J. Am. Chem. Soc. 135, 1177–1180 (2013)

Credit: © 2013 ACS

Proteins exist and function in an aqueous environment and their structures are heavily dependent on solvent interactions. Techniques such as mass spectrometry (MS) and ion-mobility spectrometry (IMS) provide much useful information on the structural and biophysical properties of macromolecules, but are gas-phase techniques that require the solvent to be evaporated off before analysis. The extent to which proteins retain their native (solvated) structure during these processes is therefore not clear. Now, Kevin Pagel and colleagues at the Fritz Haber Institute of the Max Planck Society, Berlin, have highlighted the significant structural differences that can be present between native and non-solvated proteins and have developed a technique that may lessen the effects of solvent loss.

They studied the effects of non-covalently attaching crown ethers (18-crown-6) on the structure of cytochrome c. Crown ethers (CEs) are known to co-ordinate to protonated lysine side-chains and can act as a substitute for the absent solvent molecules. Using IMS and MS they showed that the effect of adding an increasing number of CE molecules to cytochrome c differed depending on its original charge. Adding CEs to high charge (>9+) or low charge (<6+) cytochrome c caused the proteins to become progressively bigger (larger collision cross-sections) — as might be expected because of the CEs they now carry. Those of intermediate charge, however, surprisingly shrunk in size. Next the team looked at what happened when the CEs were removed using a collision-induced fragmentation technique, and saw a reversal of the behaviour — the cytochrome c proteins of intermediate charge increased in size.

Pagel and colleagues attribute the changes in size of the intermediary cytochrome c to CEs inhibiting the collapse of the protein on removal of the solvent. When no CEs are present and the solvent is removed, the groups that were previously stabilized by solvent begin to interact intramolecularly, destabilizing secondary and tertiary structures and causing partial unfolding. The CEs inhibit such intramolecular interactions, leaving the protein more compact and in a state more similar to the native protein.