Thermodynamic Analysis of a Molecular Chaperone Binding to Unfolded Protein Substrates

Abstract

Molecular chaperones are a highly diverse group of proteins that recognize and bind unfolded proteins in order to facilitate protein folding and prevent non-specific protein aggregation. The mechanisms by which chaperones bind their protein substrates have been studied for decades. However, there are few reports on the affinity of molecular chaperones for their unfolded protein substrates. Thus, little is known about the relative binding affinities of different chaperones and about the relative binding affinities of chaperones for different unfolded protein substrates. Here we describe the application of SUPREX (stability of unpurified proteins from rates of H/D exchange), an H/D exchange and MALDI-based technique, to study the binding interaction between the molecular chaperone Hsp33 and four different unfolded protein substrates including citrate synthase, lactate dehydrogenase, malate dehydrogenase, and aldolase. The results of our studies suggest that the cooperativity of the Hsp33 folding/unfolding reaction increases upon binding with denatured protein substrates. This is consistent with the burial of significant hydrophobic surface area in Hsp33 when it interacts with its substrate proteins. The SUPREX derived K d -values for Hsp33 complexes with four different substrates were found to be all within a range of 3-300 nM.Most proteins must fold into a well-defined three-dimensional structure in order to carry out their physiological roles within the cell. However, under different kinds of environmental stress conditions, such as elevated temperature, ultraviolet light, exposure of the cell to toxins (e.g., hypochlorous acid, arsenic, or trace metals) and infection, many proteins begin to lose their structure and function. Protein unfolding then often leads to non-specific aggregation, which is considered to be a largely irreversible process in vivo. Molecular chaperones are a class of proteins that function in the cell to recognize and selectively bind non-native proteins to prevent protein aggregation and facilitate their folding. It is generally believed that chaperones interact with unfolding protein substrates via hydrophobic interactions (for a review, see ref. (1)). For example, peptide binding studies using a number of different chaperones revealed that most show a greater preference toward hydrophobic peptides than to charged, hydrophilic peptides (2-4). Fluorescent hydrophobic probes have also been used to show that chaperones bind unfolded protein substrates upon exposure of hydrophobic surfaces (5-7). Finally, analysis of several X-ray crystallographic structures of chaperones indicated they preferentially bind to hydrophobic residues (4,8,9 Protein Expression and PurificationPurification of wild type Hsp33 and preparation of reduced inactive Hsp33 red was conducted as previously described (5). To activate Hsp33, 50 μM Hsp33 red was incubated with 500 μM NaOCl for 1 h at 30°C in 40 mM potassium phosphate buffer under constant shaking at 300 rpm. Excess NaOCl was removed using PD-10 desalt...

Order out of Disorder: Working Cycle of an Intrinsically Unfolded Chaperone

“…Whereas Hsp33 red failed to bind to the peptide even at the highest concentration used (i.e., 750 nM), Hsp33 ox showed specific binding with a dissociation constant (K D ) of 35 ± 0.6 nM per active Hsp33 dimer (Figures 2B and S2A and Table S2A). Note that this K D is very similar to the K D values (30–300 nM) that were previously determined using the SUPREX technique to quantify binding of Hsp33 ox to thermally unfolded CS (Xu et al, 2010). Taken together, these results strongly suggest that Pep NuoC and full-length substrates share the same binding site in Hsp33.…”

“…We therefore decided to employ the SUPREX technique to directly compare the relative changes in thermodynamic stability of Hsp33 upon oxidative activation and substrate binding (Xu et al, 2010). In SUPREX, the H/D exchange properties of globally protected amide protons in a protein are probed as a function of chemical denaturant concentration to acquire information about a protein’s conformational stability (Xu et al, 2010).…”

“…We therefore decided to employ the SUPREX technique to directly compare the relative changes in thermodynamic stability of Hsp33 upon oxidative activation and substrate binding (Xu et al, 2010). In SUPREX, the H/D exchange properties of globally protected amide protons in a protein are probed as a function of chemical denaturant concentration to acquire information about a protein’s conformational stability (Xu et al, 2010). SUPREX experiments have previously shown that Hsp33 ox is globally destabilized by 23.8 kJ/mol per dimer upon oxidative activation and that substrate binding stabilizes Hsp33 ox by 7.1–10.0 ± 0.9 kJ/mol per dimer, depending on the substrate protein (Tang et al, 2007).…”

“…SUPREX experiments have previously shown that Hsp33 ox is globally destabilized by 23.8 kJ/mol per dimer upon oxidative activation and that substrate binding stabilizes Hsp33 ox by 7.1–10.0 ± 0.9 kJ/mol per dimer, depending on the substrate protein (Tang et al, 2007). In order to map which regions of the Hsp33 structure undergo the most significant conformational changes upon oxidation and upon substrate binding, we utilized the SUPREX methodology and combined it with a pepsin digestion protocol (Xu et al, 2010). The overall coverage of Hsp33 was 60% (Figure S4B; Table S3).…”

Protein–Protein Binding Affinities in Solution Determined by Electrospray Mass Spectrometry

“…Mass spectrometry (MS) offers a number of complementary avenues for characterizing protein-protein interactions [8]. Hydrogen/deuterium exchange MS monitors changes in structure and dynamics upon binding [9][10][11]. Similarly, alterations in solvent accessibility can be probed by covalent labeling in solution, followed by a MS-based readout [12,13].…”