Accuracy and Precision of a New H/D Exchange- and Mass Spectrometry-Based Technique for Measuring the Thermodynamic Properties of Protein−Peptide Complexes

Abstract

A new H/D exchange- and MALDI mass spectrometry-based technique, termed SUPREX, was used to characterize the thermodynamic properties of a series of model protein-peptide complexes of the Abelson tyrosine kinase SH3 domain (abl-SH3) and the S-Protein (S-Pro). The SUPREX technique was employed to evaluate the folding free energies (DeltaG(f) values) of each model protein in the absence and in the presence of a series of different peptide ligands. Ultimately, these SUPREX-derived DeltaG(f) values were used to calculate dissociation constants (K(d) values) for each of the nine protein-peptide complexes in this study. As part of this work, we describe a new data collection and analysis method that allows the accurate and precise determination of protein folding m-values in the SUPREX experiment. The m-values that we determined for the abl-SH3 domain and the S-Pro system were in good agreement with those determined by conventional techniques. Our results also indicate that the SUPREX-derived K(d) values for the protein-peptide complexes in this work were in reasonably good agreement with those determined by conventional techniques.

Structural and thermodynamic characterization of a cytoplasmic dynein light chain–intermediate chain complex

“…1 as described in ref. 27. A linear least-squares analysis was used to determine the slope and y intercept of the plots of Ϫ⌬G app versus C SUPREX 1/2 that were generated for the LC8 protein and for the LC8 protein complexes.…”

“…However, we have previously shown that when SUPREX data obtained on nontwo-state folding proteins are well fit to Eq. 1 (as is the case with our LC8 data), the resulting ⌬G f values extracted for a given protein system can be used to generate accurate ⌬⌬G f values (27). Thus, whereas the SUPREX-derived ⌬G f values in Table 3 are not expected to be accurate, the ⌬⌬G f values in the table are expected to be reasonably accurate.…”

“…Our initial attempts to measure such affinity enhancement were complicated by the weak dimerization of the LCs (26) and weak intrinsic affinity of the peptide targets. Thus, we turned to an alternative method, SUPREX (27)(28)(29)(30), to analyze the thermodynamic stability of the complex in the absence and presence of a series of different peptide and protein ligands (see Table 3). This method follows the hydrogen/deuterium exchange of the amide backbone for one or more species at increasing concentrations of denaturant in deuterated buffers using mass spectrometry.…”

Throughput and efficiency of a mass spectrometry-based screening assay for protein—Ligand binding detection

“…We recently reported on a mass spectrometry-based assay for protein-ligand binding detection that utilizes an abbreviated version of stability of unpurified j;!roteins by rates of H/D exchange (SUPREX), which is an H/D exchange-and mass spectrometry-based technique capable of detecting and quantifying protein-ligand binding interactions [4][5][6][7][8][9]. In the abbreviated version of SUPREX (referred to hereafter as single-point SUPREX), binding events are detected by measuring the target protein's mass change after H/D exchange in a deuterated buffer containing a specific concentration of a chemical denaturant [10].…”

Characterization of noncovalent protein-ligand complexes and associated enzyme intermediates of GlcNAc-6-O-sulfotransferase by electrospray ionization FT-ICR mass spectrometry

“…In generating these complexes, proteins are mixed with their ligands in solution and the intact noncovalent complexes are observed in the gas phase for proteinsubstrate [9], protein-inhibitor [10], and intact multimeric proteins complexes [11]. Moreover, once these desolvated molecules are in the gas phase, molecular weight, binding stoichiometry, and relative/absolute binding strength can be determined using mass spectrometry [12][13][14][15][16]. Sulfotransferases, in particular, can be investigated in significant detail and information concerning the mechanism of catalysis can be garnered.…”