Slow Histidine H/D Exchange Protocol for Thermodynamic Analysis of Protein Folding and Stability Using Mass Spectrometry

Described here is a mass spectrometry-based protocol
to study the
thermodynamic stability of proteins and protein–ligand complexes
using the chemical denaturant dependence of the slow H/D exchange
reaction of the imidazole C2 proton in histidine side chains.
The protocol is developed using several model protein systems including:
ribonuclease (Rnase) A, myoglobin, bovine carbonic anhydrase (BCA)
II, hemoglobin (Hb), and the hemoglobin–haptoglobin (Hb–Hp)
protein complex. Folding free energies consistent with those previously
determined by other more conventional techniques were obtained for
the two-state folding proteins, Rnase A and myoglobin. The protocol
successfully detected a previously observed partially unfolded intermediate
stabilized in the BCA II folding/unfolding reaction, and it could
be used to generate a Kd value of 0.24
nM for the Hb–Hp complex. The compatibility of the protocol
with conventional mass spectrometry-based proteomic sample preparation
and analysis methods was also demonstrated in an experiment in which
the protocol was used to detect the binding of zinc to superoxide
dismutase in the yeast cell lysate sample. The yeast cell sample analyses
also helped define the scope of the technique, which requires the
presence of globally protected histidine residues in a protein’s
three-dimensional structure for successful application.