Structural Analysis using NMR Naveena Sivaram Research Report # 5.
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Transcript of Structural Analysis using NMR Naveena Sivaram Research Report # 5.
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
Disc
GARP2PeripherinPeripherinPeripherin
A1
A1
A1
B1aGARP´
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
Peripherin peptides
P1 P2
P3
Intradiscal space
Cytosol
Taken from Karin Presentation
P1: ALLKVKFDQKKRVKLAQGaa position in Protein: 1-18
P2: KICYDALDPAKYAKWKPWLKPYaa position in Protein: 79-100
P3: RYLHTALEGMANPEDPECESEGWLLEKSVPETWKAFLESVKKLGKGNQVEAEGEDAGQAPAAG
aa position in Protein: 283-345
P3A: RYLHTALEGMANPEDPECESEGWLLaa position in Protein: 283-308
P3B: KSVPETWKAFLESVKKLGKGNQVEAEGEDAGQAPAAGaa position in Protein: 309-345
Peripherin peptidesMeasured
TOCSY, COSY, ROESY/NOESY,15N & 13C HSQC
Only TOCSY & ROESY
COSY & 13C HSQC
15N HSQC & 13C HSQC
P3AS: (mixed) RYLHTALEGMANPEDPECESEGWLLaa position in Protein: 283-308
P3BS: (mixed)KSVPETWKAFLESVKKLGKGNQVEAEGEDAGQAPAAGaa position in Protein: 309-345
Peripherin peptidesMeasured
TOCSY, COSY, ROESY/NOESY,15N & 13C HSQC
TOCSY, ROESY & COSY
P1: ALLKVKFDQKKRVKLAQGaa position in Protein: 1-18
R2: VLTWLRKGVEKVVPQPAaa 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
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
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 )
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
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 SpectraKICYDALDPAKYAKWKPWLKPY
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) Peptide P1 + R2 (0.7mM)
G18
Contd…A.
Figure
A: P1 overlapped on P1R2 15N-HSQC Spectrum
B: 15N-HSQC Spectrum of Peptide R2 (Karin)
B.
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
L1 CR1 L2 CR2 JM Kinase CT
644
151 312 481 621 687 955 1186
Extracellular portion Intracellular portion
L1 CR1 L2 CR2 JM Kinase CT
644
151 312 481 621 687 955 1186
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
Epidermal Growth Factor Receptor (EGFR)the transmembrane + juxtamembrane domains
Resource from Ivan’s Presentation
615 – MHHHHHHH GPKIPSIATGMVGALLLLLVVALGIGFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETE-686
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.
• 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 availableChoowongkomon et al. (2005), J. Biol. Chem.
Important information about the tj-EGFR
Resource from Ivan’s Presentation
NMR Studies
• 15N HSQC(fhsqcf3gpph)– OG– 1%SDS– 2.5%SDS – 5%SDS
• 2D HET-NOE
• 3D NOE
F. G.
D. E. L1 CR1 L2 CR2 JM Kinase CT
644
151 312 481 621 687 955 1186
Extracellular portion Intracellular portion
L1 CR1 L2 CR2 JM Kinase CT
644
151 312 481 621 687 955 1186
Extracellular portion Intracellular portion
L1 CR1 L2 CR2 JM Kinase CT
644
151 312 481 621 687 955 1186
Extracellular portion Intracellular portion
L1 CR1 L2 CR2 JM Kinase CT
644
151 312 481 621 687 955 1186
Extracellular portion Intracellular portion
A.
B.
15 N
(ppm
)
15 N
(ppm
)
C. O
OOH
OH HO
HO
octyl glucoside
dodecyl phosphocholine
OP
O-
O
ON+
sodium dodecyl sulfate
OS
O-
O
O
Na+
615-MHHHHHHHGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVRKRTLRRLLQERELVEPLTPSGEAPNQALLRILKETE-686
Choowongkomon et al. (2005), J. Biol. Chem.
15N HSQC in OG
Figure : 1H,15N-HSQC spectrum of the transmembrane+juxtamembrane fragment in 50 mM NaPi pH 6.0, 10% D2O, 5% octyl glucoside
G
K
15N HSQC in OG + 1% SDS
Figure : 1H,15N-HSQC spectrum of the transmembrane+juxtamembrane fragment in 50 mM NaPi pH 6.0, 10% D2O, 1% sodium dodecyl sulfate
G
K
Comparison of OG & 1% SDS
R ?
Histidines
juxtamembrane domain NMR studies
Choowongkomon et al. (2005), J. Biol. Chem.
In H2O In Phosphocholine
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
• Judith Klein-Seetharaman
• Karin Abarca Heidemann
• Ivan Budyak
• David Man
Acknowledgements