Protein charge determination and implications for interactions in cell extracts
Kyne, Ciara Jordon, Kiara Filoti, Dana I. Laue, Thomas M. Crowley, Peter B.
Kyne, Ciara
Jordon, Kiara
Filoti, Dana I.
Laue, Thomas M.
Crowley, Peter B.
Repository DOI
Publication Date
2016-12-01
Type
Article
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Citation
Kyne, Ciara; Jordon, Kiara; Filoti, Dana I. Laue, Thomas M.; Crowley, Peter B. (2016). Protein charge determination and implications for interactions in cell extracts. Protein Science 26 (2), 258-267
Abstract
Decades of dilute-solution studies have revealed the influence of charged residues on protein stability, solubility and stickiness. Similar characterizations are now required in physiological solutions to understand the effect of charge on protein behavior under native conditions. Toward this end, we used free boundary and native gel electrophoresis to explore the charge of cytochrome c in buffer and in Escherichia coli extracts. We find that the charge of cytochrome c was approximate to 2-fold lower than predicted from primary structure analysis. Cytochrome c charge was tuned by sulfate binding and was rendered anionic in E. coli extracts due to interactions with macroanions. Mutants in which three or four cationic residues were replaced with glutamate were charge-neutral and inert in extracts. A comparison of the interaction propensities of cytochrome c and the mutants emphasizes the role of negative charge in stabilizing physiological environments. Charge-charge repulsion and preferential hydration appear to prevent aggregation. The implications for molecular organization in vivo are discussed.
Funder
Publisher
Wiley-Blackwell
Publisher DOI
10.1002/pro.3077
Rights
Attribution-NonCommercial-NoDerivs 3.0 Ireland