Epidermal Growth Factor Receptor (EGFR) the transmembrane + juxtamembrane domains CR1 L2 CR2 JM Kinase CT 644 151 312 481 621 687 955 1186 Extracellular portion Intracellular portion The transmembrane + juxtamembrane part (615-686 a.a.) contains the transmembrane and the regulatory juxtamembrane (JM) domain. The construct carries N-terminal 7His-tag and C-terminal StrepII-tag (WSHPQFEK)

Important information about the tj-EGFR 73 amino acid residues (without tags) carries N-terminal 7His-tag (HHHHHHH) carries C-terminal StrepII-tag (WSHPQFEK) molecular weight is about 10,152 Da pI is around 11.2 contains no Cys residues NMR structure of the juxtamembrane domain is available Choowongkomon et al. (2005), J. Biol. Chem., 280, 24043-52

tj-EGFR: why two tags? Relation to the previous studies Expression and purification MALDI-TOF m 1 2 3 4 m His-blot SDS-PAGE 20 15 10 20 15 MHHHHHHHGPKIPSIATGMVGALLLLLVVAL GIGLFMRRRH IVRKR TLRR LLQERELVEPLTPSGEAPNQALLRILKETE The results with tj-EGFR carrying ONLY 7His-tag were unsatisfactory

Expression of tj-EGFR in pET 27b+ E.coli BL21(DE3) Codon Plus RP m b 4 16 24 m b 4 16 24 m b 4 16 24 m b 4 16 24 20 15 10 20 10 Strep-blot His-blot 4 – 4 hours after induction 16 – 16 hours after induction 24 – 24 hours after induction m – marker b – before induction red – at +37°C / blue – at + 28°C The optimal expression conditions for tj-EGFR are: +28°C, 24 hours

Purification of tj-EGFR in OG on Chelating and Strep-Tactin Sepharose SDS-PAGE His-blot Strep-Blot open cells chelating Cu2+ Strep-Tactin RPC 20 15 10 tj-EGFR can be purified to homogeneity

MALDI-TOF analysis of tj-EGFR Only full-length tj-EGFR is observed: no degradation products (many thanks to Axel Niebisch, IBT-1)

CD spectra of tj-EGFR and secondary structure predictions: water and TFE α-helix β-sheet turn random water 18% 29.5% 22.5% 30% TFE 40% 13% 18.5% 28.5%

CD spectra of tj-EGFR and secondary structure predictions: detergents 50 mM NaP pH 6.0, 100 mM detergent α-helix β-sheet turn random OG 21% 28% 23% SDS 29% 20% 30% DPC 32% 27% DHPC 25% 22% LPPG 26% 19%

Sequence-based secondary structure predictions MHHHHHHHGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVR KRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEWSHPQFEK ~ 60% α-helix and ~ 40% random coil There is a discrepancy between the experimental and predicted secondary structure structure content

What is yet to be done with tj-EGFR 15N / 13C structural NMR structural investigations more detailed characterization by CD spectroscopy

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