The investigations of The investigations of structure and properties of structure and properties of

membrane receptors: membrane receptors: human EGFR and human EGFR and

halobacterial HtrIIhalobacterial HtrII

The investigations of The investigations of structure and properties of structure and properties of

membrane receptors: membrane receptors: human EGFR and human EGFR and

halobacterial HtrIIhalobacterial HtrII Ivan L. BudyakIvan L. Budyak

ForschungszentrumForschungszentrum J Jüülich, Deutschlandlich, DeutschlandUniversity of Pittsburgh, PA, USAUniversity of Pittsburgh, PA, USA

Московский физико-технический институтМосковский физико-технический институт, , РоссияРоссия

MayMay 2006 2006

PartPart 1: 1:

halobacterial transducerhalobacterial transducer II II ((HtrIIHtrII))

fromfrom Natronobacterium Natronobacterium pharaonispharaonis

PartPart 1: 1:

halobacterial transducerhalobacterial transducer II II ((HtrIIHtrII))

fromfrom Natronobacterium Natronobacterium pharaonispharaonis

Retinal-containing proteins of Retinal-containing proteins of N.pharaonisN.pharaonis

Retinal-containing proteins of Retinal-containing proteins of N.pharaonisN.pharaonis

Engelhard M. Engelhard M. et alet al. (2002), Archaeabacterial phototaxis, . (2002), Archaeabacterial phototaxis,

In In Photoreceptors and Light SignalingPhotoreceptors and Light Signaling (pp. 2-39), The Royal Society of Chemistry, UK (pp. 2-39), The Royal Society of Chemistry, UK

Two-componentTwo-component signal signal transduction system in transduction system in

N.pharaonisN.pharaonis

Two-componentTwo-component signal signal transduction system in transduction system in

N.pharaonisN.pharaonis

??

Gordeliy V.I. Gordeliy V.I. et alet al., ., NatureNature, , 419419 (2002), pp. 484-487 (2002), pp. 484-487 Oprian D.D., Oprian D.D., TIBSTIBS, , 2828 (2003), pp. 167- (2003), pp. 167-169169

transducer sequences from archaea transducer sequences from archaea including including N.pharaonisN.pharaonis are homologous are homologous to those of eubacterial chemoreceptorsto those of eubacterial chemoreceptors

both signal through the classical two-both signal through the classical two-component systemcomponent system

Structures of the cytoplasmic domains of halobacterial Structures of the cytoplasmic domains of halobacterial transducers remain unknown transducers remain unknown

open the cellsopen the cells

salting outsalting out

HIC HIC purificationpurification

gel-filtrationgel-filtration

mm1 , 1 , 2 2

33

44

55

1 1 2 2 33 44 55

Le Moual H. and Koshland D.E., Le Moual H. and Koshland D.E., J.Mol.Biol.J.Mol.Biol., , 261261 (1996), pp. (1996), pp. 568-585568-585

Tsr, T286Tsr, T286

HtrII, M234HtrII, M234

Tsr, A526Tsr, A526

HtrII, HtrII, D504D504

The cytoplasmic fragment of HtrII can be expressed in The cytoplasmic fragment of HtrII can be expressed in E.coliE.coli and purified to homogeneity and is unstructured in and purified to homogeneity and is unstructured in

solutionsolution

Design, expression, purification Design, expression, purification and and

initial characterization of HtrII-cytinitial characterization of HtrII-cyt

Design, expression, purification Design, expression, purification and and

initial characterization of HtrII-cytinitial characterization of HtrII-cyt

Structural predictions for HtrII-cytStructural predictions for HtrII-cytStructural predictions for HtrII-cytStructural predictions for HtrII-cyt

Combet C. Combet C. et al.et al., , TIBSTIBS, , 291291 (2000), pp. (2000), pp. 147-150147-150

76%76% αα-helix -helix andand 24%24% random coilrandom coil

O. Lund O. Lund et al.et al., “CPHmodels 2.0: , “CPHmodels 2.0: X3MX3M

a Computer Program to Extracta Computer Program to Extract 3D Models”, A102 abstract 3D Models”, A102 abstract

at the CASP5 conference, 2002at the CASP5 conference, 2002

Lupas A. et al., Science, 252 (1991), pp. 1162-1164

The cytoplasmic fragment of HtrII is predicted The cytoplasmic fragment of HtrII is predicted to be to be αα-helical and to form coiled coil structure-helical and to form coiled coil structure

