Mobile Protons Limit the Stability of Salt Bridges in the Gas Phase: Implications for the Structures of...

Electrosprayed protein ions can retain native-like conformations. The intramolecular contacts that stabilize these compact gas-phase structures remain poorly understood. Recent work has uncovered abundant salt bridges in electrosprayed proteins. Salt bridges are zwitterionic BH⁺/A^- contacts. The low dielectric constant in the vacuum strengthens electrostatic interactions, suggesting that salt bridges could be a key contributor to the retention of compact protein structures. A problem with this assertion is that H⁺ are mobile, such that H⁺ transfer can convert salt bridges into neutral B⁰/HA⁰ contacts. This possible salt bridge annihilation puts into question the role of zwitterionic motifs in the gas phase, and it calls for a detailed analysis of BH⁺/A^- versus B⁰/HA⁰ interactions. Here, we investigate this issue using molecular dynamics (MD) simulations and electrospray experiments. MD data for short model peptides revealed that salt bridges with static H⁺ have dissociation energies around 700 kJ mol^-1. The corresponding B⁰/HA⁰ contacts are 1 order of magnitude weaker. When considering the effects of mobile H⁺, BH⁺/A^- bond energies were found to be between these two extremes, confirming that H⁺ migration can significantly weaken salt bridges. Next, we examined the protein ubiquitin under collision-induced unfolding (CIU) conditions. CIU simulations were conducted using three different MD models: (i) Positive-only runs with static H⁺ did not allow for salt bridge formation and produced highly expanded CIU structures. (ii) Zwitterionic runs with static H⁺ resulted in abundant salt bridges, culminating in much more compact CIU structures. (iii) Mobile H⁺ simulations allowed for the dynamic formation/annihilation of salt bridges, generating CIU structures intermediate between scenarios (i) and (ii). Our results uncover that mobile H⁺ limit the stabilizing effects of salt bridges in the gas phase. Failure to consider the effects of mobile H⁺ in MD simulations will result in unrealistic outcomes under CIU conditions.