Histidine is often a key amino acid in protein function. In many protein systems, such as enzymes and metalloproteins, the protonation state of the histidine imidazole ring is especially important. The histidine pKa is usually near biological pH, allowing histidine to switch easily from positive to neutral charge states via protonation and deprotonation, often with large-scale structural consequences.  We present a technique for determining the protonation state of histidines in solvated proteins via Raman spectroscopy, using a single carbon-deuterium stretching vibration created by deuterium exchange on histidine's epsilon carbon.  This isotopic substitution can be accomplished post-translationally on solvent-exposed histidine residues.  The unique C-D stretching vibration shifts by 35 cm-1 between histidine charge states.

Raman spectroscopic probe of the protonation state of histidine

243rd National Meeting (2012)

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