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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 dodecyl phosphocholine OO 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