Context-dependent contributions of backbone hydrogen bonding to β-sheet folding energetics

Songpon Deechongkit, Houbi Nguyen, Evan T. Powers, Philip E. Dawson, Martin Gruebele, Jeffery W. Kelly

Research output: Contribution to journalArticlepeer-review


Backbone hydrogen bonds (H-bonds) are prominent features of protein structures; however, their role in protein folding remains controversial because they cannot be selectively perturbed by traditional methods of protein mutagenesis. Here we have assessed the contribution of backbone H-bonds to the folding kinetics and thermodynamics of the PIN WW domain, a small β-sheet protein, by individually replacing its backbone amides with esters. Amide-to-ester mutations site-specifically perturb backbone H-bonds in two ways: a H-bond donor is eliminated by replacing an amide NH with an ester oxygen, and a H-bond acceptor is weakened by replacing an amide carbonyl with an ester carbonyl. We perturbed the 11 backbone H-bonds of the PIN WW domain by synthesizing 19 amide-to-ester mutants. Thermodynamic studies on these variants show that the protein is most destabilized when H-bonds that are enveloped by a hydrophobic duster are perturbed. Kinetic studies indicate that native-like secondary structure forms in one of the protein's loops in the folding transition state, but the backbone is less ordered elsewhere in the sequence. Collectively, our results provide an unusually detailed picture of the folding of a β-sheet protein.

Original languageEnglish (US)
Pages (from-to)101-105
Number of pages5
Issue number6995
StatePublished - Jul 1 2004

ASJC Scopus subject areas

  • General


Dive into the research topics of 'Context-dependent contributions of backbone hydrogen bonding to β-sheet folding energetics'. Together they form a unique fingerprint.

Cite this