Structural Analysis using NMR

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Structural Analysis using NMR

• Naveena Sivaram
• Research Report 5

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Overview

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

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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
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Peripherin peptides
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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
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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

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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
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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
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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
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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 )

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

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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

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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
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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.
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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
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NMR Studies

• 15N HSQC(fhsqcf3gpph)
• OG
• 1SDS
• 2.5SDS
• 5SDS
• 2D HET-NOE
• 3D NOE

Choowongkomon et al. (2005), J. Biol. Chem.
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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
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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
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Comparison of OG 1 SDS
Histidines
R ?
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juxtamembrane domain NMR studies
In H2O
In Phosphocholine
Choowongkomon et al. (2005), J. Biol. Chem.
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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

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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

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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

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NMR Studies

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

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

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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

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Acknowledgements

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

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Thank You