Protein charge determination and implications for interactions in cell extracts.
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 ∼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.
School of Chemistry, National University of Ireland, Galway, University Road, Galway, Ireland.
Ciara
C
Kyne
Spin Analytical, 468 Portland Street, Berwick, Maine, 03901.
Kiara
K
Jordon
Centre to Advance Macromolecular Interaction Sciences University of New Hampshire, Durham, New Hampshire, 03824.
Dana I
DI
Filoti
Spin Analytical, 468 Portland Street, Berwick, Maine, 03901.|Centre to Advance Macromolecular Interaction Sciences University of New Hampshire, Durham, New Hampshire, 03824.
Thomas M
TM
Laue
School of Chemistry, National University of Ireland, Galway, University Road, Galway, Ireland.
Peter B
PB
Crowley
D045424:Complex Mixtures / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D066329:Protein Aggregates
D045424:Complex Mixtures; D029968:Escherichia coli Proteins; D066329:Protein Aggregates; D045304:Cytochromes c
cell extract; charge-inverted mutants; membrane confined electrophoresis; native gel electrophoresis; quinary interactions; size exclusion chromatography