| Thermodynamic measurements of protein stability (i.e. measurements of DeltaG f) are widely used in the field of protein biochemistry. Such measurements are essential in mutational studies of protein folding and stability, and they are commonly used to investigate the strength of protein-ligand binding interactions. Conventional methods for DeltaGf determinations include such techniques as nuclear magnetic resonance spectroscopy, calorimetry, and optical techniques such as fluorescence and circular dichroism spectroscopy.; Recently, a new method for making DeltaGf measurements on proteins was developed that utilizes matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry in conjunction with hydrogen/deuterium (H/D) exchange. This new method, which is termed SUPREX (s&barbelow;tability of u&barbelow;npurified p&barbelow;roteins from r&barbelow;ates of H/D exchange), has several experimental advantages over conventional methods for making DeltaGf measurements. These advantages include: the ability to make measurements on small quantities of protein; the ability to make measurements on unpurified protein samples; and the ability to be automated.; Described here are several significant improvements to the sample handling protocols and the data analysis procedures associated with the SUPREX technique. These improvements include: a new experimental protocol which allows SUPREX to be performed with as little as 10 pmol of protein; a new, generalized equation for the quantitative determination of DeltaGf values for both monomeric and multimeric proteins; a novel data acquisition technique that permits the accurate determination of protein-ligand dissociation constants (Kd values) and protein folding m-values; and a new, SUPREX-based methodology for the high throughput screening of protein ligands. The work described here has made SUPREX a more robust and more general analytical technique for the thermodynamic analysis of protein folding reactions. |
