Publications [#232611] of Michael C. Fitzgerald

Journal Articles

1. Tang, L; Roulhac, PL; Fitzgerald, MC, H/D exchange and mass spectrometry-based method for biophysical analysis of multidomain proteins at the domain level., Analytical chemistry, vol. 79 no. 22 (November, 2007), pp. 8728-8739, ISSN 0003-2700 [17948968], [doi]
   (last updated on 2026/01/15)

   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.