title: Probing backbone hydrogen bonds in the hydrophobic core of GCN4.
abstract: Backbone amide hydrogen bonds play a central role in protein secondary and tertiary structure. Previous studies have shown that substitution of a backbone ester (-COO-) in place of a backbone amide (-CONH-) can selectively destabilize backbone hydrogen bonds in a protein while maintaining a similar conformation to the native backbone structure. The majority of these studies have focused on backbone substitutions that were accessible to solvent. The GCN4 coiled coil domain is an example of a stable alpha-helical dimer that possesses a well-packed hydrophobic core. Amino acids in the a and d positions of the GCN4 helix, which pack the hydrophobic core, were replaced with the corresponding alpha-hydroxy acids in the context of a chemoselectively ligated heterodimer. While the overall structure and oligomerization state of the heterodimer were maintained, the overall destabilization of the ester analogues was greater (average DeltaDeltaG of 3+ kcal mol(-1)) and more variable than previous studies. Since burial of the more hydrophobic ester should stabilize the backbone and reduce the DeltaDeltaG, the increased destabilization must come from another source. However, the observed destabilization is correlated with the protection factors for individual amide hydrogens from previous hydrogen exchange experiments. Therefore, our results suggest that backbone engineering through ester substitution is a useful approach for probing the relative strength of backbone hydrogen bonds.
authors.affiliation: Department of Cell Biology, Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, CVN-6, La Jolla, CA 92037, USA.
authors.forename: John W
authors.initials: JW
authors.lastname: Blankenship
authors.forename: Rema
authors.initials: R
authors.lastname: Balambika
authors.forename: Philip E
authors.initials: PE
authors.lastname: Dawson
mesh_terms: D000817:Anilino Naphthalenesulfonates / Q000737:chemistry; D001665:Binding Sites; D002942:Circular Dichroism; D004268:DNA-Binding Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D019281:Dimerization; D005456:Fluorescent Dyes / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D006358:Hot Temperature; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D008024:Ligands; D010452:Peptide Biosynthesis; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011494:Protein Kinases / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D013440:Sulfides / Q000737:chemistry; D013816:Thermodynamics; D014461:Ultracentrifugation
chemical_list: D000817:Anilino Naphthalenesulfonates; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D008024:Ligands; D029701:Saccharomyces cerevisiae Proteins; D013440:Sulfides; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D011494:Protein Kinases; D019791:Guanidine