Protein hydrogen exchange studied by the fragment separation method

Englander, Joan J.; Rogero, J.R.; Englander, S. Walter

doi:10.1016/0003-2697(85)90033-8

Cited by 155 publications

(184 citation statements)

References 29 publications

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“…More recently a proteolytic fragmentation method (Englander et al 1985) together with analysis by mass spectrometry (Zhang & Smith, 1993;Eyles & Kaltashov, 2004) makes it possible to obtain segment-resolved HX information for proteins that are too large for NMR and even to move toward amino-acid resolution in favorable cases Englander, 2006).…”

Section: Hx Measurementmentioning

confidence: 99%

Protein folding and misfolding: mechanism and principles

Englander

Mayne

Mallela

2007

Quart. Rev. Biophys.

172

267

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Two fundamentally different views of how proteins fold are now being debated. Do proteins fold through multiple unpredictable routes directed only by the energetically downhill nature of the folding landscape or do they fold through specific intermediates in a defined pathway that systematically puts predetermined pieces of the target native protein into place? It has now become possible to determine the structure of protein folding intermediates, evaluate their equilibrium and kinetic parameters, and establish their pathway relationships. Results obtained for many proteins have serendipitously revealed a new dimension of protein structure. Cooperative structural units of the native protein, called foldons, unfold and refold repeatedly even under native conditions. Much evidence obtained by hydrogen exchange and other methods now indicates that cooperative foldon units and not individual amino acids account for the unit steps in protein folding pathways. The formation of foldons and their ordered pathway assembly systematically puts native-like foldon building blocks into place, guided by a sequential stabilization mechanism in which prior native-like structure templates the formation of incoming foldons with complementary structure. Thus the same propensities and interactions that specify the final native state, encoded in the amino-acid sequence of every protein, determine the pathway for getting there. Experimental observations that have been interpreted differently, in terms of multiple independent pathways, appear to be due to chance misfolding errors that cause different population fractions to block at different pathway points, populate different pathway intermediates, and fold at different rates. This paper summarizes the experimental basis for these three determining principles and their consequences. Cooperative native-like foldon units and the sequential stabilization process together generate predetermined stepwise pathways. Optional misfolding errors are responsible for 3-state and heterogeneous kinetic folding. The protein folding problemThe search for protein folding pathways and the principles that guide them has proven to be one of the most difficult problems in all of structural biology. Biochemical pathways have almost universally been solved by isolating the pathway intermediates and determining their structures. This approach fails for protein folding pathways. Folding intermediates only live for <1 s and they cannot be isolated and studied by the usual structural methods.The protein folding problem has been attacked by the entire range of fast spectroscopic methods which are able to track folding in real time. Much valuable information has been obtained but mainly of a kinetic nature. Kinetic methods do not describe the structure of the

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Section: Hx Measurementmentioning

confidence: 99%

Protein folding and misfolding: mechanism and principles

Englander

Mayne

Mallela

2007

Quart. Rev. Biophys.

172

267

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“…These represent the best fits for any sequences in the protein. N-terminal amino acids corroborated the compositional analysis and the proposed cut sites were evaluated for the probability of hydrolysis by pepsin due to its kinetic specificity (Englander et al, 1985).…”

Section: Thioredoxin Proteolysis and Peptide Identificationmentioning

confidence: 93%

“…To determine the amount of tritium originally present on each peptide as it existed before fragmentation of the protein, the losses of the analytical procedures were calculated from the intrinsic rate constants for the fully solvent-exposed amide protons at each position (Molday et al, 1972;Englander et al, 1985). Due to the addition of different components (urea, acetonitrile) that affect hydrogen exchange rates, the losses during the digestion and chromatography steps were calculated separately.…”

