Transcript Structural analysis of GARP

Structural Analysis using
NMR
Naveena Sivaram
Research Report # 5
Overview
• NMR studies were performed in
– Peripherin peptides
– Epidermal Growth factor Receptor
– Transducer
• Results
• Conclusions & Outlook
Interaction GARP2/Peripherin
Peripherin
GARP2
A1
GARP´
B1a
A1
A1
Disc
Peripherin/rds (retinal degeneration slow):
• highly conserved in both rod and cone photoreceptors of all vertebrates
• 4 TM glycoprotein (39 kDa) present in photoreceptor outer segment discs
• forms homodimers in rods (covalently bonded), heterodimers with ROM-1
• are located at the disc rim and may play a role in anchoring the
disc to the cytoskeletal system of the outer segment
Taken from Karin Presentation
Peripherin peptides
P3
P1
P2
Cytosol
Intradiscal space
Taken from Karin Presentation
Peripherin peptides
Measured
TOCSY, COSY, ROESY/NOESY,15N & 13C HSQC
P1:
ALLKVKFDQKKRVKLAQG
COSY & 13C HSQC
aa position in Protein: 1-18
P2:
KICYDALDPAKYAKWKPWLKPY
15N HSQC & 13C HSQC
aa position in Protein: 79-100
P3:
RYLHTALEGMANPEDPECESEGWLLEKSVPETWKAFLESVKKLGKGNQVEAEGED
AGQAPAAG
aa position in Protein: 283-345
P3A:
RYLHTALEGMANPEDPECESEGWLL
aa position in Protein: 283-308
P3B:
P
KSVPETWKAFLESVKKLGKGNQVEAEGEDAGQAPAAG Only TOCSY & ROESY
aa position in Protein: 309-345
Peripherin peptides
Measured
TOCSY, COSY, ROESY/NOESY,15N & 13C HSQC
P3AS:
(mixed) RYLHTALEGMANPEDPECESEGWLL TOCSY, ROESY & COSY
aa position in Protein: 283-308
P3BS:
(mixed)KSVPETWKAFLESVKKLGKGNQVEAEGEDAGQAPAAG
aa position in Protein: 309-345
P1:
ALLKVKFDQKKRVKLAQG
15N HSQC
aa position in Protein: 1-18
R2:
P
VLTWLRKGVEKVVPQPA
aa position in Protein: 100-116
Missing Experiments :
P3AS : 15N and 13C – HSQC’s
P3B : COSY,15N and 13C – HSQC’s
P3A : Have to rerun everything
COSY ( cosydfesgpph )
• COrrelation SpectroscopY
KICYDALDPAKYAKWKPWLKPY
• Each pair of coupled spins shows up
as a cross-peak in a 2D COSY
spectrum.
•
The diagonal peaks correspond to
the 1D spectrum.
• Cross peaks are useful for assigning
individual amino acid “spin systems”
TOCSY ( dipsi2esgpph )
• Total Correlation Spectroscopy
• Relies on scalar or J couplings
• J coupling between nuclei that are
more than 3 bond lengths away is
very weak
• Number of protons that can be
linked up in a 2D TOCSY spectrum
is therefore limited to all those
protons within an amino acid
KICYDALDPAKYAKWKPWLKPY
ROESY/NOESY ( noesyesgpph )
• Nuclear Overhauser Enhancement
Spectroscopy
• Each cross peak in a NOESY
spectrum indicates that the nuclei
resonating at the 2 frequencies are
within 5 Å in space.
• Intensity of cross peaks is related to
internuclear distance
KICYDALDPAKYAKWKPWLKPY
HSQC
• Heteronuclear Single-Quantum Coherence
• spectrum contains the signals of the HN protons
in the protein backbone
• Each signal in a HSQC spectrum represents a
proton that is bound to a nitrogen atom
• use of these hetero nuclei facilitates the
structure determination
• 15N – HSQC (fhsqcf3gpph) and 13C – HSQC (
hsqcetgpsi2 )
HSQC Spectra
KICYDALDPAKYAKWKPWLKPY
ALLKVKFDQKKRVKLAQG
Figure
A: 1H,15N-HSQC Spectrum of Peptide P1
B: 1H,13C-HSQC Spectrum of Peptide P2
Per_P1 & Garp_R2 interaction
Peptide P1 (1.5mM)
G18
Peptide P1 + R2 (0.7mM)
Contd…
B.
A.
