title: H/D exchange and mass spectrometry-based method for biophysical analysis of multidomain proteins at the domain level.
abstract: A protocol was developed to characterize the domain-specific thermodynamic stabilities of multidomain proteins using SUPREX (Stability of Unpurified Proteins from Rates of H/D Exchange). The protocol incorporates a protease digestion step into the conventional SUPREX protocol and enables folding free energy (DeltaGf) and cooperativity (m-value) measurements to be made on the individual domains of multidomain proteins in their native context (i.e., in the intact protein). Three multidomain protein systems (calmodulin, a Fyn construct, and transferrin) were used to validate the SUPREX-protease digestion protocol. The DeltaGf and m-value of each domain in the intact test proteins were measured in the absence and presence of ligands using the new protocol. Domain-specific thermodynamic parameters were obtained on each system; and the measured parameters were consistent with known biophysical properties of the test proteins. The known stabilization of the N-terminal domain of CaM in the context of the intact protein and the known binding affinity of a proline-rich peptide to the SH3 domain in the Fyn construct were successfully quantified using the new protocol. Qualitative information about the relative calcium binding affinities of the N- and C-terminal domains of CaM and about the relative iron binding affinities of the N- and C-terminal domains of transferrin was also obtained using the new protocol.
authors.affiliation: Department of Chemistry, Duke University, Durham, North Carolina 27708, USA.
authors.forename: Liangjie
authors.initials: L
authors.lastname: Tang
authors.forename: Petra L
authors.initials: PL
authors.lastname: Roulhac
authors.forename: Michael C
authors.initials: MC
authors.lastname: Fitzgerald
mesh_terms: D000818:Animals; D055592:Biophysical Phenomena; D001703:Biophysics; D002417:Cattle; D041961:Deuterium Exchange Measurement / Q000295:instrumentation* / Q000379:methods*; D006736:Horses; D011506:Proteins / Q000032:analysis* / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000295:instrumentation* / Q000379:methods*; D013816:Thermodynamics
chemical_list: D011506:Proteins