Ion-specific modulation of protein interactions: Anion-induced, reversible oligomerization of a fusion protein
Ion-specific modulation of protein interactions: Anion-induced, reversible oligomerization of a fusion protein
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DOI：
10.1002/pro.20
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发表时间：
2009-01-01
期刊：
影响因子：
8
通讯作者：
Brems, David N.
中科院分区：
文献类型：
--
作者：
Gokarn, Yatin R.;Fesinmeyer, R. Matthew;Brems, David N.
Ions can significantly modulate the solution interactions of proteins. We aim to demonstrate that the salt-dependent reversible heptamerization of a fusion protein called peptibody A or PbA is governed by anion-specific interactions with key arginyl and lysyl residues on its peptide arms. Peptibody A, an E. coli expressed, basic (pI = 8.8), homodimer (65.2 kDa), consisted of an IgG1-Fc with two, C-terminal peptide arms linked via penta-glycine linkers. Each peptide arm was composed of two, tandem, active sequences (SEYQGLCTRWPWMCPPQGWK) separated by a spacer (GSGSATGGSGGGASSGSGSATG). PbA was monomeric in 10 mM acetate, pH 5.0 but exhibited reversible self-association upon salt addition. The sedimentation coefficient (s(w)) and hydrodynamic diameter (D-H) versus PbA concentration isotherms in the presence of 140 mM NaCl (A5N) displayed sharp increases in sw and DH, reaching plateau values of 9 s and 16 nm by 10 mg/mL PbA. The D-H and sedimentation equilibrium data in the plateau region (>12 mg/mL) indicated the oligomeric ensemble to be monodisperse (PdI = 0.05) with a z-average molecular weight (M-z) of 433 kDa (stoichiometry 5 7). There was no evidence of reversible self-association for an IgG1-Fc molecule in A5N by itself or in a mixture containing fluorescently labeled IgG1-Fc and PbA, indicative of PbA self-assembly being mediated through its peptide arms. Self-association increased with pH, NaCl concentration, and anion size (I- > Br- > Cl- > F-) but could be inhibited using soluble Trp-, Phe-, and Leu-amide salts (Trp > Phe > Leu). We propose that in the presence of salt (i) anion binding renders PbA self-association competent by neutralizing the peptidyl arginyl and lysyl amines, (ii) self-association occurs via aromatic and hydrophobic interactions between the ..xxCTRWPWMC..xxx..CTRWPWMCxx.. motifs, and (iii) at >10 mg/mL, PbA predominantly exists as heptameric clusters.
