Design of a heterotetrameric coiled coil.
We have successfully designed a simple peptide sequence that forms highly stable coiled-coil heterotetramers. Our model system is based on the GCN4-pLI parallel coiled-coil tetramer, first described by Kim and coworkers (Harbury et al., Science 1993;262:1401-1407). We introduced glutamates at all of the e and c heptad positions of one sequence (ecE) and lysines at the same positions in a second sequence (ecK). Based on a modeling study, these sidechains are close enough in space to form structure-stabilizing salt bridges. We show that ecE and ecK are highly unstable by themselves but form very stable parallel helical tetramers when mixed, as judged by circular dichroism, analytical ultracentrifugation, and disulfide crosslinking studies. The origin of the difference in stabilities between the homomeric structures and the heteromeric structures comes from a combination of the relief of electrostatic repulsions with concomitant formation of electrostatic attractive interactions based on pH and NaCl screening experiments. We quantify the stability of the heterotetrameric coiled coil from a thermodynamic analysis and compare the finding to other similar coiled-coil systems.
Root
Benjamin C
BC
Department of Biology, Haverford College, Pennsylvania 19041, USA.
Pellegrino
Laurel D
LD
Crawford
Emily D
ED
Kokona
Bashkim
B
Fairman
Robert
R
D000817:Anilino Naphthalenesulfonates; D002942:Circular Dichroism; D004220:Disulfides / Q000378:metabolism*; D005456:Fluorescent Dyes; D018698:Glutamic Acid / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D055503:Protein Multimerization; D055550:Protein Stability; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012965:Sodium Chloride; D055672:Static Electricity; D013816:Thermodynamics; D014461:Ultracentrifugation
D000817:Anilino Naphthalenesulfonates; D004220:Disulfides; D005456:Fluorescent Dyes; D010455:Peptides; D011506:Proteins; D018698:Glutamic Acid; D012965:Sodium Chloride; C027132:1-anilino-8-naphthalenesulfonate; D008239:Lysine