Picomole-scale characterization of protein stability and function by quantitative cysteine reactivity

Abstract

The Gibbs free energy difference between native and unfolded states ("stability") is one of the fundamental characteristics of a protein. By exploiting the thermodynamic linkage between ligand binding and stability, interactions of a protein with small molecules, nucleic acids, or other proteins can be detected and quantified. Determination of protein stability can therefore provide a universal monitor of biochemical function. Yet, the use of stability measurements as a functional probe is underutilized, because such experiments traditionally require large amounts of protein and special instrumentation. Here we present the quantitative cysteine reactivity (QCR) technique to determine protein stabilities rapidly and accurately using only picomole quantities of material and readily accessible laboratory equipment. We demonstrate that QCRderived stabilities can be used to measure ligand binding over a wide range of ligand concentrations and affinities. We anticipate that this technique will have broad applications in high-throughput protein engineering experiments and functional genomics. Macromolecular stability is therefore one of the most fundamental thermodynamic measures in biochemistry by quantitatively reporting on structure-function relationships to provide a universal monitor for biochemical function.There are two distinct approaches for determining protein stability (4). The first measures the free energy of protein (un)folding under equilibrium conditions by assessing the fraction of the native state using spectroscopy, hydrodynamic observations, functional assays, or calorimetry. The second exploits the relationship between protein dynamics and stability by monitoring the differential reactivity of internal chemical groups in native and unfolded states. This second approach measures conformational free energies, which under appropriate conditions corresponds to global protein stability. Amide proton exchange is used most commonly to monitor such differential reactivity (5-9), but its widespread use to assess biological function typically is limited by the need for specialized instrumentation and relatively large amounts of protein. Recently, cysteine reactivity (10-14) and proteolysis (15) have emerged as alternative means to determine rates of protein (un)folding and estimate protein stabilities. Here we present a method, quantitative cysteine reactivity (QCR), in which protein stability is determined by monitoring the reactivity of cysteine residues buried in the hydrophobic core of proteins. This approach has the advantage over more traditional methods for measuring protein stability in that it requires only picomoles (nanograms) of protein, uses simple instrumentation accessible to any lab, can be reasonably high throughput, and can provide site-specific thermodynamic information. QCR can be used to determine apparent protein stabilities rapidly and accurately, construct Gibbs-Helmholtz stability profiles, measure ligand binding over a large range of ligand concentrations and affinities, and infer e...

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