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