The Role of Backbone Hydrogen Bonds in the Transition State for Protein Folding of a PDZ Domain

Abstract

Backbone hydrogen bonds are important for the structure and stability of proteins. However, since conventional site-directed mutagenesis cannot be applied to perturb the backbone, the contribution of these hydrogen bonds in protein folding and stability has been assessed only for a very limited set of small proteins. We have here investigated effects of five amide-to-ester mutations in the backbone of a PDZ domain, a 90-residue globular protein domain, to probe the influence of hydrogen bonds in a β-sheet for folding and stability. The amide-to-ester mutation removes NH-mediated hydrogen bonds and destabilizes hydrogen bonds formed by the carbonyl oxygen. The overall stability of the PDZ domain generally decreased for all amide-to-ester mutants due to an increase in the unfolding rate constant. For this particular region of the PDZ domain, it is therefore clear that native hydrogen bonds are formed after crossing of the rate-limiting barrier for folding. Moreover, three of the five amide-to-ester mutants displayed an increase in the folding rate constant suggesting that the hydrogen bonds are involved in non-native interactions in the transition state for folding.

Probing Backbone Hydrogen Bonds in Proteins by Amide‐to‐Ester Mutations

“…Taken together, these studies highlight how the H‐bond networks are pivotal for interactions of peptide ligands and that H‐bonds in PDZ domain interactions can contribute to fine‐tuning the specificity, which, in general, is dictated by side chains . The A‐to‐E variants of the PSD‐95 PDZ2 domain have also been used to probe the folding mechanism of this PDZ domain by employing Φ value analysis, for which a value of 0 indicates that the H‐bond is not involved in the rate‐limiting transition state for folding and a value of 1 shows full H‐bond formation in the transition state . The analysis yields low Φ values, which demonstrates that the H‐bonds related to the backbone conformation of the PDZ β2 strand and the carboxylate binding loop form late in the folding reaction.…”

“…One Φ value is negative, which suggests a non‐native backbone interaction during the folding reaction. Such frustration is expected in regions optimized for function rather than stability …”

Targeting SARS-CoV-2 Nonstructural Protein 15 Endoribonuclease: an in silico Perspective

“…These halogen bonds contribute to the binding affinity with the macromolecular systems [60]. The screened drugs, valrubicin and aprepitant, provide more hydrogen bonds with SARS-CoV-2 Nsp15, where this bonding pattern impacts the overall complex stability and molecular recognition of the system [61]. Some of the interacting residues in Figure 2 are surface accessible (gray shadowed circles) that reflect its surface exposed property.…”

Molecular and clinical profile of congenital fibrinogen disorders in Iran, identification of two novel mutations

“…The HB, a strong intermolecular force, plays an important role in the formation, stabilization, and function of proteins. 25 With the aim of determining whether substitution of Arg by Thr influences the number of HBs, HB analysis was performed for the WT and MT structures during MD simulations (Figure 1C). Based on the HB average calculation, the WT system appears to form an average number of HB in the range of 8, whereas the mutant appears to form fewer, around 5.…”