Predictions of dynamic properties of Predictions of dynamic properties of HtrIIHtrII

Predictions of dynamic properties of Predictions of dynamic properties of HtrIIHtrII

Predictions of structural parameters are Predictions of structural parameters are contradictory contradictory

suggesting the possibility of structural transitionssuggesting the possibility of structural transitions

Romero P. et al., Proteins: Struct. Funct. Gen., 42 (2001), pp. 38-48

The cytoplasmic domain of HtrII is predicted to be The cytoplasmic domain of HtrII is predicted to be disordereddisordered

CONTIN algorithm: Van Stokkum I.H.M. CONTIN algorithm: Van Stokkum I.H.M. et al.et al., , Anal.Biochem.Anal.Biochem., , 191191 (1990), pp. (1990), pp. 110-119110-119

KClKCl NaClNaCl glycerolglycerolConformational transitionsConformational transitionsConformational transitionsConformational transitions

KCl, NaCl and glycerol induceKCl, NaCl and glycerol induceconformational transitions from mainly random coil to conformational transitions from mainly random coil to -helix-helix

CONTIN algorithm: Van Stokkum I.H.M. CONTIN algorithm: Van Stokkum I.H.M. et al.et al., , Anal.Biochem.Anal.Biochem., , 191191 (1990), pp. (1990), pp. 110-119110-119

sucrosesucrose ammonium sulfateammonium sulfate TFETFEConformational transitionsConformational transitionsConformational transitionsConformational transitions

Sucrose, ammonium sulfate and TFE also induceSucrose, ammonium sulfate and TFE also induceconformational transitions from mainly random coil to conformational transitions from mainly random coil to -helix-helix

FTIR spectroscopyFTIR spectroscopyFTIR spectroscopyFTIR spectroscopy

wavenumber, cm-1 assignment

1621-1640, 1671-1679 β-structure

1641-1647 random coil

1651-1657 α-helix

1658-1671, 1681-1690 turns and bends

1644164416541654

adapted from adapted from Stuart B. Stuart B. (1997), (1997),

Biological Biological Applications Applications of Infrared of Infrared

SpectroscopySpectroscopy, , University of University of Greenwich, Greenwich,

UKUK

FTIR indicates random coil in solution and FTIR indicates random coil in solution and -helix in dry -helix in dry filmfilm

10 mM Tris-HCl pH 9.0 in D10 mM Tris-HCl pH 9.0 in D22OO dry filmdry film

NMR spectroscopyNMR spectroscopyNMR spectroscopyNMR spectroscopy10 mM NaP pH 6.010 mM NaP pH 6.0

NMR data supportNMR data supportstructural transitions in glycerolstructural transitions in glycerol

minimal spectral dispersionminimal spectral dispersion negative het-NOE signalsnegative het-NOE signals

(data not shown)(data not shown)

10mM NaP pH 6.0 + 70% 10mM NaP pH 6.0 + 70% glycerolglycerol

peaks shifted and broadenedpeaks shifted and broadened strong Trosy effectstrong Trosy effect

Red:1H-15N HSQCGreen: Trosy-HSQC

15

15 N

chem

ical sh

ift,

ppm

N c

hem

ical sh

ift,

ppm

9.0 8.0 7.09.0 8.0 7.011H chemical shift, ppmH chemical shift, ppm

12

5 1

20

1

15

1

10

12

5 1

20

1

15

1

10

10 9 8 7 6 5 4 3 2 1 010 9 8 7 6 5 4 3 2 1 0

11H chemical shift, H chemical shift, ppmppm

8.5 8.0 7.5 7.0 6.58.5 8.0 7.5 7.0 6.5

12

5 1

20

1

15

12

5 1

20

1

15

11

01

10

15

15 N

chem

ical sh

ift,

N

chem

ical sh

ift,

ppm

ppm

AnalyticalAnalytical gel-filtration gel-filtration chromatography (AGFC)chromatography (AGFC)AnalyticalAnalytical gel-filtration gel-filtration chromatography (AGFC)chromatography (AGFC)

