1. cTnC-L29Q
Does a Leu to Glu
heart 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 cTnI
Finley 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 units
Dong, 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
WT
Baryshnikova, 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
filaments
Liang, 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 differences
Dweck, 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
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 measurements
Berg 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
restraints
Berg J.M., 2002
30. Structural Statistics for L29Q
R.m.s.d. from the average structure Backbone atoms Heavy Atoms
a
Ordered residues (Å) 0.94 ± 0.18 1.40 ± 0.16
Total Distance Restraints 1692
Intra Residual NOEs 1033
Short range (|i-j|=1) NOEs 307
Medium range (1<|i-j|<5) NOEs 191
Long range (|i-j|≥5) NOEs 153
2+
Ca distance restraints 8
Dihedral restraints 175
φ/ψ 154 (72/72)
χ1 21
b
NOE violations/Structure
> 0.5 Å 0.0
> 0.3 Å 0.0
> 0.1 Å 3.35
Dihedral Violations/Structure (> 5º) 0.0
Ramachadran plot statistics c
φ/ψ in most favored regions (%) 96.6
φ/ψ in additionally allowed regions 3.4
(%)
φ/ψ generously allowed regions (%) 0.0
φ/ψ in disallowed regions (%) 0.0
a
Residues 3-49, 52-85; as calculated by psvs
b
Violations are for the 20 NMR lowest energy
structures
c
Procheck for ordered residues listed above.
33. Alignment with other ‘closed’
structures
L29Q (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.
37. S of L29Q compared with WT
2
• 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
38. 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
39. Does a Leu to Glu
heart function undo?
…I still have no clue!
40. Acknowledgments
University of Alberta
Brian Sykes
Monica Li
Leo Spyracopoulos
Simon Fraser University
Glen Tibbits
King’s College London
Malcolm Irving
Yin-Biao Sun
(and everyone else)
Hinweis der Redaktion
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:
