Visualizing Protein “Functional Structures” in Aqueous Solutions via Mass Spectrometry

Electrospray ionization mass spectrometry can transfer intact proteins and noncovalent protein complexes from aqueous solution into the gas phase for mass analysis. Because molecular mass changes when proteins bind metal ions, substrates, or other molecules, ESI-MS can distinguish different binding states and determine complex stoichiometry. It can also monitor how populations of complexes change with time.

This provides information that complements structural techniques such as X-ray crystallography and cryo-EM. Those methods can produce highly detailed structures but may have difficulty capturing reversible, transient states occurring while a protein is functioning in solution. ESI-MS sacrifices direct atomic-level structural visualization but can reveal which molecular components are associated with one another and how those associations change during processes such as metal incorporation or enzymatic catalysis.

A major challenge is preserving weak noncovalent interactions as a protein complex moves from solution into the gas phase and through the mass spectrometer. During ESI, droplets undergo desolvation, and the resulting ions experience collisions with gas molecules. Weakly bound complexes or complexes retaining many solvent molecules may dissociate before detection.

Instrument design also matters. Although high-resolution mass analyzers can be advantageous for large complexes, long ion residence times or complicated ion trajectories can increase the possibility of complex dissociation. In addition, proteins generally need buffer solutions to preserve their native structures, while ESI-MS requires volatile aqueous buffers compatible with ionization. These factors mean that the structures detected by ESI-MS depend partly on whether the relevant interactions can survive the measurement process.

The order in which Cu²⁺ and Zn²⁺ are introduced strongly influences the resulting metal configuration of SOD1. When both ions were added simultaneously to metal-depleted SOD1, multiple forms containing different combinations of copper and zinc developed. The resulting enzyme showed approximately 80% of the activity of native SOD1.

Copper can readily occupy SOD1's zinc-binding sites, whereas zinc does not readily occupy its copper-binding sites. Consequently, simultaneous addition can produce copper-rich forms with lower activity. When Zn²⁺ was added first and Cu²⁺ subsequently, nearly native Cu,Zn-SOD1 was produced. This behavior supports a stepwise mechanism in which appropriate metal incorporation is important for establishing the enzyme's functional structure.

ESI-MS can distinguish different SOD1 metal-binding states through the mass differences associated with bound metal ions. It can also determine whether SOD1 remains a homodimer and reveal how metal-bound populations evolve after ions are introduced.

Experiments with metal-depleted SOD1 showed that its binding sites do not simply accept Cu²⁺ and Zn²⁺ interchangeably. Copper can occupy the sites normally associated with zinc more readily than zinc can occupy the copper-binding sites. The resulting distributions of copper- and zinc-containing complexes provide direct evidence of this selectivity. Monitoring these distributions over time also showed changes consistent with subunit exchange between dimers, demonstrating that metal binding and protein assembly are dynamic processes in aqueous solution.

RNase H cleaves the RNA strand of a DNA/RNA hybrid and requires Mg²⁺ or Mn²⁺ for activity. ESI-MS measurements performed soon after initiating a reaction with Mn²⁺ detected a ternary complex consisting of RNase H, the DNA/RNA substrate, and two Mn²⁺ ions. Molecular mass provided direct evidence for the number of metal ions associated with the active complex.

As the reaction progressed, the ternary complex declined while free enzyme and complexes containing cleavage products increased. The product complexes did not retain Mn²⁺. These observations show that two divalent metal ions can bind transiently during the active stage of the reaction and subsequently dissociate, providing direct in-solution evidence supporting a two-metal-ion catalytic mechanism.

Mg²⁺ is considered the primary physiological cofactor for RNase H in E. coli because of its relatively high intracellular concentration. However, its moderate affinity for the enzyme and the extremely rapid catalytic turnover make the Mg²⁺-bound functional complex difficult to capture directly.

Mn²⁺ has similar coordination properties and can serve as an in vitro surrogate while sufficiently stabilizing the transient complex for ESI-MS detection. Using Mn²⁺ made it possible to observe a ternary enzyme–substrate complex containing two metal ions and follow its disappearance as RNA cleavage proceeded. The experiment therefore provides a practical way to investigate the transient metal-binding state associated with RNase H catalysis while recognizing that Mg²⁺ is the more physiologically relevant cofactor.

Yes. If measurements can begin quickly enough after a reaction is initiated, ESI-MS can follow changes in the relative abundance of enzyme, substrate, intermediate, and product complexes over time. In the RNase H experiment, conventional sample handling was initially too slow because the reaction was largely complete before measurement began.

A modified nanoESI setup reduced the delay to approximately 1.5 minutes. Continuous monitoring then detected the metal-containing ternary complex early in the reaction and showed its signal decreasing as RNA cleavage progressed. At the same time, free enzyme and product-containing binary complexes increased. This demonstrates how molecular mass can function as a direct indicator of changing complex composition and provide a time dimension for studying protein function in solution.