Tracking lysozyme unfolding during salt-induced precipitation with hydrogen exchange and mass spectrometry

Tobler, Scott A.; Sherman, Nicholas E.; Fernandez, Erik J.

doi:10.1002/1097-0290(2000)71:3<194::aid-bit1009>3.0.co;2-q

Cited by 15 publications

(15 citation statements)

References 66 publications

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“…Because the oligomeric state of SecA depends on the KCl concentration, and the intrinsic exchange rate of amide protons is largely unaffected by different salt concentrations, 55 we prepared samples of SecA containing predominantly the monomer or dimer, at the same protein concentration, by simply varying the KCl concentration. Any measured changes in HDX rates would then reliably reflect any dimerization-induced changes in the protein.…”

Section: Resultsmentioning

confidence: 99%

Analysis of SecA Dimerization in Solution

et al. 2014

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The Sec pathway mediates translocation of protein across the inner membrane of bacteria. SecA is a motor protein that drives translocation of preprotein through the SecYEG channel. SecA reversibly dimerizes under physiological conditions, but different dimer interfaces have been observed in SecA crystal structures. Here, we have used biophysical approaches to address the nature of the SecA dimer that exists in solution. We have taken advantage of the extreme salt sensitivity of SecA dimerization to compare the rates of hydrogen–deuterium exchange of the monomer and dimer and have analyzed the effects of single-alanine substitutions on dimerization affinity. Our results support the antiparallel dimer arrangement observed in one of the crystal structures of Bacillus subtilis SecA. Additional residues lying within the preprotein binding domain and the C-terminus are also protected from exchange upon dimerization, indicating linkage to a conformational transition of the preprotein binding domain from an open to a closed state. In agreement with this interpretation, normal mode analysis demonstrates that the SecA dimer interface influences the global dynamics of SecA such that dimerization stabilizes the closed conformation.

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Section: Resultsmentioning

confidence: 99%

Analysis of SecA Dimerization in Solution

et al. 2014

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“…The dilution into GdnDCl was intended to dissociate any noncovalent aggregates. We have found this approach effective for dissolving and analyzing such labeled samples of aggregates with low solubility 22. Figure 5A shows the mass spectra for GdnDCl‐redissolved IFN‐γ after a pulsed label step after incubation times of up to 8 days.…”

Section: Resultsmentioning

confidence: 99%

Benzyl Alcohol‐Induced Destabilization of Interferon‐γ: A Study by Hydrogen‐Deuterium Isotope Exchange

Tobler

Holmes

Cromwell³

et al. 2004

Journal of Pharmaceutical Sciences

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“…Indeed the ionic strength can be used to stimulate the salt induced precipitation: sodium chloride reduces the electrostatic repulsive 395 interaction between the charged proteins, promoting in this way attractive hydrophobic interaction (Tobler et al, 2000;Vonhippel and Wong, 1964). However the increase of ionic strength was limited by the solubility of the salt used (NaCl 2.175 M) and by the formation of artefacts during the mixing.…”

Section: Discussionmentioning

confidence: 99%

“…However the increase of ionic strength was limited by the solubility of the salt used (NaCl 2.175 M) and by the formation of artefacts during the mixing. Moreover, excess salt-induced precipitation may induce proteins to incorrectly fold or to form insoluble aggregates upon re-dissolution (Tobler et al, 2000). 400…”

Section: Discussionmentioning

confidence: 99%

Nanoprecipitated catestatin released from pharmacologically active microcarriers (PAMs) exerts pro-survival effects on MSC

Angotti

Venier-Julienne

Penna

et al. 2017

International Journal of Pharmaceutics

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Catestatin (CST), a fragment of Chromogranin-A, exerts angiogenic, arteriogenic, vasculogenic and cardioprotective effects. CST is a very promising agent for revascularization purposes, in "NO-OPTION" patients. However, peptides have a very short half-life after administration and must be conveniently protected. Fibronectin-coated 30 pharmacologically active microcarriers (FN-PAM), are biodegradable and biocompatible polymeric microspheres that can convey mesenchymal stem cell (MSCs) and therapeutic proteins delivered in a prolonged manner. In this study, we first evaluated whether a small peptide such as CST could be nanoprecipitated and incorporated within FN-PAMs.

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Tracking lysozyme unfolding during salt-induced precipitation with hydrogen exchange and mass spectrometry

Cited by 15 publications

References 66 publications

Analysis of SecA Dimerization in Solution

Analysis of SecA Dimerization in Solution

Benzyl Alcohol‐Induced Destabilization of Interferon‐γ: A Study by Hydrogen‐Deuterium Isotope Exchange

Nanoprecipitated catestatin released from pharmacologically active microcarriers (PAMs) exerts pro-survival effects on MSC

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