2dx - gluconate transporter / permease; 2D-crystallization

Properties

• Amino acid sequence:

• 461 amino acids
• 47.6 kDa
• pI ~ 9.14
• 21 negatively charged residues
• 25 positively charged residues
• extinction coefficient: 23950
• instability index: 20.72
• alipathic index: 131.43
• GRAVY: 1.001
• TM helices: 12 (Phoebius) or 10 (TMHMM)
• amino acids in the TM domain: 255 or 227 (LPR(m/m) expected: 8-178 (16-63 peak ~30) ~ 0.13-2.92 w/w (0.26-1.04, peak at 0.49); with LPR(m/m)=24>>LPR(w/w)=0.4

X145CD - X146 - Buffer and lipid screening

• DMPC and E.coli in DDM and DOPC in C12E8
• LPR 0.35, 0.7, 1.4 and 2.2 (0.86 mg/ml protein)
• 1 ul of 4 M NaCl added (so final [ NaCl] ~ 225 mM)

X146: in microdialysis plate (conditions: X146-Statistics_from_2dxTable.xlsx)

• DMPC and E.coli in DDM and DOPC in C12E8
• LPR 0.3, 0.6, 1.2 (and 2 for some conditions)
• Buffers (all with 5 mM NaN3): selection based on k-mean applied on buffers used in the litterature with DMPC, E.coli or other lipids:
  • for DMPC only:
    • 20 mM Tris pH8, 250 mM NaCl, 0 mM MgCl2
    • 20 mM TES pH7.5, 50 mM NaCl, 2 mM MgCl2
  • for E.coli only:
    • 20 mM TES pH7.5, 100 mM NaCl, 5 mM MgCl2
    • 20 mM TES pH7.5, 200 mM NaCl, 0 mM MgCl2
  • for all lipids:
    • 20 mM MES pH6, 100 mM NaCl, 20 mM MgCl2
    • 20 mM TES pH7.5, 280 mM NaCl, 5 mM MgCl2
    • 20 mM Tris pH8, 50 mM NaCl, 0 mM MgCl2
• 2 mM MBCD added in all buffers after 1 week

Results, X146

• Peak was close to void volume and broad. Iban suggests it's not a good sign
• Many conditions crashed.

• Set ALLOWTOPICVIEW =

-- NicolasCoudray - 19 Sep 2012