Effects of Multidentate Metal Interactions on the Structure of Collisionally Activated Proteins: Insights from Ion Mobility Spectrometry and Molecular Dynamics Simulations

Abstract

Much remains to be learned about the way in which bound metal ions modulate the response of electrosprayed proteins and protein complexes to collisional excitation. Nonspecific metal adducts can affect the extent of collision-induced unfolding (CIU) and collision-induced dissociation (CID). Here, we examine how Na(+) and Ca(2+) adducts alter the CIU response of monomeric proteins under native electrospray conditions. Both of these metals are commonly encountered in biological samples. Measured collision cross sections are largely independent of metal adduction as long as in-source excitation is minimized. In contrast, under CIU conditions, the metal-adducted proteins are markedly more compact than their metal-free counterparts. This phenomenon is particularly pronounced for Ca(2+) binding, but Na(+) adducts have significant effects as well. Molecular dynamics simulations reproduce the experimentally observed trends. The simulations show that structural expansion of the collisionally unfolded proteins is limited by multidentate metal contacts that restrict the conformational freedom of the polypeptide chains. Multidentate interactions with carboxylates and other electron-rich moieties are to be anticipated for divalent metals such as Ca(2+). It is surprising that Na(+) also engages in multidentate ligation. Electrostatic mapping reveals that the propensity of both Na(+) and Ca(2+) to interact with multiple electron-rich groups is caused by ineffective charge shielding during ion pairing. Despite their compactness, the CIU structures of metalated proteins do not retain native-like elements. Instead, CIU generates inside-out conformations where previously surface-exposed hydrophilic side chains get buried along with most of the metal ions. Our findings caution that the observation of compact conformers after collisional excitation does not imply the survival of solution-like structural features. We also discuss possible implications of adduct-mediated effects for CIU fingerprinting studies.

Replacing H+by Na+or K+in phosphopeptide anions and cations prevents electron capture dissociation

“… 54 To test this hypothesis, we used collisional activation in the source region of the mass spectrometer to disrupt noncovalent bonds within the peptide anions prior to niECD. Previous studies have shown that vibrational activation of gaseous peptide and protein ions by collisions or absorption of infrared photons can result in partial or full unfolding, 52 , 62 , 83 and that the stability of intramolecular interactions generally follows the order salt bridges > ionic hydrogen bonds > neutral hydrogen bonds > hydrophobic bonds. 44 , 45 , 53 , 55 , 84 Instead of the 10 V skimmer potential in the experiments for Fig.…”

Effects of Detergent Micelles on Lipid Binding to Proteins in Electrospray Ionization Mass Spectrometry

“… 5 , 9 The tight association can be rationalized by multivalent charge interactions between serum albumin and ligand. 29 In fact, this protein contains a protected phospholipid-binding pocket, in addition to several low-affinity sites, in which the lipid is bound via contacts between basic residues and the ionic phosphate headgroup. 30 Our data suggest that lipid interactions with selected binding sites on soluble proteins can mirror effects observed for membrane proteins.…”

ATP Kinetically Modulates Pathogenic Tau Fibrillations

“…The high concentration of ammonium acetate (20 mM, pH 7.4) added for native MS caused the ammonium adduction of K18. [36][37] The ESI-MS spectra of K18 with ATP (K18:ATP = 1:1 and 1:10) indicated that the number of ATP molecules bound to a K18 ion increased with increase in the ATP concentration ( Figure 3a). The property of K18-ATP binding was investigated from the relative abundance of K18 and K18-ATP complex peaks in the mass spectrum of K18:ATP = 1:10 ( Figure 3b).…”