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I.Introduction2A.  The Protein‐Folding Problem2B.  Protein‐Folding Mechanisms3C.  The Role of Folding Intermediates3 II.Studies on Protein‐Folding Intermediates by Isotopic Pulse‐Labeling5A.  Continuous Isotopic Labeling6B.  Isotopic Pulse‐Labeling7    1.  Pulse‐Labeling in Quench‐Flow Experiments7    2.  Pulse Intensity9    3.  Possible Artifacts in Pulse‐Labeling Experiments9C.  Obligatory Intermediates and Parallel Folding Pathways: Studies by Quench‐Flow Pulsed HDX and ESI‐MS10    1.  Lysozyme10    2.  Interleukin‐1β10    3.  Apo‐Myoglobin10D.  Analysis of Isotopically Pulse‐Labeled Proteins by Proteolytic Digestion/MS12    1.  Principles12    2.  Cytochrome c12E.  Pulse‐Labeling with On‐Line ESI‐MS Analysis13    1.  ESI‐MS as a Probe for Conformational Changes and Non‐Covalent Interactions13    2.  Time‐Resolved ESI‐MS14    3.  Time‐Resolved ESI‐MS with On‐Line Isotopic Pulse‐Labeling14    4.  The Mechanism of Myoglobin Reconstitution14III.Other Pulse‐Labeling Methods17A.  Covalent Labeling of Cysteinyl Residues17B.  Synchrotron X‐Ray Radiolysis Techniques18 IV.Conclusions and Outlook18A.  Ultra‐Rapid Folding Triggers19B.  MALDI‐MS19C.  Gas‐Phase Fragmentation Methods19D.  “Quasi‐Instantaneous” Analysis of Pulse‐Labeled Proteins19Acknowledgments19References20 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 H...

A novel technique for studying protein folding kinetics is presented. It is based on a continuous-flow setup that is coupled to an electrospray (ESI) mass spectrometer and allows initiation of a folding reaction, followed by isotopic pulse labeling. The protein is electrosprayed "quasi-instantaneously" after exposure to the deuterated solvent. This approach yields structural information from the ESI charge state distribution and from the H/D exchange levels of individual protein states, while at the same time noncovalent interactions can be monitored. This technique is used to study the reconstitution of holomyoglobin (hMb) from unfolded apomyoglobin (aMb) and free heme. MS/MS is used to establish that a short-lived folding intermediate with two heme groups attached represents a protein-bound heme dimer. This state appears to have a compactness close to that of native hMb; however, isotopic labeling indicates a significantly perturbed structure. Another intermediate is bound to a single heme group and shows a charge state distribution similar to that of unfolded aMb. Exchange levels exhibited by this state are lower than for unfolded aMb, indicating that fewer hydrogens are exposed to the solvent and/or that more of them are involved in hydrogen bonding. Native hMb leads to the formation of low charge state ions (hMb(9+), hMb(8+)) and shows low exchange levels. However, early during reconstitution, a slightly unfolded form of the heme-protein complex contributes to the observed hMb(9+) ions. A peak width analysis reveals that the structural heterogeneity of some of the observed protein species decreases as reconstitution proceeds.

We report the use of electrospray ionization (ESI) mass spectrometry (MS) in conjunction with online rapid mixing to monitor the kinetics of acid-induced ferrihemoglobin denaturation. Under equilibrium conditions, the hemoglobin mass spectrum is dominated by the intact heterotetramer. Dimeric and monomeric species are also observed at lower intensities. In addition, ionic signals corresponding to hexameric (tetramer-dimer) and octameric (tetramer x 2) hemoglobin species are observed. These complexes may represent weak solution-phase assemblies. The acid-induced denaturation process was monitored for reaction time ranging from 9 ms to approximately 3 s. The data obtained were subjected to a global analysis procedure which simultaneously fit all kinetic (ESI-MS intensity vs time) profiles to multiexponential expressions. Results of the global analysis are consistent with the coexistence of two subpopulations of tetrameric hemoglobin which differ in their disassembly rates and ESI charge states. The higher-charge state tetramer ions preferentially dissociate via a rapid pathway (tau(1) = 51 ms), resulting in the transient formation of a heme-saturated dimer, holo-alpha-globin, and a heme-deficient dimer. The latter is shown by MS/MS to be comprised of a heme-bound alpha-subunit complexed with an apo-beta-chain. The slow-decaying tetramer population, apparent at a slightly lower average charge state, breaks down into its monomeric constituents with no observable intermediate species (tau(2) = 390 ms). Surprisingly, unfolded apo-alpha-globin is formed more rapidly than unfolded apo-beta-globin. The appearance of the latter occurs with a relaxation time tau(3) of 1.2 s. It is postulated that accumulation of unfolded apo-beta-globin is delayed by transient population of an undetected unfolding intermediate.

It has been suggested that the bacterial GroEL chaperonin accommodates only one substrate at any given time, due to conformational changes to both the cis and trans ring that are induced upon substrate binding. Using electrospray ionization mass spectrometry, we show that indeed GroEL binds only one molecule of the model substrate Rubisco. In contrast, the capsid protein of bacteriophage T4, a natural GroEL substrate, can occupy both rings simultaneously. As these substrates are of similar size, the data indicate that each substrate induces distinct conformational changes in the GroEL chaperonin. The distinctive binding behavior of Rubisco and the capsid protein was further investigated using tandem mass spectrometry on the intact 800-914 kDa GroEL-substrate complexes. Our data suggest that even in the gas phase the substrates remain bound inside the GroEL cavity. The analysis revealed further that binding of Rubisco to the GroEL oligomer stabilizes the chaperonin complex significantly, whereas binding of one capsid protein did not have the same effect. However, addition of a second capsid protein molecule to GroEL resulted in a similar stabilizing effect to that obtained after the binding of a single Rubisco. On the basis of the stoichiometry of the GroEL chaperonin-substrate complex and the dissociation behavior of the two different substrates, we hypothesize that the binding of a single capsid polypeptide does not induce significant conformational changes in the GroEL trans ring, and hence the unoccupied GroEL ring remains accessible for a second capsid molecule.