Abnormal retention volumesAbnormal retention volumes ofof the cytoplasmic the cytoplasmic fragmentfragment of HtrII evidence its non-globular shapeof HtrII evidence its non-globular shape;;

ammonium sulfate induces hydrophobic interactions ammonium sulfate induces hydrophobic interactions with the column up to complete retentionwith the column up to complete retention

ammonium ammonium sulfatesulfate

KClKCl andand NaClNaCl

AnalyticalAnalytical gel-filtration gel-filtration chromatography (AGFC) and chemical chromatography (AGFC) and chemical

cross-linkingcross-linking

AnalyticalAnalytical gel-filtration gel-filtration chromatography (AGFC) and chemical chromatography (AGFC) and chemical

cross-linkingcross-linking

Cross-linking data evidence HtrII-cyt dimerization in Cross-linking data evidence HtrII-cyt dimerization in 4 M KCl4 M KCl

1xPB

1xPB

SS1

xPB

S +

10

%

1xPB

S +

10

%

asas

1xPB

S +

40

%

1xPB

S +

40

%

asas

1xPB

S +

4 M

1

xPB

S +

4 M

K

Cl

KC

l

Small-angle neutron scattering Small-angle neutron scattering and atomic force microscopyand atomic force microscopy

Small-angle neutron scattering Small-angle neutron scattering and atomic force microscopyand atomic force microscopy

HtrII-cyt has characteristic size ofHtrII-cyt has characteristic size of ~180-~180-200 200 ÅÅ and elongated and elongated shape in solutionshape in solution

SANS withSANS with HtrII-HtrII-cytcyt

AFMAFM withwith HtrII-cyt in dry filmHtrII-cyt in dry film

(many thanks to Dirk Mayer, ISG-(many thanks to Dirk Mayer, ISG-2)2)

Conclusions, partConclusions, part 1 1Conclusions, partConclusions, part 1 1

The cytoplasmic The cytoplasmic domaindomain of HtrII from of HtrII from N.pharaonisN.pharaonis (HtrII-cyt) (HtrII-cyt) can be expressed in can be expressed in E.coliE.coli in soluble form and then in soluble form and then successfully purifiedsuccessfully purified

HtrII-cyt is shown to be unstructured (disordered) in common HtrII-cyt is shown to be unstructured (disordered) in common aqueous solutionsaqueous solutions

Drying and certain additives render HtrII-cyt Drying and certain additives render HtrII-cyt αα-helical with -helical with different efficacydifferent efficacy

HtrII-cyt exists in solution in monomeric form, 4 M NaCl and HtrII-cyt exists in solution in monomeric form, 4 M NaCl and KCl induce oligomerization with dimers being the most KCl induce oligomerization with dimers being the most abundant speciesabundant species

HtrII-cyt has a rod-like shape of ~200 HtrII-cyt has a rod-like shape of ~200 Å long and ~14 Å in Å long and ~14 Å in diameter for the monomeric form, and diameter for the monomeric form, and ~250 ~250 Å long and ~20 Å long and ~20 Å in diameter as a dimerÅ in diameter as a dimer

PartPart 2: 2:

human Epidermal Growth human Epidermal Growth Factor Receptor Factor Receptor ((hEGFRhEGFR))

PartPart 2: 2:

human Epidermal Growth human Epidermal Growth Factor Receptor Factor Receptor ((hEGFRhEGFR))

Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor (EGFR):(EGFR):

general informationgeneral information

Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor (EGFR):(EGFR):

general informationgeneral information found in a number of epithelial tissues in found in a number of epithelial tissues in

humanhuman transmembrane, 1186 a.a. long, 170 transmembrane, 1186 a.a. long, 170

kDa, 8 domainskDa, 8 domains (precursor peptide – 1212 a.a. with 26 (precursor peptide – 1212 a.a. with 26

a.a. signal sequence)a.a. signal sequence) has 3 homologous proteins in humans has 3 homologous proteins in humans

(ErbB2, ErbB3 and ErbB4) and one each (ErbB2, ErbB3 and ErbB4) and one each from from D.melanogasterD.melanogaster and and C.elegansC.elegans

posttranslationally glycosylated (20% of posttranslationally glycosylated (20% of protein mass)protein mass)

binds EGF, TGF-binds EGF, TGF-α and neuregulinsα and neuregulins exists both as monomers and dimersexists both as monomers and dimers implicated in a variety of human cancers implicated in a variety of human cancers

(e.g. mammary carcinoma, (e.g. mammary carcinoma, glioblastomas etc.)glioblastomas etc.)

