Ethanol and acetonitrile induces conformational changes in porcine pepsin at alkaline denatured state

Shanmugam, Ganesh; Selvi, C. Chinnarul; Mandal, Asit Baran

doi:10.1016/j.ijbiomac.2012.06.026

Cited by 9 publications

(2 citation statements)

References 37 publications

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“…To rationalize the SETA incorporation trends of the lysines for each protein, two features of the chemical probe methodology must be considered: the factors that influence the reaction of the lysines with the SETA probe and the effects of acetonitrile denaturation on the tertiary structure of the proteins. In the context of the latter factor, acetonitrile has been shown to be a mild-denaturant at lower concentrations, disrupting hydrophobic interactions and causing loss of tertiary structure while maintaining secondary structural features, such as hydrogen bonding, until high concentrations of acetonitrile have been reached. ,− For some proteins, alpha helical content has been shown to be retained even in solutions containing more than 50% ACN. ,− With respect to the intrinsic reactivity of a primary amine with the SETA chemical probe, two factors are expected to be most influential: (i) the relative accessibility of the amine to the chemical probe which impacts its reaction probability, and (ii) the local p K a of each lysine amine which determines its nucleophilicity. Residues with higher p K a values are more basic and therefore are expected to more significantly favor the protonated (unreactive) form in solution.…”

Section: Resultsmentioning

confidence: 99%

Probing the Unfolding of Myoglobin and Domain C of PARP-1 with Covalent Labeling and Top-Down Ultraviolet Photodissociation Mass Spectrometry

et al. 2014

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Ultraviolet photodissocation (UVPD) mass spectrometry was used for high mass accuracy top-down characterization of two proteins labeled by the chemical probe, S-ethylacetimidate (SETA), in order to evaluate conformational changes as a function of denaturation. The SETA labeling/UVPD-MS methodology was used to monitor the mild denaturation of horse heart myoglobin by acetonitrile, and the results showed good agreement with known acetonitrile and acid unfolding pathways of myoglobin. UVPD outperformed electron transfer dissociation (ETD) in terms of sequence coverage, allowing the SETA reactivity of greater number of lysine amines to be monitored and thus providing a more detailed map of myoglobin. This strategy was applied to the third zinc-finger binding domain, domain C, of PARP-1 (PARP-C), to evaluate the discrepancies between the NMR and crystal structures which reported monomer and dimer forms of the protein, respectively. The trends reflected from the reactivity of each lysine as a function of acetonitrile denaturation in the present study support that PARP-C exists as a monomer in solution with a close-packed C-terminal α helix. Additionally, those lysines for which the SETA reactivity increased under denaturing conditions were found to engage in tertiary polar contacts such as salt bridging and hydrogen bonding, providing evidence that the SETA/UVPD-MS approach offers a versatile means to probe the interactions responsible for conformational changes in proteins.

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Section: Resultsmentioning

confidence: 99%

Probing the Unfolding of Myoglobin and Domain C of PARP-1 with Covalent Labeling and Top-Down Ultraviolet Photodissociation Mass Spectrometry

et al. 2014

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“…The intensity of the tryptophan fluorescence is directly dependent not only on the degree of hydrophobicity of the immediate solvent environment but the proximity of certain amino acid side chains such as lysine and tyrosine have also been shown to quench tryptophan fluorescence [54]. As shown in previous studies with papain [55] and pepsin [56], fluorescence of these proteins will increase quite linearly as the percentage of organic solvent such as MeCN increases in water. We are primarily interested in the fluorescence peak area ratio of the weakly retained (likely native protein with less hydrophobic exposed areas) to more retained peaks (protein denatured to some degree) as a function of increasing IPAF and MeCN in water.…”

Section: Resultsmentioning

confidence: 99%

The ionic liquid isopropylammonium formate as a mobile phase modifier to improve protein stability during reversed phase liquid chromatography

Zhou

Danielson

2013

Journal of Chromatography B

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The room temperature ionic liquid isopropylammonium formate (IPAF) is studied as a reversed phase HPLC mobile phase modifier for separation of native proteins using a polymeric column and this protein stability is compared to that using acetonitrile (MeCN) as the standard organic mobile phase modifier. A variety of important proteins with different numbers of subunits is investigated, including non-subunit proteins: albumin, and amyloglucosidase (AMY); a two subunit protein: thyroglobulin (THY); and four subunit proteins: glutamate dehydrogenase (GDH) and lactate dehydrogenase (LDH). A significant enhancement in protein stability is observed in the chromatograms upon using IPAF as a mobile phase modifier. The first sharper peak at about 2 min represented protein in primarily the native form and a second broader peak more retained at about 5–6 min represented substantially denatured or possibly aggregated protein. The investigated proteins (except LDH) could maintain the native form within up to 50% IPAF, while a mobile phase, with as low as 10% MeCN, induced protein denaturation. The assay for pyruvate using LDH has further shown that enzymatic activity can be maintained up to 30% IPAF in water in contrast to no activity using 30% MeCN.

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Correlating structure and activity of pepsin enzyme in H2O and D2O for the study of gastric digestion

Bayrak,

Han,

Greaves

et al. 2024

Food Chemistry Advances

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Ethanol and acetonitrile induces conformational changes in porcine pepsin at alkaline denatured state

Cited by 9 publications

References 37 publications

Probing the Unfolding of Myoglobin and Domain C of PARP-1 with Covalent Labeling and Top-Down Ultraviolet Photodissociation Mass Spectrometry

Probing the Unfolding of Myoglobin and Domain C of PARP-1 with Covalent Labeling and Top-Down Ultraviolet Photodissociation Mass Spectrometry

The ionic liquid isopropylammonium formate as a mobile phase modifier to improve protein stability during reversed phase liquid chromatography

Correlating structure and activity of pepsin enzyme in H2O and D2O for the study of gastric digestion

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