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

Abstract I. Introduction 2 A. The Protein‐Folding Problem 2 B. Protein‐Folding Mechanisms 3 C. The Role of Folding Intermediates 3 II. Studies on Protein‐Folding Intermediates by Isotopic Pulse‐Labeling 5 A. Continuous Isotopic Labeling 6 B. Isotopic Pulse‐Labeling 7 1. Pulse‐Labeling in Quench‐Flow Experiments 7 2. Pulse Intensity 9 3. Possible Artifacts in Pulse‐Labeling Experiments 9 C. Obligatory Intermediates and Parallel Folding Pathways: Studies by Quench‐Flow Pulsed HDX and ESI‐MS 10 1. Lysozyme 10 2. Interleukin‐1β 10 3. Apo‐Myoglobin 10 D. Analysis of Isotopically Pulse‐Labeled Proteins by Proteolytic Digestion/MS 12 1. Principles 12 2. Cytochrome c 12 E. Pulse‐Labeling with On‐Line ESI‐MS Analysis 13 1. ESI‐MS as a Probe for Conformational Changes and Non‐Covalent Interactions 13 2. Time‐Resolved ESI‐MS 14 3. Time‐Resolved ESI‐MS with On‐Line Isotopic Pulse‐Labeling 14 4. The Mechanism of Myoglobin Reconstitution 14 III. Other Pulse‐Labeling Methods 17 A. Covalent Labeling of Cysteinyl Residues 17 B. Synchrotron X‐Ray Radiolysis Techniques 18 IV. Conclusions and Outlook 18 A. Ultra‐Rapid Folding Triggers 19 B. MALDI‐MS 19 C. Gas‐Phase Fragmentation Methods 19 D. “Quasi‐Instantaneous” Analysis of Pulse‐Labeled Proteins 19 Acknowledgments 19 References 20 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. © 2003 Wiley Periodicals, Inc., Mass Spec Rev 22:1–26, 2003; Published online in Wiley InterScience ( www.interscience.wiley.com )

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