Redesigning the topology of a four-helix-bundle protein: monomeric Rop.
The topology of alpha-helices and beta-sheets in folded proteins is largely specified by the connectivities of the loops and turns which join them. We have used the protein Rop to test the feasibility of using short glycine-rich linkers to reconnect the alpha-helices within a four-helix-bundle protein. In wild-type Rop the four-helix-bundle structure is formed by the association of two identical helix-turn-helix monomers. Our redesigns encode Rop as a single chain to create a monomeric, rather than a dimeric, form of the protein. Characterization of a series of such variants demonstrates that new connections of this type can be used to generate stable, native-like proteins. The length of the connections is of key importance; if the loops are too short, correct association of the helices is prevented, and misfolded, higher order oligomers occur. Designs with sufficiently long loop connections, however, generate exclusively the desired monomeric form of the protein. Moreover, the successful monomeric designs bind Rop's RNA substrate with affinities that are equal to that of the wild-type protein. This result provides strong confirmation that the positioning of the helices in the monomeric variants is closely similar to that in wild-type Rop.
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Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, USA.
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D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D016296:Mutagenesis; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012313:RNA / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry
D001426:Bacterial Proteins; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; C054243:Rop protein, ColE1 plasmid; D012313:RNA