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  • Incidently, if you are trying to get a visa to go to the states, don’t say you’re a nuclear physicist…they don’t understand
  • In part to develop a protocol for determing structures in the Sykes group
  • The structures were all aligned by their secondary structural elements (residues 5-10,15-27, 35-37, 40-48, 54-64, 71-73, and 64-86). The structures overlaid are:
  • Transcript

    • 1. cTnC-L29Q Does a Leu to Gluheart function undo?
    • 2. Topics• Introduce NMR spectroscopy• Review L29Q history and literature• Structure calculation by NMR• Dynamics measurement by NMR• Conclusions
    • 3. What is NMR?• Nuclear Magnetic Resonance spectroscopy• Similar to other forms of spectroscopy – A photon of light causes a transition from a ground state to an excited state• In visible spectroscopy an electron absorbs the energy• In NMR, the absorbed photon promotes a transition of nuclear spin from ground to excited state
    • 4. What is NMR?• Lifetime is ~109 times longer than conventional spectroscopies• Ground and excited states in NMR arise from the interaction of a nuclear magnetic dipole moment with an intense external magnetic field• The magnetic dipole arises from spin angular momentum – The spin angular momentum of a ½ nuclei can be either: +½ħ or -½ħ – The magnetic moment of s nuclear spin is proportional to its gyromagnetic ratio (γ)
    • 5. What is NMR?• As the strength of the field increases so does the energy separation • The net absorption depends on population difference • Since NMR is insensitive need lots of material (i.e. mM concentrations) • Going from 14.1 T (600 MHz) to 21.2 T (900 MHz) increases the ∆E = (h / 2π )γ Bo signal to noise by ca. 84% and even from 18.8 T (800 MHz) to Nβ (h / 2π )γ Bo 900 MHz increases it by 20% ≈ 1− Nα kT S / N ∝ B0 3/2
    • 6. What is NMR?• In a magnetic field nuclei precess about B0 at the resonance frequency (600 MHz = γ(1H atoms)*14.1 Tesla)• Pulse sample with a second magnetic field oscillating at the resonance frequency perpendicular to B0• Spins precess about B0 at their resonance frequency (bulk is in the transverse plane)• Measure the evolution speed of the spins (chemical shift; represented by p.p.m., but really defined as hz/mhz) – 10 ppm in a 600 MHz instrument represents 6000 Hz off from 600 MHz
    • 7. What is NMR?• Coupling: if nucleus A is near another nonequivalent nucleus B than when nucleus B is +½ and -½ nucleus A will experience different magnetic fields, and thus will have different chemical shifts• J-coupling: through bonds• Dipolar coupling: through space
    • 8. The N-HSQC15• 1 H , 15N-HSQC correlates amide 1H with amide 15N• Spectra will change if magnetic environment changes• Can be used to obtain binding constants and predict binding sites
    • 9. First FHC mutation in cTnC• In 2001, Hoffmann B, et al. identified in a 60 year old male patient – ECG revealed he had concentric hypertrophy of the left ventricle• Did not find it in 96 healthy volunteers, but authors were not willing to rule it out as “simple coincidence”• L29 serves to stabilize the A- helix Hoffmann B, et al. (2001) Human Mutation 17, 524
    • 10. Function of L29• Differences in chemical shift of cTnC when cTnI1-80DD vs. cTnI1-80• L29 may be involved in binding to the cardiac specific N-terminal extension of cTnIFinley et al., 1999
    • 11. Function of L29• Deletion of 16-29 mimic phosphorylated state of contraction (Ward et al. 2002)• Cross-linking implicate cTnI1-64 interacts with I18C and R26C to cTnC (Ward et al. 2003)• Ward et al. (2004a) proved by looking at cTnI1-64 NMR spectrum that when it is bisphosphorylated it does not bind to cNTnC, but does so when unphosphorylated – Observed by monitoring broadening of 1D signals of cTnI1-64 as cNTnC was titrated in – Binds via Y25, Y28, and H33 of cTnI• Ward et al. (2004b) used 15N-HSQC data of cTnI1-64 to show that residues that flank the S22 and S23 are less perturbed by cTnC when phosphorylated
    • 12. Rosevear/Solaro Model• Rosevear and Solaro (Howarth et al., 2007) solved the NMR structure of cTnI1-32pp and proposed a mechanism – Model suggested that R21 and R27 of cTnI interacts with E32 and D33 in site I and P11 forms a hydrophobic interaction with L29• Model also supported by cross-linking data(Warren et al. 2009 – Also implicates cTnI147-163 (bound to cNTnC) as a binding partner of the N-terminal extension of cTnI
