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.

Conserved thermodynamic contributions of backbone hydrogen bonds in a protein fold

“…The m values determined for the ester bond-containing analogues were on average Ϸ20% larger than the values determined for the wild-type controls. Such increased m values of ester bond-containing analogues have been previously observed in other protein systems (3,5,7,9,10). It is particularly noteworthy in this work that similarly increased m values were observed at each ester-bond position.…”

“…2 Clearly, the physical environment (e.g., buried surface area) surrounding backbone-backbone hydrogen bonds can influence their apparent ''strength,'' or measured ⌬⌬G f value. This is likely one contributing factor to the variability of the measured ⌬⌬G f values determined for the ester bond-containing analogues in this study and other studies (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). For example, in this study the ⌬⌬G f value observed in the ␤-a position, which is only partially buried, was smaller than the values observed in the ␣-I-b, ␣-II-a, and ␣-II-b positions, which are all completely buried in the structure.…”

“…The relative contributions of different backbone-backbone hydrogen-bonding interactions in protein-folding reactions have been explored in a series of recent studies on several different protein systems (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). These studies have typically relied on total chemical synthesis methods (13) or specialized in vitro translation techniques (14) to incorporate amide-to-ester bond mutations into the polypeptide backbone of proteins and modulate the hydrogen-bonding properties of a protein's polypeptide backbone.…”

“…These studies have typically relied on total chemical synthesis methods (13) or specialized in vitro translation techniques (14) to incorporate amide-to-ester bond mutations into the polypeptide backbone of proteins and modulate the hydrogen-bonding properties of a protein's polypeptide backbone. The structural and thermodynamic consequences of introducing such amide-to-ester-bond mutations into ␤-sheet and ␣-helical regions of different protein folds have been reported (3)(4)(5)(6)(7)(8)(9)(10)(11)(12). In most cases reported to date, the amide-toester bond mutation has not significantly altered the threedimensional structure of the protein under study; however, it has been observed that the thermodynamic stabilities of protein analogues containing ester-bond mutations in the ␤-sheet and ␣-helical regions of different protein folds are generally reduced 0.5-3.0 kcal͞mol per ester bond compared with the wild-type protein.…”

Estersubstitutionen in α‐helicalen Coiled‐Coil‐Peptiden: Effekt der Eliminierung von Wasserstoffbrücken auf die Struktur von Proteinen

“…[1][2][3][4][5] "Amid-zu-Ester"-Substitutionen haben sich hierbei als nützliches Hilfsmittel erwiesen, um den Effekt der Wasserstoffbrücken im Peptidrückgrat auf die Strukturbildung und Stabilität von Peptiden und Proteinen zu untersuchen. [6][7][8][9][10][11][12][13][14][15][16] Um den umgebungsabhängigen Einfluss von Wasserstoffbrücken zu studieren, wurde ein kombinierter Ansatz aus experimentellen Techniken und Moleküldyna-mik(MD)-Simulationen angewendet.…”