Burgess A. Burgess A. et al.et al., , Mol.CellMol.Cell,,

1212 (2003), pp. 541-552 (2003), pp. 541-552

Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor (EGFR):(EGFR):

extracellular and kinase domainsextracellular and kinase domains

Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor (EGFR):(EGFR):

extracellular and kinase domainsextracellular and kinase domains

Burgess A. Burgess A. et al.et al., , Mol.CellMol.Cell, ,

1212 (2003), pp. 541-552 (2003), pp. 541-552

Ogiso H. Ogiso H. et al.et al., , CellCell, ,

110110 (2002), pp. 775- (2002), pp. 775-787787

residues 1-619 + EGF residues 1-619 + EGF

Stamos J. Stamos J. et al.et al., , J.Biol.Chem.J.Biol.Chem., ,

277277 (2002), pp. 46265- (2002), pp. 46265-4627246272

residues 672-998 +residues 672-998 +ATP / kinase inhibitorATP / kinase inhibitor

Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor (EGFR):(EGFR):

trans- and juxtamembrane domainstrans- and juxtamembrane domains

Epidermal Growth Factor Receptor Epidermal Growth Factor Receptor (EGFR):(EGFR):

trans- and juxtamembrane domainstrans- and juxtamembrane domains

Burgess A. Burgess A. et al.et al., , Mol.CellMol.Cell, ,

1212 (2003), pp. 541-552 (2003), pp. 541-552

Rigby A. Rigby A. et al.et al., , Biochim.Biophys.ActaBiochim.Biophys.Acta, ,

13711371 (1998), pp. 241- (1998), pp. 241-253253

residues 621-654 residues 621-654

Choowongkomon K. Choowongkomon K. et alet al., ., J.Biol.Chem.J.Biol.Chem.,,280 280 (2005), pp. 24043-52(2005), pp. 24043-52

residues 645-697residues 645-697

Important information about the tj-Important information about the tj-EGFREGFR

Important information about the tj-Important information about the tj-EGFREGFR

73 amino acid residues (615-686 a.a.) (without 73 amino acid residues (615-686 a.a.) (without tags)tags)

carries N-terminal 7His-tag (HHHHHHH)carries N-terminal 7His-tag (HHHHHHH) carries C-terminal carries C-terminal StrepStrepII-tag (WSHPQFEK)II-tag (WSHPQFEK) molecular weight is about 10,152 Damolecular weight is about 10,152 Da pI is around 11.2pI is around 11.2 contains no Cys residues contains no Cys residues

L1 CR1 L2 CR2 JM Kinase CT

644

151 312 481 621 687 955 1186

Extracellular portion Intracellular portion

L1 CR1 L2 CR2 JM Kinase CT

644

151 312 481 621 687 955 1186

Extracellular portion Intracellular portion

MHHHHHHHGPKIPSIATGMVGALLLLLVVAL GIGLFMRRRH IVRKR TLRR LLQERELVEPLTPSGEAPNQALLRILKETE

tj-EGFR: why two tags?tj-EGFR: why two tags?Relation to the previous studiesRelation to the previous studies

tj-EGFR: why two tags?tj-EGFR: why two tags?Relation to the previous studiesRelation to the previous studies

Expression and Expression and purificationpurification

The results with tj-EGFR carrying ONLY The results with tj-EGFR carrying ONLY 7His-tag were unsatisfactory7His-tag were unsatisfactory

MALDI-TOFMALDI-TOF

15151010

2020

m 1 2 3 4m 1 2 3 4

1515

2020

m His-blot SDS-PAGEm His-blot SDS-PAGE

1957.0 3810.8 5664.6 7518.4 9372.2 11226.0

Mass (m/z)

0

1.1E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tensi

ty

Voyager Spec #1=>BC=>NR(2.00)[BP = 5741.6, 10904]

