Stability study of Rhodobacter capsulatus ferrocytochrome c2 wild‐type and site‐directed mutants using hydrogen/deuterium exchange monitored by electrospray ionization mass spectrometry

Jaquinod, Michel; Guy, Philippe A.; Halgand, Frédéric; Caffrey, Michael; Fitch, John; Cusanovich, Michael A.; Forest, Éric

doi:10.1016/0014-5793(96)00004-x

Cited by 15 publications

(13 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The H/D exchange experiments showed that all labile hydrogens were fully exchanged at pH 7 compared to the case for pH 3 where four hydrogens were found to be unexchanged. These results are in contrast to those described previously for other cases,26,, 44 in which more hydrogens were exchanged at ‘infinite time’ under acidic conditions (more denaturing conditions). This discrepancy might be linked to the presence of the three disulfide bridges.…”

Section: Discussioncontrasting

confidence: 99%

“…The charge state distribution (CSD) of multicharged ions1–12 reveals the solvent‐accessible potentially protonated basic residues, and consequently (by omission) those buried in the solvent‐inaccessible cores located in folded native structures. The studies of hydrogen/deuterium (H/D) isotopic exchange6,, 16–31 give information on the accessibility of the labile hydrogens to the solvent. All these measurements need strict control of the percentage of cosolvent,4–10 of pH values,4–12 of the ion‐source temperature13,, 15 and of the extraction cone voltage 16.…”

Section: Methodsmentioning

confidence: 99%

“…Changes in the CSD reflect variations of the higher‐order structure of the protein, but this information is not always sufficient. Hydrogen/deuterium (H/D) isotopic exchange, monitored by ES‐MS,6,, 7,, 17–28 has been shown to provide a complementary approach to conformational studies using techniques such as circular dichroism (CD) or NMR. Such exchange is dependent on the accessibility of the labile hydrogens, such as the backbone amide hydrogens and the side‐chain hydrogens (Ser and Thr, for hydroxylic function, Asp and Glu for carboxylic function, Arg, Lys, and His for primary and secondary amine functions, and Asn and Gln for amide functions).…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

Conformational study of a new SGTx1 neurotoxin from the spider Scodra griseipes using mass spectrometry

Marvin

Lange

2001

Rapid Comm Mass Spectrometry

View full text Add to dashboard Cite

SGTx1 is a new neurotoxin from the venom of Scodra griseipes. Because of the small quantity of this natural peptide available, mass spectrometry was used to obtain information on its higher-order structure. The kinetics of reduction by 1,4-dithiothreitol (DTT) was monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), and showed that one of the three disulfide bridges was appreciably more accessible to the DTT. Studies based on the charge state distribution (CSD) and H/D exchange of the non-reduced peptide, under neutral and acidic conditions, were performed using electrospray mass spectrometry (ES-MS). In neutral solution, SGTx1 showed a maximum charge state of four compared with seven potentially protonated basic residues, and all labile hydrogens were exchanged. However, under acidic conditions, a maximum charge state of only five was observed, and four of the labile hydrogens could not be deuterated. These observations are interpreted in terms of a rigid structure maintained by the disulfide bridges, which can be temporarily relaxed by disulfide bridge scrambling only at higher pH values.

show abstract

Section: Discussioncontrasting

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Conformational study of a new SGTx1 neurotoxin from the spider Scodra griseipes using mass spectrometry

Marvin

Lange

2001

Rapid Comm Mass Spectrometry

View full text Add to dashboard Cite

show abstract

“…The described results indicate that this method is suitable for the initial characterization of site‐directed mutants. Our results are similar to those obtained by electrospray ionization mass spectrometry [31] but have the advantages of simplicity of operation and much higher tolerance to the presence of salts and chaotropes in the samples. Moreover, the back‐exchange problem is more easily avoided [32].…”

Section: Resultssupporting

confidence: 85%

Hydrogen exchange monitored by MALDI‐TOF mass spectrometry for rapid characterization of the stability and conformation of proteins

Villanueva¹,

Canals²,

Villegas³

et al. 2000

FEBS Letters

View full text Add to dashboard Cite

Matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) has been used to monitor hydrogen exchange on entire proteins. Two alternative methods have been used to carry out the hydrogen exchange studies, exchanging deuteron (H to D experiments) or proton (D to H experiments). In the former case, the use of a deuterated matrix has made possible to overcome back-exchange problems and attain reproducible results. The methods presented have been used to determine the slow exchange core of the potato carboxypeptidase inhibitor in different folding states, and to differentially compare the activation domain of human procarboxypeptidase A2 versus three site-directed mutants of different conformational stability. In this work, we show that by using MALDI-TOF MS to monitor hydrogen exchange in entire proteins, it is possible to rapidly check the folding state of a protein and characterize mutational effects on protein conformation and stability, while requiring minimal amounts of sample.z 2000 Federation of European Biochemical Societies.

show abstract

“…Hydrogen-exchange monitored by ESI MS (HX-MS) has shown that a number of 'H are protected from exchange with solvent protons in the native state of cytochrome C (Katta & Chait, 1991). HX-MS has also been used to study lysozyme during protein folding experiments (Miranker et al, 1993) and to compare the relative stabilities of ferrocytochrome c2 and site-directed mutants (Jaquinod et al, 1996). Different exchange rates are observed for myoglobin in its apo and holo forms (Johnson & Walsh, 1994) and the solution dynamics of P-sheet and a-helical peptides have also been measured by HX-MS (Wagner et al, 1994).…”

mentioning

confidence: 99%

Hydrogen exchange properties of proteins in native and denatured states monitored by mass spectrometry and NMR

et al. 1997

View full text Add to dashboard Cite

The extent of deuterium labeling of hen lysozyme, its three-disulfide derivative, and the homologous a-lactalbumins, has been measured by both mass spectrometry and NMR. Different conformational states of the proteins were produced by varying the solution conditions. Alternate protein conformers were found to contain different numbers of ' H atoms. Furthermore, measurement in the gas phase of the mass spectrometer or directly in solution by NMR gave consistent results. The unique ability of mass spectrometry to distinguish distributions of ' H atoms in protein molecules is exemplified using samples prepared to contain different populations of 'H-labeled protein. A comparison of the peak widths of bovine a-lactalbumin in alternate solution conformations but containing the same average number of ' H atoms showed dramatic differences due to different ' H distributions in the two protein conformers. Measurement of ' H distributions by ESI-MS enabled characterization of conformational averaging and structural heterogeneity. In addition, a time course for hydrogen exchange was examined and the variation in distributions of ' H atom compared with simulations for different hydrogen exchange models. The results clearly show that exchange from the native state of bovine a-lactalbumin at 15 "C is dominated by local unfolding events.

show abstract

Stability study of Rhodobacter capsulatus ferrocytochrome c₂ wild‐type and site‐directed mutants using hydrogen/deuterium exchange monitored by electrospray ionization mass spectrometry

Cited by 15 publications

References 38 publications

Conformational study of a new SGTx1 neurotoxin from the spider Scodra griseipes using mass spectrometry

Conformational study of a new SGTx1 neurotoxin from the spider Scodra griseipes using mass spectrometry

Hydrogen exchange monitored by MALDI‐TOF mass spectrometry for rapid characterization of the stability and conformation of proteins

Hydrogen exchange properties of proteins in native and denatured states monitored by mass spectrometry and NMR

Contact Info

Product

Resources

About