Thermodynamic stability measurements on multimeric proteins using a new H/D exchange‐ and matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry‐based method

Abstract

We recently reported on a new H/D exchange-and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-based technique, termed SUPREX, that removes several important limitations associated with measuring the thermodynamic stability of proteins. In contrast to conventional spectroscopy-based techniques for characterizing the equilibrium unfolding behavior of proteins, SUPREX is amenable to the thermodynamic analysis of both purified and unpurified proteins using mg to ng quantities of material. Here we report on the application of SUPREX to the analysis of multimeric protein systems. Included in this work are the SUPREX results we obtained in studies on six model multimeric proteins including the GCN4p1 dimer, the coil-V a L d trimer, the 4-oxalocrotonate tautomerase (4-OT) hexamer, the Trp repressor (TrpR) dimer, the Arc repressor (ArcR) dimer, and an ArcR mutant (the (DOA20)ArcR) dimer which contained two destabilizing mutations including an Asp to Ala mutation at position 20 and an amide to ester bond mutation between amino acid (aa) residues 19 and 20. As part of the work described here, we present a new method for the analysis of SUPREX data that is generally applicable to both monomeric and multimeric protein systems. Our results on the model proteins in this study indicate that this new method can be used to determine folding free energies for proteins with the accuracy and precision of conventional spectroscopybased methods.Keywords: H/D exchange; MALDI; thermodynamic stability; protein folding Conventional, spectroscopy-based methods for measuring the thermodynamic stability of proteins have the disadvantage that they require relatively large amounts of pure protein. This limits the thermodynamic analysis of proteins to those that can be purified in large quantities. We recently reported a new H/D exchange-and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-based technique, termed SUPREX, that can be used to quantitate the thermodynamic stability of proteins (Ghaemmaghami et al. 2000). In contrast to conventional methods, the SUPREX technique can be used to quantitate the stability of mg to ng quantities of both purified and unpurified proteins (Powell and Fitzgerald 2001). In SUPREX, protein samples are subjected to H/D exchange by dilution into a series of deuterated exchange buffers containing different concentrations of a chemical denaturant such as guanidinium chloride (GdmCl). After a specified exchange time, the deuterium content of each protein sample is determined using MALDI mass spectrometry. Ultimately, the change in mass relative to the fully protonated sample is plotted as a function of [GdmCl] to generate a SUPREX curve.SUPREX curves can be used to extract accurate thermodynamic parameters for a protein's folding reaction provided that the protein's equilibrium unfolding behavior is

Protein‐folding kinetics and mechanisms studied by pulse‐labeling and mass spectrometry

“…Only very recently has it been shown that the analysis of isotopically labeled proteins by MALDI‐MS represents a viable alternative to ESI‐MS (Henry, 1998; Mandell, Falick, & Komives, 1998). Following this discovery, MALDI‐MS has quickly become a standard tool for folding experiments carried out under equilibrium conditions (Figueroa & Russell, 1999; Ghaemmaghami, Fitzgerald, & Oas, 2000; Powell & Fitzgerald, 2001; Powell, Wales, & Fitzgerald, 2002). However, so far all MS‐based studies on short‐lived folding intermediates have exclusively employed ESI‐MS.…”

Studies of biomolecular conformations and conformational dynamics by mass spectrometry

“…Studies of protein equilibrium unfolding induced by chaotropes usually require extensive sample clean-up and thus cannot be carried out ''on-line,'' although Powell et al have managed successfully to measure protein stability by urea-induced unfolding with HDX MALDI MS (Powell & Fitzgerald, 2001;Powell, Wales, & Fitzgerald, 2002). Thermal denaturation is another way to study protein equilibrium unfolding that can be implemented with an ''on-line'' experimental set-up, as demonstrated by the Deinzer group (Maier, Schimerlik, & Deinzer, 1999).…”

Detection of early unfolding events in a dimeric protein by amide proton exchange and native electrospray mass spectrometry

“…Both H/D exchange with mass spectrometry [13][14][15][16][17][18] and native electrospray mass spectrometry 19 have been successfully applied for the study of protein folding. H/D exchange allows monitoring phase transition (unfolding), and the measuring of the specific kinetics as unfolding processes heralds itself in the appearance of EX1 exchange pattern.…”

“…Yet, dimer interchange assays, such as one we have developed here, do provide this type of information. Although study of non-labeled proteins would not be of help with this type of analysis (as dissociated/re-associated dimers are not discernable from never-dissociated dimers), 15 N-labeled proteins allowed us to monitor the dynamics of dimer dissociation. Upon dissociation, normoisotopic and 15 N-labeled monomers re-associate randomly with the formation of heterodimers, and by measuring the area under each of the mass spectrometric peaks, we were able to quantify the rate of dimer dissociation.…”