5742.33

4819.71

4557.40

5757.75 9109.28

4564.952111.82 2874.41

5216.334533.963390.85

1957.0 3810.8 5664.6 7518.4 9372.2 11226.0

Mass (m/z)

0

1.1E+4

0

10

20

30

40

50

60

70

80

90

100

% In

tensi

ty

Voyager Spec #1=>BC=>NR(2.00)[BP = 5741.6, 10904]

5742.33

4819.71

4557.40

5757.75 9109.28

4564.952111.82 2874.41

5216.334533.963390.85

Expression of tj-EGFR in pET Expression of tj-EGFR in pET 27b+ 27b+

E.coliE.coli BL21(DE3) Codon Plus RP BL21(DE3) Codon Plus RP

Expression of tj-EGFR in pET Expression of tj-EGFR in pET 27b+ 27b+

E.coliE.coli BL21(DE3) Codon Plus RP BL21(DE3) Codon Plus RP

m – markerm – markerb – before inductionb – before inductionredred – at +37 – at +37°C / °C / blueblue – at + – at + 28°C28°C

4 – 4 hours after induction4 – 4 hours after induction16 – 16 hours after 16 – 16 hours after inductioninduction24 – 24 hours after 24 – 24 hours after inductioninduction

1515

1010

2020

2020

1010

Strep-Strep-blotblot His-blotHis-blot

m b m b 4 16 244 16 24 m b m b 4 16 244 16 24 m b m b 4 16 244 16 24 m b m b 4 16 244 16 24

The optimal expression conditions for tj-EGFR are:The optimal expression conditions for tj-EGFR are:+28+28°C, 24 hours°C, 24 hours

Purification of tj-EGFR in OG on Purification of tj-EGFR in OG on Chelating and Strep-Tactin Chelating and Strep-Tactin

SepharoseSepharose

Purification of tj-EGFR in OG on Purification of tj-EGFR in OG on Chelating and Strep-Tactin Chelating and Strep-Tactin

SepharoseSepharose

SDS-PAGE His-blot Strep-BlotSDS-PAGE His-blot Strep-Blot

open open cellscells

chelating chelating CuCu2+2+

Strep-Strep-TactinTactin

RPCRPC1515

1010

2020

tj-EGFR can be purified to tj-EGFR can be purified to homogeneityhomogeneity

MALDI-TOF analysis of tj-MALDI-TOF analysis of tj-EGFREGFR

MALDI-TOF analysis of tj-MALDI-TOF analysis of tj-EGFREGFR

999.0 3799.4 6599.8 9400.2 12200.6 15001.0

Mass (m/z)

0

7313.8

0

10

20

30

40

50

60

70

80

90

100

% I

nte

ns

ity

Voyager Spec #1=>RSM2000[BP = 10161.8, 7314]

10161.32

10217.60

5077.00

10274.985104.76

10057.235135.56 10028.83

4954.85 10013.10

4428.86

999.0 3799.4 6599.8 9400.2 12200.6 15001.0

Mass (m/z)

0

7313.8

0

10

20

30

40

50

60

70

80

90

100

% I

nte

ns

ity

Voyager Spec #1=>RSM2000[BP = 10161.8, 7314]

10161.32

10217.60

5077.00

10274.985104.76

10057.235135.56 10028.83

4954.85 10013.10

4428.86

(many thanks to Axel Niebisch, (many thanks to Axel Niebisch, IBT-1)IBT-1)

Only full-length tj-EGFR is observed: no degradation productsOnly full-length tj-EGFR is observed: no degradation products

CD spectra of tj-EGFR and CD spectra of tj-EGFR and secondary structure predictions: secondary structure predictions:

water and TFEwater and TFE

CD spectra of tj-EGFR and CD spectra of tj-EGFR and secondary structure predictions: secondary structure predictions:

water and TFEwater 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 CD spectra of tj-EGFR and secondary structure predictions: secondary structure predictions:

detergentsdetergents

CD spectra of tj-EGFR and CD spectra of tj-EGFR and secondary structure predictions: secondary structure predictions:

detergentsdetergents

50 mM NaP pH 50 mM NaP pH 6.0,6.0,100 mM 100 mM detergentdetergent

α-helix β-sheet turn random

OG 21% 28% 23% 28%

SDS 21% 29% 20% 30%

DPC 32% 21% 20% 27%

DHPC 23% 25% 22% 30%

LPPG 25% 26% 19% 30%

~ 60% ~ 60% αα-helix and ~ 40% random coil-helix and ~ 40% random coil

Sequence-basedSequence-basedsecondary structure secondary structure

predictionspredictions

Sequence-basedSequence-basedsecondary structure secondary structure

predictionspredictions

MHHHHHHHGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEWSHPQFEK

There is a discrepancy between the experimental and There is a discrepancy between the experimental and predicted secondary structure structure contentpredicted secondary structure structure content

