Protocol for the thermodynamic analysis of some proteins using an H/D exchange- and mass spectrometry-based technique.
SUPREX (stability of unpurified proteins from rates of H/D exchange) is a new H/D exchange- and mass spectrometry-based technique for the measurement of protein folding free energies (i.e., DeltaG values) and protein folding m values (i.e., deltaDeltaG/delta[denaturant]). Robust protocols for the acquisition and analysis of SUPREX data have been established and shown to be useful for the analysis of a number of different protein systems. Here we report on the SUPREX behavior of a special class of proteins that are not amenable to conventional SUPREX analyses using previously established protocols. This class of proteins includes protein systems that require an extended time to reach a folding/unfolding equilibrium in chemical denaturant-induced equilibrium unfolding experiments. As part of this work we use ubiquitin as a model system to highlight the complications that can arise in the conventional SUPREX analysis of such protein systems, and we describe a modified SUPREX protocol that can be used to eliminate these complications.
Dai
Susie Y
SY
Department of Chemistry, Duke University, Durham, North Carolina 27708, USA.
Gardner
Myles W
MW
Fitzgerald
Michael C
MC
D041961:Deuterium Exchange Measurement / Q000379:methods*; D011489:Protein Denaturation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013816:Thermodynamics; D013995:Time; D025801:Ubiquitin / Q000737:chemistry*
D011506:Proteins; D025801:Ubiquitin