    • 13. Back to L29Q (Jaquet) • Signal was reduced by ca. 14% at 208 and 222 nm. • Results suggested that secondary structure contained ~2% less alpha helix for both apo and Ca2+ bound • Found by peptide arrays that L29Q did not bind the N- terminal extension of cTnI, regardless of phosphorylation level (wt did, except for cTnIpp)Schmidtmann A, et al. (2005) FEBS J. 6087-6097
    • 14. Schmidtmann A, et al. Continued• ATPase assays and in vitro motility assays• pCa50 of L29Q was reduced when compared to WT (by ca. 0.1 units)• Found that phosphorylation had less of an impact on L29Q than WT
    • 15. L29Q (Cheung)• FRET measurements in cTnC(L12W/N51C- IAEDANS) reconstituted thin filaments• No structural change in L29Q versus WT• Calcium sensitivity decreased for L29Q by 0.1 unit• No further decrease as a function of phosphorylation – Whereas wt decreased by approximately 0.2 unitsDong, W-J, et al. (2008) JBC 3424-3432
    • 16. L29Q (Sykes)• No affect on Calcium binding• cTnI147-163 affinity was not altered by cTnI1-29 or cTnI-pp – Not true for WT-cTnC (as shown by OKB and Abbott et al)• And relaxation studies indicated that cTnI1-29 bound less efficiently to L29Q than WTBaryshnikova, O, et al. (2008) JMB 735-751
    • 17. L29Q (trout cardiac troponin C)• Trout troponin has an increased calcium affinity (2-3 fold) – Residues responsible are: N2, I28, Q29 and D30 (Gillis et al., 2005) – Human cardiac cTnC: D2, V28, L29, G30 – When cardiac contained these residues Ca sensitivty increased by 2-fold• Coordinate a second calcium weakly? – Not actually observed experimentally – Structure not much different than human cardiac (Blumenschein et al., 2004)• Trout cardiac troponin I lacks the N-terminal extension – Found that trout cTn is less sensitive to PKA than human cTn (Kirkpatrick et al., 2011)
    • 18. L29Q (Davis and Tibbits)• Florescence Measurements: – Half maximal Ca2+ for cTnCF27W: 3.7 ± 0.2 μM – L29Q: 2.8 ± 0.3 μM – NIQD: 2.0 ± 0.1 μM• Force pCa curves of skinned murine cardiomyocytes – WT: EC50 = 4.1 ± 0.5 μM – L29Q: EC50 = 3.0 ± 0.5 μM – NIQD: EC50 = 2.1 ± 0.5 μM• Stress that skinned cardiomyocytes are a better representation of reality than isolated thin filamentsLiang, B, et al. (2008) Physiol Genomics 257-266
    • 19. L29Q (Potter)• Did not see a statistically significant increase in calcium sensitivity with skinned fibers, cardiac myofibrils, or regulated thin filaments (fluorescence) – although all had a “trend” towards a slight increase in calcium sensitivity• Porcine instead of murine muscle• Both Potter and Davis not controlling for phosphorylation levels, so may explain differencesDweck, D, et al. (2008) JBC 33119-33128
    • 20. L29Q (Pfitzer)• pCa50 unaffected by L29Q• Nor did PP1c treatment followed by PKA treatment yield any differences between wt and L29Q• Not just phosphorylating S22/S23 anymore…• Unfortunately, they do not address differences between their results and Davis’s; actually they mention them as if they agree!Neulen, A, et al. (2009) Basic Res Cardiol 751-760
    • 21. Structure-function relationship
    • 22. Assignment of Chemical shifts• Easy for a single amino acid or even a small polypeptide…
    • 23. Three-dimensional spectroscopy• Need to increase resolution for larger molecules
    • 24. Assignmentof HSQC and Cα and Cβ
    • 25. Structure prediction by ORBplus AB interhelical angle (°) CD interhelical angle (°) cNTnC(WT) 134 ± 3 118 ± 4 cNTnC(Acys) 142 ± 3 109 ± 4cNTnC(WT)-cTnI(147-163) 102 ± 4 95 ± 6 L29Q (ORBplus) 143 118.3
    • 26. NMR experiment Purpose Time15 N-HSQC 1 HN,15N pairs 30 min – 24 hours13 C-HSQC 1 H,13C pairs (aromatic 30 min – 24 hours and/or aliphatic)HNCACB 1 HN,13C (a and b; i, 48 – 64 hours i-1)),15NCBCA(CO)NNH 1 HN,13C (a and b; i-1),15N 48 – 64 hoursHC(CO)NH 1 HN,1H (i-1), 15N 72 hours • Only represent a fractionC(CO)NH 1 HN,13C (i-1),15N 72 hours of the NMR experiments15 N-HSQC-NOESY 1 HN, 1H (j), 15N 72 – 144 hours to help determine a15 N-HSQC-TOCSY 1 HN, 1H (i), 15N 72 hours protein structure • Different for other13 C-HSQC-NOESY 1 H (aliphatic), 1H (j), 13C 100 – 144 hours biological samples, such(aliphatic) (aliphatic) as DNA13 C-HSQC-NOESY 1 H (aromatic), 1H (j), 13C 48 – 72 hours • Also need to add more(aromatic) (aromatic) experiments, if interestedHNHA 1 HN, 1H (a some b), 15N to 48 – 64 hours in solving a protein-ligand obtain φ structureHNHB 1 HN, 1H (b), 15N to obtain 48 – 64 hours χ1HN(CO)HB 1 HN, 1H (b), 15N to obtain 48 – 64 hours χ1Total 677 (28 days) – 944 (40 days)
