Evaluation of direct and cooperative contributions towards the strength of buried hydrogen bonds and salt bridges

Shira Albeck, Ron Unger, Gideon Schreiber

Research output: Contribution to journalArticlepeer-review

60 Scopus citations


An experimental approach to evaluate the net binding free energy of buried hydrogen bonds and salt bridges is presented. The approach, which involves a modified multiple-mutant cycle protocol, was applied to selected interactions between TEM-1-β-lactamase and its protein inhibitor, BLIP. The selected interactions (two salt bridges and two hydrogen bonds) all involving BLIP-D49, define a distinct binding unit. The penta mutant, where all side-chains constructing the binding unit were mutated to Ala, was used as a reference state to which combinations of side-chains were introduced. At first, pairs of interacting residues were added allowing the determination of interaction energies in the absence of neighbors, using double mutant cycles. Addition of neighboring residues allowed the evaluation of their cooperative effects on the interaction. The two isolated salt bridges were either neutral or repulsive whereas the two hydrogen bonds contribute 0.3 kcal mol-1 each. Conversely, a double mutant cycle analysis of these interactions in their native environment showed that they all stabilize the complex by 1-1.5 kcal mol-1. Examination of the effects of neighboring residues on each of the interactions revealed that the formation of a salt bridge triad, which involves two connected salt bridges, had a strong cooperative effect on stabilizing the complex independent of the presence or absence of additional neighbors. These results demonstrate the importance of forming networks of buried salt bridges. We present theoretical electrostatic calculations which predict the observed mode of cooperativity, and suggest that the cooperative networking effect results from the favorable contribution of the protein to the interaction. Furthermore, a good correlation between calculated and experimentally determined interaction energies for the two salt bridges, and to a lesser extent for the two hydrogen bonds, is shown. The data analysis was performed on values of ΔΔG(Kd)/(+)) which reflect the strength of short range interactions, while ΔΔG((O)/(KD)) values which include the effects of long range electrostatic forces that alter specifically ΔΔG((Ka)/(+)) were treated separately. (C) 2000 Academic Press.

Original languageEnglish
Pages (from-to)503-520
Number of pages18
JournalJournal of Molecular Biology
Issue number3
StatePublished - 5 May 2000
Externally publishedYes

Bibliographical note

Funding Information:
We thank Tzvia Selzer for her help with all the electrostatic calculations and Dr Amnon Horovitz and Dr Jacob Piehler for their critical reading of the manuscript. G. S. is incumbent of the Dewey David Stone and Harry Levine career development chair. This research was supported by the Israel Science Foundation founded by the Israel Academy of Sciences and Humanities-Charles H. Revson Foundation (106/97-1).


  • Cooperativity
  • Interaction energy
  • Protein-protein interaction
  • Salt-bridge
  • β-lactamase


Dive into the research topics of 'Evaluation of direct and cooperative contributions towards the strength of buried hydrogen bonds and salt bridges'. Together they form a unique fingerprint.

Cite this