Section: Calculation Of Tritium Lossesmentioning

confidence: 99%

“…(1) For the digestion step, the presence of 4 M urea required a correction of the rate constants by a factor determined from the data given by Lofthus et al (1986). (2) Tritium loss during the chromatographic separation was determined using the procedure of Englander et al (1985). This accounts for effects on exchange rates due to the presence of an increasing concentration of organic solvents in the elution buffer.…”

Section: Calculation Of Tritium Lossesmentioning

confidence: 99%

“…Medium resolution hydrogen exchange experiments (Rosa & Richards, 1979, 1981Englander et al, 1985) were then used to localize the affected areas in the structure. It was found that significant changes occur in two regions of the protein, at the active site, residues 32-35, and within residues 1-13 at the amino terminal end.…”

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confidence: 99%

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Differences in hydrogen exchange behavior between the oxidized and reduced forms of Escherichia coli thioredoxin

Kaminsky

Richards

1992

Protein Science

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Amide proton exchange of thioredoxin is used to monitor the structural effects of reduction of its single disulfide. An effective 3–5‐proton difference between the oxidized and reduced protein form is observed early in proton out‐exchange of the whole protein, which is independent of temperature in the range of 5–45 °C, indicating that redox‐sensitive changes are probably not due to low‐energy structural fluctuations. Medium resolution hydrogen exchange experiments have localized the redox‐sensitive amide protons to two parts of the sequence that are distant from each other in the three‐dimensional structure: the active‐site turn and the first β‐strand. The sum of the proton differences observed in the peptides from these regions is equal to that of the whole protein, indicating that all redox‐sensitive hydrogen exchange effects are observed in the peptide experiments. A model combining structural changes within the protein matrix with changes in the surface hydration properties is proposed as a mechanism for the communication between distant sites within the protein. Sound velocity and density measurements of reduced and oxidized thioredoxin are presented in the accompanying paper (Kaminsky, S.M. & Richards, F.M., 1992, Protein Sci. 1, 22–30).

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Probing the Non-covalent Structure of Proteins by Amide Hydrogen Exchange and Mass Spectrometry

1997

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The rates at which hydrogens located at peptide amide linkages in proteins undergo isotopic exchange when a protein is exposed to D2O depend on whether these amide hydrogens are hydrogen bonded and whether they are accessible to the aqueous solvent. Hence, amide hydrogen exchange rates are a sensitive probe for detecting changes in protein conformation and dynamics. Hydrogen exchange rates in proteins are most often measured by NMR or Fourier transform IR spectroscopy. After a brief introduction to model kinetics used to relate amide hydrogen exchange rates to protein structure and dynamics, information required to understand and implement a new method that uses acid proteases and mass spectrometry to determine amide hydrogen exchange rates in proteins is presented. Structural and dynamic features affecting isotopic exchange rates can be detected and localized from the deuterium levels detected by mass spectrometry in proteolytic fragments of the protein. Procedures used to adjust for isotopic exchange occurring during the analysis, to extract isotope exchange rate constants from mass spectra and to link bimodal isotope patterns to protein unfolding and structural heterogeneity are also discussed. In addition, the relative merits of using mass spectrometry or NMR combined with amide hydrogen exchange to study protein structure and dynamics are discussed. The spatial resolution of hydrogen exchange results obtained by this method is typically in the range of 1–10 residues, which is substantially less than that obtained by high‐resolution NMR, but sufficient to detect many functionally significant structural changes. Advantages in the areas of sensitivity, protein solubility, detection of correlated exchange and high molecular mass proteins make this approach particularly attractive for a wide range of studies. © 1997 by John Wiley & Sons, Ltd.

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Protein hydrogen exchange studied by the fragment separation method

Cited by 155 publications

References 29 publications

Protein folding and misfolding: mechanism and principles

Protein folding and misfolding: mechanism and principles

Differences in hydrogen exchange behavior between the oxidized and reduced forms of Escherichia coli thioredoxin

Probing the Non-covalent Structure of Proteins by Amide Hydrogen Exchange and Mass Spectrometry

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