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Structural Analysis using NMR

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highly conserved in both rod and cone photoreceptors of all vertebrates ... Phototaxis system is a complex consisting of the Sensory rhodopsin II (SRII) and ... – PowerPoint PPT presentation

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Title: Structural Analysis using NMR


1
Structural Analysis using NMR
  • Naveena Sivaram
  • Research Report 5

2
Overview
  • NMR studies were performed in
  • Peripherin peptides
  • Epidermal Growth factor Receptor
  • Transducer
  • Results
  • Conclusions Outlook

3
Interaction GARP2/Peripherin
  • 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
4
Peripherin peptides
5
Peripherin peptides
Measured TOCSY, COSY, ROESY/NOESY,15N 13C HSQC
COSY 13C HSQC
P1 ALLKVKFDQKKRVKLAQG aa position in Protein
1-18 P2 KICYDALDPAKYAKWKPWLKPY aa position in
Protein 79-100 P3 RYLHTALEGMANPEDPECESEGWLLEKSV
PETWKAFLESVKKLGKGNQVEAEGED AGQAPAAG aa position
in Protein 283-345 P3A RYLHTALEGMANPEDPECESEGWL
L aa position in Protein 283-308
P3B KSVPETWKAFLESVKKLGKGNQVEAEGEDAGQAPAAG aa
position in Protein 309-345
15N HSQC 13C HSQC
P
Only TOCSY ROESY
6
Peripherin peptides
Measured TOCSY, COSY, ROESY/NOESY,15N 13C HSQC
P3AS (mixed) RYLHTALEGMANPEDPECESEGWLL aa
position in Protein 283-308 P3BS (mixed)KSVPET
WKAFLESVKKLGKGNQVEAEGEDAGQAPAAG aa position in
Protein 309-345
TOCSY, ROESY COSY
P
P1 ALLKVKFDQKKRVKLAQG aa position in Protein
1-18 R2 VLTWLRKGVEKVVPQPA aa position in
Protein 100-116
15N HSQC
  • Missing Experiments
  • P3AS 15N and 13C HSQCs
  • P3B COSY,15N and 13C HSQCs
  • P3A Have to rerun everything

7
COSY ( cosydfesgpph )
  • COrrelation SpectroscopY
  • 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

KICYDALDPAKYAKWKPWLKPY
8
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
9
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
10
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 )

11
HSQC Spectra
Figure A 1H,15N-HSQC Spectrum of Peptide P1 B
1H,13C-HSQC Spectrum of Peptide P2
12
Per_P1 Garp_R2 interaction
Peptide P1 (1.5mM)
Peptide P1 R2 (0.7mM)
G18
13
Contd
B.
A.
Figure A P1 overlapped on P1R2 15N-HSQC Spectrum
B 15N-HSQC Spectrum of Peptide R2 (Karin)
14
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

15
Future Work
  • Running the missing expts 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

16
Epidermal Growth Factor Receptor (EGFR)the
transmembrane juxtamembrane domains
The transmembrane juxtamembrane part (615-686
a.a. N-terminal 7His-tag) contains the
transmembrane and the regulatory juxtamembrane
(JM) domain
615 MHHHHHHH GPKIPSIATGMVGALLLLLVVALGIGFMRRRHIVR
KRTLRRLLQERELVEPLTPSGEAPNQALLRILKETE-686
Resource from Ivans Presentation
17
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.
18
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 Ivans Presentation
19
NMR Studies
  • 15N HSQC(fhsqcf3gpph)
  • OG
  • 1SDS
  • 2.5SDS
  • 5SDS
  • 2D HET-NOE
  • 3D NOE

Choowongkomon et al. (2005), J. Biol. Chem.
20
15N HSQC in OG
G
K
Figure 1H,15N-HSQC spectrum of the
transmembranejuxtamembrane fragment in 50 mM
NaPi pH 6.0, 10 D2O, 5 octyl glucoside
21
15N HSQC in OG 1 SDS
G
K
Figure 1H,15N-HSQC spectrum of the
transmembranejuxtamembrane fragment in 50 mM
NaPi pH 6.0, 10 D2O, 1 sodium dodecyl sulfate
22
Comparison of OG 1 SDS
Histidines
R ?
23
juxtamembrane domain NMR studies
In H2O
In Phosphocholine
Choowongkomon et al. (2005), J. Biol. Chem.
24
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 Hs and Rs

25
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

26
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

27
NMR Studies
  • 1H-15N HSQC fhsqcf3gpph
  • 1H-15N HSQC (Ammonium Sulphate)
  • 1H-15N HSQC (Ammonium Sulphate)
  • 20oC
  • 37oC
  • 8oC
  • 2oC

28
HtrII_15N HSQC
Figure 1H,15N-HSQC spectrum of the htrII
fragment in 20 mM NaPi pH 6.0, 10 D2O
29
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.
30
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

31
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

32
Acknowledgements
  • Judith Klein-Seetharaman
  • Karin Abarca Heidemann
  • Ivan Budyak
  • David Man

33
Thank You
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