Thermodynamic analysis of protein folding and stability using a tryptophan modification protocol

Area of Science:

• Biochemistry
• Proteomics
• Chemical Biology

Background:

• Protein folding free energies (ΔG(f) values) are crucial for understanding protein function and stability.
• Existing methods for measuring ΔG(f) values have limitations in coverage and precision.
• Tryptophan (Trp) residues are key indicators of protein structure and dynamics.

Purpose of the Study:

• To develop and validate a mass spectrometry-based covalent labeling protocol using HNSB for accurate ΔG(f) value determination.
• To assess the protocol's performance in measuring protein-ligand interactions.
• To evaluate the integration of the HNSB protocol with the SPROX technique for enhanced proteome-wide analysis.

Main Methods:

• Development of a mass spectrometry protocol utilizing dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium bromide (HNSB) for covalent labeling of tryptophan (Trp) residues.
• Evaluation of chemical denaturant dependence of HNSB reaction rates using MALDI-TOF analysis or quantitative proteomics with isobaric mass tags.
• Integration of the HNSB protocol with the Stability of Proteins from Rates of Oxidation (SPROX) technique.

Main Results:

• Accurate ΔG(f) values were obtained for two-state folding proteins (lysozyme, cytochrome c).
• The protocol successfully measured the dissociation constant (K(d)) for lysozyme-chitotriose binding and detected brinzolamide binding to BCA II.
• Combining HNSB with SPROX increased peptide and protein coverage by 50% and 25% in proteome-wide experiments, respectively.

Conclusions:

• The HNSB protocol provides an accurate and versatile method for measuring protein folding free energies and binding affinities.
• Integration with SPROX significantly enhances the scope and efficiency of proteome-wide protein stability measurements.
• This approach offers a powerful tool for studying protein folding dynamics and interactions across biological systems.