title: Slow histidine H/D exchange protocol for thermodynamic analysis of protein folding and stability using mass spectrometry.
abstract: 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 C(2) 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 K(d) 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.
authors.affiliation: Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27708, USA.
authors.forename: Duc T
authors.initials: DT
authors.lastname: Tran
authors.forename: Sambuddha
authors.initials: S
authors.lastname: Banerjee
authors.forename: Abdu I
authors.initials: AI
authors.lastname: Alayash
authors.forename: Alvin L
authors.initials: AL
authors.lastname: Crumbliss
authors.forename: Michael C
authors.initials: MC
authors.lastname: Fitzgerald
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D024402:Carbonic Anhydrase II / Q000737:chemistry / Q000378:metabolism; D002417:Cattle; D041961:Deuterium Exchange Measurement; D006639:Histidine / Q000737:chemistry; D013058:Mass Spectrometry; D009211:Myoglobin / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D012259:Ribonuclease, Pancreatic / Q000737:chemistry / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D009211:Myoglobin; D011506:Proteins; D006639:Histidine; D013482:Superoxide Dismutase; D012259:Ribonuclease, Pancreatic; D024402:Carbonic Anhydrase II