    • 27. Dihedral Restarints
    • 28. NOESY • The NOESY experiment measures the dipolar interaction between nuclei • The intensity of an NOE is proportional to 1/r6 and can therefore provide distance measurementsBerg J.M., 2002
    • 29. Structure calculation• Energy minimization: move atoms around to try and minimize energy – Define experimental restraints (and non-experimental, such as covalent bonds) as having energy – The higher the energy the greater the divergence a model is from the constraints• To avoid the structure from becoming trapped in a local minima simulated annealing is employed – Atoms are given a kinetic energy (associated with a high temperature and then cooled slowly• The ensemble represents a set of structures that satisfy the experimental restraintsBerg J.M., 2002
    • 30. Structural Statistics for L29QR.m.s.d. from the average structure Backbone atoms Heavy Atoms aOrdered residues (Å) 0.94 ± 0.18 1.40 ± 0.16Total Distance Restraints 1692Intra Residual NOEs 1033Short range (|i-j|=1) NOEs 307Medium range (1<|i-j|<5) NOEs 191Long range (|i-j|≥5) NOEs 153 2+Ca distance restraints 8Dihedral restraints 175φ/ψ 154 (72/72)χ1 21 bNOE violations/Structure> 0.5 Å 0.0> 0.3 Å 0.0> 0.1 Å 3.35Dihedral Violations/Structure (> 5º) 0.0Ramachadran plot statistics cφ/ψ in most favored regions (%) 96.6φ/ψ in additionally allowed regions 3.4(%)φ/ψ generously allowed regions (%) 0.0φ/ψ in disallowed regions (%) 0.0a Residues 3-49, 52-85; as calculated by psvsb Violations are for the 20 NMR lowest energy structuresc Procheck for ordered residues listed above.
    • 31. cNTnC(L29Q) structure AB interhelical angle (°) CD interhelical angle (°) cNTnC(WT) 134 ± 3 118 ± 4 cNTnC(Acys) 142 ± 3 109 ± 4 cNTnC(WT)-cTnI(147-163) 102 ± 4 95 ± 6 sNTnC(WT) 90 ± 3 69 ± 5 L29Q (ORBplus) 143 118.3 L29Q (NMR) 139 ± 5 122 ± 7
    • 32. cNTnC(L29Q) structure
    • 33. Alignment with other ‘closed’ structuresL29Q (slate); cNTnC(WT), pdb code:1AP4 (magenta); cNTnC(Acys), pdb code: 2CTN (grey);trout NTnC at 30°C, pdb code: 1R2U (orange); trout NTnC at 7°C, pdb code:1R6P (yellow);sNTnC(E41A), pdb code: 1SMG (Green)
    • 34. Alignment of loop 1• The structures were aligned between residues 15-27 and 40-48 and the r.m.s.d. of the flexible loop in site 1 (residues 28-34) was determined to be (A) 3.5 Å, (B) 2.1 Å, and (C) 1.6 Å.• Loop 1 of cNTnC(L29Q) superimposes much better with cNTnC- cTnI(147-163) than cNTnC(Acys)
    • 35. Dynamics of loop 1• Can determine the mobility of a backbone amide by determining its relaxation rates – T1 is the relaxation time to return to thermal equilibrium – T2 is the time it takes for transverse magnetization to be lost – 1 H-15N NOE measures how altering the ground and excited state of one spin can affect the ground and excited state of another spin• Relaxation is caused by magnetic field fluctuations – Can be caused by rapid internal (or external) motion – direct interactions with nearby magnetic nuclei (DD), chemical shift effects (CSA), quadrupole-electric field gradient interaction (QR) and rapid modulation of J-coupling (SC)
    • 36. S of L29Q compared with WT2 • Since relaxation values are difficult to interpret on their own, it is often useful to use their values to calculate the order parameter, S2 – Related to the amplitude of internal motion – If all orientations of the 1H-15N vector are equally probable than S2 = 0; if motion is rigid, than S2 = 1. • The data suggest that the order of the loop is relatively unchanged • There may be a slight increase in the order at the end of the loop
    • 37. Conclusions• L29Q did not alter the global structure of cNTnC• May slightly change the orientation of loop 1 – New conformation may destabilize binding to cTnI1-29 – Or may function just simply by destabilizing necessary hydrophobic interactions between cTnI1-29 and L29Q – Dynamics of loop were not significantly altered when compared to cNTnC• Maybe L29Q does nothing…• Limitations: – Only N-domain – Only Ca2+-bound (apo was not analyzed) – No cTnI147-163 or cTnI1-29 bound in structure – Low resolution of NMR so it’s difficult to be certain of change in the conformation of loop 1
    • 38. Does a Leu to Gluheart function undo?…I still have no clue!
    • 39. AcknowledgmentsUniversity of AlbertaBrian SykesMonica LiLeo SpyracopoulosSimon Fraser UniversityGlen TibbitsKing’s College LondonMalcolm IrvingYin-Biao Sun(and everyone else)