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 P1P2 P3 Intradiscal space Cytosol Taken from Karin Presentation

5. P1: ALLKVKFDQKKRVKLAQG aa position in Protein: 1-18 P2: KICYDALDPAKYAKWKPWLKPY aa position in Protein: 79-100 P3: RYLHTALEGMANPEDPECESEGWLLEKSVPETWKAFLESVKKLGKGNQVEAEGED AGQAPAAG aa position in Protein: 283-345 P3A: RYLHTALEGMANPEDPECESEGWLL aa position in Protein: 283-308 P3B: KSVPETWKAFLESVKKLGKGNQVEAEGEDAGQAPAAG aa position in Protein: 309-345 Peripherin peptides Measured TOCSY, COSY, ROESY/NOESY,15N & 13C HSQC  Only TOCSY & ROESY COSY & 13C HSQC 15N HSQC & 13C HSQC

6. P3AS:(mixed) RYLHTALEGMANPEDPECESEGWLL aa position in Protein: 283-308 P3BS:(mixed) KSVPETWKAFLESVKKLGKGNQVEAEGEDAGQAPAAG aa position in Protein: 309-345 Peripherin peptides Measured TOCSY, COSY, ROESY/NOESY,15N & 13C HSQC  TOCSY, ROESY & COSY P1: ALLKVKFDQKKRVKLAQG aa position in Protein: 1-18 R2:VLTWLRKGVEKVVPQPA aa position in Protein: 100-116 15N HSQC Missing Experiments :  P3AS : 15N and 13C – HSQC’s  P3B : COSY,15N and 13C – HSQC’s  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 ) KICYDALDPAKYAKWKPWLKPY 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

10. HSQC Heteronuclear Single-Quantum Coherence spectrum contains the signals of the H N 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 15 N – HSQC (fhsqcf3gpph) and 13 C – HSQC ( hsqcetgpsi2 )

11. HSQC Spectra KICYDALDPAKYAKWKPWLKPY ALLKVKFDQKKRVKLAQG 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… A. Figure A: P1 overlapped on P1R2 15N-HSQC Spectrum B: 15N-HSQC Spectrum of Peptide R2 (Karin) B.

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 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

16. L1CR1L2CR2 JM KinaseCT 644 1513124816216879551186 Extracellular portionIntracellular portion L1CR1L2CR2 JM KinaseCT 644 1513124816216879551186 Extracellular portionIntracellular portion The transmembrane + juxtamembrane part (615-686 a.a. + N-terminal 7His-tag) contains the transmembrane and the regulatory juxtamembrane (JM) domain Epidermal Growth Factor Receptor (EGFR) the transmembrane + juxtamembrane domains Resource from Ivan’s Presentation 615 – MHHHHHHH GPKIPSIATGMVGALLLLLVVALGIGFMRRRHIVRKRTLRRLLQER ELVEPLTPSGEAPNQALLRILKETE-686

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. 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. Important information about the tj-EGFR Resource from Ivan’s Presentation

19. NMR Studies 15N HSQC(fhsqcf3gpph) –OG –1%SDS –2.5%SDS –5%SDS 2D HET-NOE 3D NOE Choowongkomon et al. (2005), J. Biol. Chem.

20. 15N HSQC in OG Figure : 1H,15N-HSQC spectrum of the transmembrane+juxtame mbrane fragment in 50 mM NaPi pH 6.0, 10% D2O, 5% octyl glucoside G K

21. 15N HSQC in OG + 1% SDS Figure : 1H,15N-HSQC spectrum of the transmembrane+juxtame mbrane fragment in 50 mM NaPi pH 6.0, 10% D2O, 1% sodium dodecyl sulfate G K

22. Comparison of OG & 1% SDS R ? Histidines

23. juxtamembrane domain NMR studies Choowongkomon et al. (2005), J. Biol. Chem. In H 2 O In Phosphocholine

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 H’s and R’s

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) –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. Judith Klein-Seetharaman Karin Abarca Heidemann Ivan Budyak David Man Acknowledgements