Protein‐folding kinetics and mechanisms studied by pulse‐labeling and mass spectrometry

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Abstract

The "protein-folding problem" refers to the question of how and why a denatured polypeptide chain can spontaneously fold into a compact and highly ordered conformation. The classical description of this process in terms of reaction pathways has been complemented by models that describe folding as a biased conformational diffusion on a multidimensional energy landscape. The identification and characterization of short-lived intermediates provide important insights into the mechanism of folding. Pulsed hydrogen/deuterium exchange (HDX) methods are among the most powerful tools for studying the properties of kinetic intermediates. Analysis of pulse-labeled proteins by mass spectrometry (MS) provides information that is complementary to that obtained in nuclear magnetic resonance (NMR) studies; NMR data represent an average of entire protein ensembles, whereas MS can detect co-existing protein species. MS-based pulse-labeling experiments can distinguish between folding scenarios that involve parallel pathways, and those where folding is channeled through obligatory intermediates. The proteolytic digestion/MS technique provides spatially resolved information on the HDX pattern of folding intermediates. This method is especially important for proteins that are too large to be studied by NMR. Although traditional pulsed HDX protocols are based on quench-flow techniques, it is also possible to use electrospray (ESI) MS to analyze the reaction mixture on-line and "quasi-instantaneously" after labeling. This approach allows short-lived protein conformations to be studied by their HDX level, their ESI charge-state distribution, and their ligand-binding state. Covalent labeling of free cysteinyl residues provides an alternative approach to pulsed HDX experiments. Another promising development is the use of synchrotron X-rays to induce oxidation at specific sites within a protein for studying their solvent accessibility during folding.

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