Integration of hydrogen/deuterium exchange and cyanylation-based methodology for conformational studies of cystinyl proteins

LI, X; Chou, Yi-Te; Husain, Rhonda; Watson, Jennifer

doi:10.1016/s0003-2697(04)00268-4

Cited by 5 publications

(4 citation statements)

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“…Therefore, these experiments have allowed us to study much earlier events in the folding of onconase. The formation of secondary structure monitored by H/D exchange and mass spectroscopy on a longer time scale during disulfide-dependent folding has also been observed in an all-α-helical protein . In that study, intermediates with different numbers of disulfide bonds were isolated from the oxidative folding of LR 3 IGF-I.…”

Section: Discussionmentioning

confidence: 97%

“…The formation of secondary structure monitored by H/D exchange and mass spectroscopy on a longer time scale during disulfide-dependent folding has also been observed in an all-α-helical protein. 18 In that study, intermediates with different numbers of disulfide bonds were isolated from the oxidative folding of LR 3 IGF-I. As the number of disulfide bonds increased in each intermediate, less H/D exchange occurred, which is indicative of an increasing level of secondary structure.…”

Section: ■ Discussionmentioning

confidence: 99%

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Identification of Formation of Initial Native Structure in Onconase from an Unfolded State

2011

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In the oxidative folding of onconase, the stabilization of intermediates early in the folding process gives rise to efficient formation of its biologically active form. To identify the residues responsible for initial formation of structured intermediates, the transition from an ensemble of unstructured three-disulfide species, 3SU, to a single structured three-disulfide intermediate species, des-[30–75] or 3SF, at pH 8.0, 25 °C was examined. This transition was first monitored by far-UV CD spectroscopy at pH 8.0, 25 °C, showing that it occurs with formation of secondary structure, presumably due to native interactions. The time-dependence of formation of native-like structure was then followed by NMR spectroscopy after arresting the transition at different times by lowering the pH to 3 and then acquiring 1H,15N – HSQC spectra at pH 3, 16 °C to identify amide hydrogens that become part of native-like structure. H/D exchange was utilized to reduce the intensity of resonances from backbone amide hydrogens not involved in structure, without allowing exchange of backbone amide hydrogens involved in initial structure. Six hydrogen-bonding residues, namely, Tyr38, Lys49, Ser82, Cys90, Glu91, and Ala94 were identified as involved in the earliest detectable native-like structure before complete formation of des-[30–75], and are further stabilized later in the formation of this intermediate through S-S/SH interchange. By observing the stabilization of the structures of these residues by their neighboring residues, the initial, native-like structural elements formed in this transition have been identified, providing details of the initial events in the oxidative folding of onconase.

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Section: Discussionmentioning

confidence: 97%

Section: ■ Discussionmentioning

confidence: 99%

Identification of Formation of Initial Native Structure in Onconase from an Unfolded State

2011

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“…Therefore, the disulfide bonds need to be reduced for effective HDX MS analysis. [15][16][17][18] For example, Kattat and Chait reduced intact chicken egg lysozyme by adding dithiothreitol (DTT) to the protein in H 2 O, 15 followed by H/D exchange for both the reduced and nonreduced proteins. However, if HDX is to probe the native conformation of a protein or complex, reduction cannot be performed before or during the H/D exchange reaction, because the disulfide bond reduction can change the protein conformation.…”

mentioning

confidence: 99%

“…However, disulfide linkage(s) can render extensive segments of the protein inaccessible to proteolysis, and the available proteolytic segments are too long to allow localization of the site(s) of H/D exchange. Therefore, the disulfide bonds need to be reduced for effective HDX MS analysis. − For example, Kattat and Chait reduced intact chicken egg lysozyme by adding dithiothreitol (DTT) to the protein in H 2 O, followed by H/D exchange for both the reduced and nonreduced proteins.…”

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confidence: 99%

Simultaneous Reduction and Digestion of Proteins with Disulfide Bonds for Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry

et al. 2010

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Proteolyzed peptides provide the basis for mass-analyzed hydrogen/deuterium exchange (HDX) for mapping solvent access to various segments of solution-phase proteins. Aspergillus saitoi protease type XIII and porcine pepsin can generate peptides of overlapping sequences and high sequence coverage. However, if disulfide bonds are present, proteolysis can be severely limited, particularly in the vicinity of the disulfide linkage(s). Disulfide bonds cannot be reduced before or during the H/ D exchange reaction without affecting the protein higher-order structure. Here, we demonstrate simultaneous quench/digestion/reduction following H/D exchange, for subsequent mass analysis. Proteolysis is conducted in the presence of TCEP·HCl and urea, and all other steps of the H/D exchange and analysis are maintained. This method yields dramatically increased sequence coverage and localization of solvent exposed segments for mass-analyzed solution-phase H/D exchange of proteins containing disulfide bonds.

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Hydrogen exchange mass spectrometry for the analysis of protein dynamics

Wales

Engen

2005

Mass Spectrometry Reviews

780

908

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Hydrogen exchange coupled to mass spectrometry (MS) has become a valuable analytical tool for the study of protein dynamics. By combining information about protein dynamics with more classical functional data, a more thorough understanding of protein function can be obtained. In many cases, protein dynamics are directly related to specific protein functions such as conformational changes during enzyme activation or protein movements during binding. The method is made possible because labile backbone hydrogens in a protein will exchange with deuterium atoms when the protein is placed in a D2O solution. The subsequent increase in protein mass over time is measured with high-resolution MS. The location of the deuterium incorporation is determined by monitoring deuterium incorporation in peptic fragments that are produced after the labeling reaction. In this review, we will summarize the general principles of the method, discuss the latest variations on the experimental protocol that probe different types of protein movements, and review other recent work and improvements in the field.

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Integration of hydrogen/deuterium exchange and cyanylation-based methodology for conformational studies of cystinyl proteins

Cited by 5 publications

References 0 publications

Identification of Formation of Initial Native Structure in Onconase from an Unfolded State

Identification of Formation of Initial Native Structure in Onconase from an Unfolded State

Simultaneous Reduction and Digestion of Proteins with Disulfide Bonds for Hydrogen/Deuterium Exchange Monitored by Mass Spectrometry

Hydrogen exchange mass spectrometry for the analysis of protein dynamics

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