn10 and(c) TNfn3 are shown projected onto the protein structure. The three structuresare in approximately the same orientation with the C-terminus at the top of themolecule. S2 ranges from zero to unity,with higher values of S2 indicatinggreater conformational restriction. FNoTNc has a cluster of highly mobileresidues in the core as does TNfn3. Figures created using MacPyMOL. (d) Acomparison of S2 values of identicalside chains in FNoTNc and TNfn3. (A similar comparison has not been shown forFNfn10 and FNoTNc since very few core residues have the same identity and so theside-chain dynamics per residue are not directly comparable.) Data for FNfn10and TNfn3 were taken from Ref. 22.
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fig6: Side-chain dynamics. The methylS2 values of (a) FNoTNc (b) FNfn10 and(c) TNfn3 are shown projected onto the protein structure. The three structuresare in approximately the same orientation with the C-terminus at the top of themolecule. S2 ranges from zero to unity,with higher values of S2 indicatinggreater conformational restriction. FNoTNc has a cluster of highly mobileresidues in the core as does TNfn3. Figures created using MacPyMOL. (d) Acomparison of S2 values of identicalside chains in FNoTNc and TNfn3. (A similar comparison has not been shown forFNfn10 and FNoTNc since very few core residues have the same identity and so theside-chain dynamics per residue are not directly comparable.) Data for FNfn10and TNfn3 were taken from Ref. 22.
Mentions: The relaxation of the operator terms IzCzDz, IzCzDy and IzCz was measured to give deuterium relaxation time constantsT1(D) andT2(D)31 (Supplementary Table5). Order parameters,S2, were determined for eachmethyl group using the standard model-free formalism as previouslydescribed22 (Supplementary Table6). The dynamics data for those methyl groups that hadoverlapping peaks were treated with caution, as the contribution fromthe overlapped peaks cannot be separated. Nevertheless, we have someconfidence in these results due to the agreement of the order parametersfor residues that have both overlapped and well-resolved methyl groups.S2 ranges from zero tounity, with higher values of S2indicating greater conformational restriction. The methylS2 values are shownprojected onto the protein structure and compared to the parent proteinsin Fig. 6. Within the core of FNoTNc is a cluster of deeplyburied residues, which have unusually low order parameters, as haspreviously been observed in TNfn3.

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