2012 · Hopper et al. — Evidence for the preservation of native inter- and intra-molecular hydrogen bonds in the desolvated FK-binding protein·FK506 complex produced by electrospray ionization.
Super-Abstract
This biophysical study used mass spectrometry and ion mobility spectrometry to investigate whether protein-ligand hydrogen bonds are preserved when protein complexes are transferred from solution into the gas phase by electrospray ionisation. „Hydrogen bonds“ here refers to structural inter-atomic bonds in proteins — not to molecular hydrogen (H₂) gas or therapy. This paper has no connection to H₂ medicine.
Commentary
FK506-binding protein (FKBP) is a well-studied protein important in immunosuppression (it binds the drug FK506/tacrolimus). The question of whether native non-covalent interactions — especially hydrogen bonds — are preserved in gas-phase mass spectrometry is fundamental to native mass spectrometry methodology. The study used site-directed mutagenesis (removing specific residues that form H-bonds) to show that the loss of native H-bonds destabilises the complex in the gas phase, providing evidence that these bonds survive the electrospray process. Throughout, „hydrogen bonds“ (H-bonds) are the classical weak inter-atomic bonds (N–H···O, O–H···N etc.) found in all proteins — these are entirely unrelated to molecular hydrogen gas (H₂) as used in H₂ medicine. This paper is indexed in this database due to keyword overlap on „hydrogen bonds.“
Key quotes
- „removal of native protein-ligand interactions formed between residues Asp37, Tyr82, and FK506 significantly destabilized the complex.“ — key finding: specific H-bonds maintain complex stability in the gas phase
- „destabilization of the FKBP·FK506 complex, resulting from targeted removal of specific H-bonds, provides evidence for the preservation of these interactions in the desolvated wild-type complex.“ — conclusion: native H-bonds are preserved during electrospray ionisation
- „removal of the basic Arg42 residue was found to induce significant structural weakening of the [M + 7H](7+) complex when raised to dissociation-level energies.“ — structural evidence from ion mobility: H-bond removal alters gas-phase conformation
Our assessment
This is a biophysics / native mass spectrometry methodology paper with no relevance to molecular hydrogen therapy. The term „hydrogen bonds“ refers to the fundamental structural interactions between electronegative atoms and hydrogen within proteins — standard physical chemistry. Honest note: this paper is catalogued here due to keyword overlap ("hydrogen bonds"). It contributes to native mass spectrometry methodology and protein structural biology, not to H₂ medicine.
Study design
- Type: in-vitro biophysics study · Method: electrospray ionisation mass spectrometry (ESI-MS), collision-induced dissociation (CID), ion mobility spectrometry (IMS); site-directed mutagenesis of FKBP
- H₂ relevance: none — „hydrogen bonds“ are classical inter-atomic bonds (N–H···O etc.) in proteins, not molecular H₂ gas
- Result: loss of specific H-bond-forming residues destabilises FKBP·FK506 complex in gas phase; native H-bonds are preserved during electrospray desolvation; ion mobility shows compact structure
Abstract
It is now well established that electrospray ionization (ESI) is capable of introducing noncovalent protein assemblies into a desolvated environment, thereby allowing their analysis by mass spectrometry. The degree to which native interactions from the solution phase are preserved in this environment is less clear. Site-directed mutagenesis of FK506-binding protein (FKBP) has been employed to probe specific intra- and inter-molecular interactions within the complex between FKBP and its ligand FK506. Collisional activation of wild-type and mutant-FKBP•FK506 ions, generated by ESI, demonstrated that removal of native protein-ligand interactions formed between residues Asp37, Tyr82, and FK506 significantly destabilized the complex. Mutation of Arg42 to Ala42, or Tyr26 to Phe26 also resulted in lower energy dissociation of the FKBP·FK506 complex. Although these residues do not form direct H-bonds to FK506, they interact with Asp37, ensuring its correct orientation to associate with the ligand. Comparison with solution-based affinity measurements of these mutants has been discussed, including the stabilization afforded by ordered water molecules. Ion mobility spectrometry (IMS) has been employed to provide gas-phase structural information on the unfolding of the complexes. The [M + 6H](6+) complexes of the wild-type and mutants have been shown to resist unfolding and retain compact conformations. However, removal of the basic Arg42 residue was found to induce significant structural weakening of the [M + 7H](7+) complex when raised to dissociation-level energies. Overall, destabilization of the FKBP·FK506 complex, resulting from targeted removal of specific H-bonds, provides evidence for the preservation of these interactions in the desolvated wild-type complex.
Source & links
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