Protein charge determination and implications for interactions in cell extracts

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• 作者：Ciara Kyne ; Kiara Jordon ; Dana I. Filoti ; Thomas M. Laue and Peter B. Crowley
• 刊名：Protein Science
• 出版年：2017
• 出版时间：February 2017
• 年：2017
• 卷：26
• 期：2
• 页码：258-267
• 全文大小：2671K
• ISSN：1469-896X

文摘

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 m>cm> in buffer and in m>Escherichia colim> extracts. We find that the charge of cytochrome m>cm> was ∼2-fold lower than predicted from primary structure analysis. Cytochrome m>cm> charge was tuned by sulfate binding and was rendered anionic in m>E. colim> 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 m>cm> 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 m>in vivom> are discussed.