Figure
A: P1 overlapped on P1R2 15N-HSQC Spectrum
B: 15N-HSQC Spectrum of Peptide R2 (Karin)
Conclusions
• Spectra obtained show well resolved
resonances - teritiary structure
• Chemical shifts of two residues in P1 have
shown to shift by more than 0.05 ppm in
15N dimension
Future Work
• Running the missing expt’s to get the
complete data for all Peripherin Peptides
• Analysing chemical shifts and determining
the structure for the Peripherin Peptides
• Trying out the different combinations of
Peripherin and GARP Peptides
Epidermal Growth Factor Receptor (EGFR)
the transmembrane + juxtamembrane domains
151
L1
312
CR1
481
L2
621
CR2
687
JM
955
Kinase
1186
CT
644
Extracellular portion
Intracellular portion
The transmembrane + juxtamembrane part (615-686 a.a. + N-terminal
7His-tag) contains the transmembrane and the regulatory
juxtamembrane (JM) domain
615 – MHHHHHHH
GPKIPSIATGMVGALLLLLVVALGIGFMRRRHIVRKRTLRRLLQER
ELVEPLTPSGEAPNQALLRILKETE-686
Resource from Ivan’s Presentation
Figure : EGFR-EGF complex view with the two-fold axis oriented vertically (taken
from den Hartigh JC etal,J Cell Biol 1992 ). Domains I and III correspond to L1 and
L2, domains II and IV - to CR1 and CR2, respectively.
Important information about the tj-EGFR
•
•
•
•
•
73 amino acid residues (without tag)
carries N-terminal 7His-tag
molecular weight is about 9,112 Da
contains no Cys residues
contains no aromatic residues (Trp, Tyr or Phe)
• NMR structure of the juxtamembrane domain is
available
Choowongkomon et al. (2005), J. Biol. Chem.
Resource from Ivan’s Presentation
Extracellular portion
Intracellular po
NMR Studies
615-MHHHHHHH
GPKIPSIATGMVGALLLLLVVALGIGLFMRR
VRKRTLRRLLQERELVEPLTPSGEAPNQA
LKETE-686
• 15N HSQC(fhsqcf3gpph)
B.
O
OH
O
HO
octyl glucoside
OH
O
N+
O
• 2D HET-NOE
P
O
O-
dodecyl phosphocholine
O
O
• 3D NOE
HO
– OG
– 1%SDS
– 2.5%SDS
– 5%SDS
S
O- +
Na
O
sodium dodecyl sulfate
Choowongkomon et al. (2005), J. Biol. Chem.
15N HSQC in OG
G
K
Figure : 1H,15N-HSQC
spectrum of the
transmembrane+juxtame
mbrane fragment in 50
mM NaPi pH 6.0, 10%
D2O, 5% octyl glucoside
15N HSQC in OG + 1% SDS
G
K
Figure : 1H,15N-HSQC
spectrum of the
transmembrane+juxtame
mbrane fragment in 50
mM NaPi pH 6.0, 10%
D2O, 1% sodium dodecyl
sulfate
Comparison of OG & 1% SDS
Histidines
R?
juxtamembrane domain NMR
studies
In H2O
In Phosphocholine
Choowongkomon et al. (2005), J. Biol. Chem.
Conclusions
• 1H,15N HSQC studies in OG shows limited spectral
dispersion suggesting little stable tertiary structure
• 1H,15N-HSQC spectrum in OG has a qualitatively
similar appearance as the one in phosphocholine
• In the presence of SDS, the spectral dispersion
significantly increased
• Increasing in SDS concentrations after some point did
not show significant effect
• Quick analyses of chemical shifts suggested that some
of the new peaks in HSQC are from H’s and R’s
Future Work
• Analysing chemical shifts inorder to quantify the
claim of increase in spectral dispersion induced
by SDS compared to that of OG sample and to
find ideal SDS concentration
• Analyzing & Assigning of the resonance peaks in
1H,15N-HSQC spectrum of tj-hegfr sample in
SDS, to find out if the new peaks in the spectrum
are resulting from the +vely charged residues
Transducer in N.Pharaonis
• Phototaxis system is a complex
consisting of the Sensory
rhodopsin II (SRII) and the
transducer protein HtrII
• Light-activation of SRII induces
structural changes in HtrII
– 2-helical membrane protein
with a long cytoplasmic
extension
– structure of cytoplasmic
fragment of HtrII (HtrII-cyt),
playing an important role in
information relay, remains
unknown
NMR Studies
• 1H-15N HSQC – fhsqcf3gpph
• 1H-15N HSQC (Ammonium Sulphate)
• 1H-15N HSQC (Ammonium Sulphate)
– 20oC
– 37oC
– 8oC
– 2oC
HtrII_15N HSQC
Figure : 1H,15N-HSQC spectrum of the htrII fragment in 20 mM NaPi pH 6.0,
10% D2O
HtrII_15N HSQC(Ammonium Sulphate)
Figure : 1H,15N-HSQC spectrum of the htrII fragment in 20 mM NaPi pH 6.0,
10% D2O & 5% Ammonium Sulfate.
Conclusions
• Observed that the signals intensities were
varying under different buffer conditions
• The high peak intensities suggests that
their be a localized structure
• 1H,15N-HSQC spectrum performed at
different temparatures suggest that the
transducer may not be in an aggregated
state
Future Work
• Analysis and investigation of AA involved
in changes and their occurrence in the
crystal structure
• Changes in spectrum and chemical shifts
at different temperatures
Acknowledgements
• Judith Klein-Seetharaman
• Karin Abarca Heidemann
• Ivan Budyak
• David Man