NMR spectra of tj-EGFR in SDS and NMR spectra of tj-EGFR in SDS and DPCDPC

NMR spectra of tj-EGFR in SDS and NMR spectra of tj-EGFR in SDS and DPCDPC

2D HSQC NMR spectra look promising in terms of peak 2D HSQC NMR spectra look promising in terms of peak assignmentassignment

10 10 mM NaP pH 6.0 + mM NaP pH 6.0 + SDSSDS

10 10 mM NaP pH 6.0 + mM NaP pH 6.0 + DPCDPC

Conclusions, partConclusions, part 2 2Conclusions, partConclusions, part 2 2

The transmembrane The transmembrane + + juxtamembrane domainjuxtamembrane domain of EGFRof EGFR fromfrom HH.sapiens.sapiens ( (tjtj--EGFREGFR) can be expressed in ) can be expressed in E.coliE.coli and and then successfully purifiedthen successfully purified

tj-EGFR is prone to oligomerization/aggregationtj-EGFR is prone to oligomerization/aggregation

The secondary structure of tj-EGFR is almost independent The secondary structure of tj-EGFR is almost independent of the type of detergentof the type of detergent

The tertiary structure of tj-EGFR strongly depends on the The tertiary structure of tj-EGFR strongly depends on the type of detergent, e.g. the presence of charged headstype of detergent, e.g. the presence of charged heads

AcknowledgementsAcknowledgementsAcknowledgementsAcknowledgements

FIRST FIRST mymy BIG BOSSES BIG BOSSES:: Prof. Judith Klein-SeetharamanProf. Judith Klein-Seetharaman ( (University of PittsburghUniversity of Pittsburgh)) Prof. Georg Büldt (Forschungszentrum JProf. Georg Büldt (Forschungszentrum Jülich, IBI-2ülich, IBI-2)) Dr. Ramona Schlesinger (Forschungszentrum JDr. Ramona Schlesinger (Forschungszentrum Jülich, IBI-ülich, IBI-

22)) Dr.Dr. Valentin Gordeliy Valentin Gordeliy ((MIPTMIPT))

... and then my ... and then my NICE COLLEAGUESNICE COLLEAGUES:: Dr.Dr. Olga Mironova (HtrII-cyt, cloning & purification)Olga Mironova (HtrII-cyt, cloning & purification) Vijayalaxmi ManoharanVijayalaxmi Manoharan ( (HtrII-cyt, NMRHtrII-cyt, NMR)) Naveena YanamalaNaveena Yanamala ( (tjtj--EGFR, NMREGFR, NMR)) Prof. Joe Zaccai and Dr. Vitaliy PipichProf. Joe Zaccai and Dr. Vitaliy Pipich ( (HtrII-cyt, SANSHtrII-cyt, SANS))

What is yet to be done?What is yet to be done?– I’m not leaving you right now!– I’m not leaving you right now!

What is yet to be done?What is yet to be done?– I’m not leaving you right now!– I’m not leaving you right now!

HtrII-cyt project:HtrII-cyt project:- finalize the papers;finalize the papers;- mutagenesis (if necessary);mutagenesis (if necessary);- try to obtain diffracting crystals.try to obtain diffracting crystals.

EGFR project:EGFR project:- tj-EGFR: final CD in lipids;tj-EGFR: final CD in lipids;- tj-EGFR: cross-linking in lipids and detergents;tj-EGFR: cross-linking in lipids and detergents;- write up the paper;write up the paper;- prepare 13C, 15N sample (if necessary);prepare 13C, 15N sample (if necessary);

- N-EGFR: express in COS-1 and develop purification N-EGFR: express in COS-1 and develop purification strategy.strategy.