Hydrogen/Deuterium Exchange Measurements May Provide an Incomplete View of Protein Dynamics: a Case Study on Cytochrome c

Many aspects of protein function rely on conformational fluctuations. Hydrogen/deuterium exchange (HDX) mass spectrometry (MS) provides a window into these dynamics. Despite the widespread use of HDX-MS, it remains unclear whether this technique provides a truly comprehensive view of protein dynamics. HDX is mediated by H-bond-opening/closing events, implying that HDX methods provide an H-bond-centric view. This raises the question if there could be fluctuations that leave the H-bond network unaffected, thereby rendering them undetectable by HDX-MS. We explore this issue in experiments on cytochrome c (cyt c). Compared to the Fe(II) protein, Fe(III) cyt c shows enhanced deuteration on both the distal and proximal sides of the heme. Previous studies have attributed the enhanced dynamics of Fe(III) cyt c to the facile and reversible rupture of the distal M80-Fe(III) bond. Using molecular dynamics (MD) simulations, we conducted a detailed analysis of various cyt c conformers. Our MD data confirm that rupture of the M80-Fe(III) contact triggers major reorientation of the distal Omega loop. Surprisingly, this event takes place with only miniscule H-bonding alterations. In other words, the distal loop dynamics are almost HDX-silent. Moreover, distal loop movements cannot account for enhanced dynamics on the opposite (proximal) side of the heme. Instead, enhanced deuteration of Fe(III) cyt c is attributed to sparsely populated conformers where both the distal (M80) and proximal (H18) coordination bonds have been ruptured, along with opening of numerous H-bonds on both sides of the heme. We conclude that there can be major structural fluctuations that are only weakly coupled to changes in H-bonding, making them virtually impossible to track by HDX-MS. In such cases, HDX-MS may provide an incomplete view of protein dynamics.

Automated summary

The study suggests that there may be major structural fluctuations in proteins that are weakly correlated with changes in hydrogen bonding, making them difficult to track using HDX-MS, potentially leading to an incomplete view of protein dynamics.