Fret Studies into the Dynamics of the N

FRET studies into the dynamics of the N-terminal extension of cardiac troponin I in
the presence of cTnT disease mutations.
By Kenneth Brooks
1
Abstract:
The Sarcomere is the functional unit of the cardiac myocyte, which contains
overlapping regions of thin and thick filaments. The thin filament of the cardiac
sarcomere is composed of proteins cardiac troponin (cTn), Actin and cardiac
tropomyosin (cTm). The troponin complex consists of three individual proteins
termed as cardiac troponin C (cTnC), cardiac troponin I (cTnI), and cardiac troponin
T (cTnT). cTnC binds to Ca2+_ and Mg2+ ions, cTnI interacts to inhibit cross bridge
cycling on actin at low levels of Ca2+. cTnT interacts with tropomyosin (2). When
hearts of mice carrying the mutation on cTnT (R92L, del160E) were subjected to
isoproterenol stimulation, it was observed that in the presence of TnT (R92L)
mutation the levels of protein kinase A (PKA) were not elevated. Whether the PKA
substrates on the N-terminal extension of cTnI were occluded because of cTnT
(R92L) mutation was further examined. FRET studies revealed that in the presence
of cTnT disease mutations the N-terminal extension of cTnI does experience nonharmonic fluctuations. This may be associated with an inaccessibility of PKA to its
substrates on cTnI. The inability to phosphorylate sites Ser23 and Ser24 of HcTnI by
PKA may be associated with effects of the disease mutation.
Introduction:
The heart contains four chambers, which act to facilitate the movement of blood into
the systemic circulation in order to perfuse the tissues with oxygen (3). In the
presence of oxygen cells will generate ATP through cellular respiration, which will
facilitate normal daily activity. Returning blood will pass from the superior and
inferior Vena Cava into the Right Atrium, followed by the Right Ventricle before
moving through the Pulmonary Artery into the lungs where CO2 and Oxygen will be
exchanged converting previously deoxygenated blood into oxygenated blood. Blood
flow will then travel through the two remaining chambers of the heart and will be
2
pumped out of the left ventricle via a significant pressure gradient against the aortic
valve.
The left ventricle will generate the greatest amount of pressure in order to push the
blood into the systemic circulation (3). During high metabolic necessity the rate at
which blood will be oxygenated and dispersed throughout the body (as well as heart
rate) will increase because the body will require a greater amount of oxygen in
order to maintain increased physical activity. This increased metabolic activity is
associated with an increased heart rate. However, when the left ventricle becomes
thickened due to cardiomyopathy resulting from disease causing mutations the
ability to pump out a sufficient amount of blood per beat becomes compromised
resulting in an insufficient oxygen supply to the tissues leading to sudden cardiac
death (1). Although the left ventricle remodeling may contribute to alterations in
normal cardiac output the magnitude of remodeling of the left ventricle has not
been clearly linked to a degree of clinical severity (1). This is interesting in that it
suggests that although patients with irregular left ventricles may be expected to
function abnormally (based on the magnitude of left ventricle remodeling) in the
presence of high metabolic necessity the magnitude by which patients may be
affected varies between each individual.
The Cardiac muscle consists of cardiac myocytes containing contractile units
known as sarcomeres. Within the sarcomere, overlapping regions of thin and thick
filaments (in the presence of activating pathways) facilitate in contraction when
Myosin binding sites of the cardiac Thin filament are exposed allowing for cross
bridge formation with Myosin heads of the cardiac Thick Filament (). The Thin
filament of the cardiac sarcomere is composed of proteins including Troponin,
Tropomyosin and Actin (1:1:7 ratio). The Troponin complex acts as a regulatory
complex between Actin and Tropomyosin, which consists of proteins HcTnC, HcTnI
and HcTnT (2). HcTnC acts as a calcium sensitive attachment site, which allows for
regulation of cross bridge formation via activation/ deactivation of cross bridge
formation in the presence or absence of calcium. HcTnI acts as a regulatory subunit
with direct attachment to actin facilitating in modulation of Myosin head binding
3
tendencies during normal cross bridge formations. The last protein, which the
troponin complex consists of, is cTnT, which anchors cTnC and cTnI to the Actin
complex while also having a direct interaction with regulatory subunit
Tropomyosin. Calcium binding to cTnC will result in protein-protein interactions
within the troponin complex and retraction of HcTnI from Actin (2). The retraction
will modulate the position of tropomyosin, which in turn exposes myosin-binding
sites on actin. The interaction between the thick and thin filament results in
contraction of the sarcomere. However, the presence of disease mutations
associated with damage to the thin filament of the cardiac sarcomere will contribute
to dis-regulation of the cardiac sarcomere leading to abnormal behavior often linked
to clinical implications (1).
Our study focused on how a single amino acid mutation within the thin
filament troponin complex may alter the cardiac sarcomeres structural stability
resulting in dis-regulation of the Cardiac Sarcomere with possible implications
towards Hypertrophic cardiomyopathy (1). In particular our study focused on
whether HcTnT disease amino acid mutation R92L (Arginine to Leucine) as well as
Del 160E led to upstream distance changes between HcTnC and HcTnI which may
contribute to alterations in normal sarcomere activation pathways (in particular
PKA phosphorylation at sites 23, 24 of HcTnI). This inability to phosphorylate at
sites 23 and 24 by PKA may lead to possible mechanisms of thin filament disregulation and Hypertrophic Cardiomyopathy. In order to measure distance changes
between HcTnI and HcTnC in the presence and absence of disease mutation R92L,
Del 160E, protein synthesis and purifications (review methods section for additional
information) were performed in order to facilitate in the reconstitution of the thin
filament for FRET (Förster resonance energy transfer) measurements (to be
performed following in-silico predictions).
FRET is a technique in which distances are measured between an excited
donor molecule and a non-excited acceptor molecule. The magnitude of energy
transfer provides information on the relative position of a given donor and its
acceptor pair and thus may contribute to the identification of distance changes in
4
the presence and absence of disease mutations. Typical distances which FRET based
analysis are able to pick up range from 10Å-100Å. The degree by which FRET based
studies may be considered credible depends significantly on the donor acceptor pair
distances being studied. For instance, Fret based donor Aedans with relationship to
FRET acceptor DDPM will allow for distance analysis between proteins of 28A.
However, other Fret pairs including AEDANS (donor) and DABMI (acceptor) allow
for a greater distance analysis (40A), which may provide a better perspective of
distance changes relative to Aedans-DDPM Fret pairs.
Techniques of FRET (5)

Forster distance Expression:
The equation below expresses the relationship between the transfer of energy of
two given probes and their respective distances from one another (Ro).

Ro expression below describes the maximum distance that two probes
(donor-acceptor pair) may be from each other while still allowing for energy
transfer

Fret Efficiency: The efficiency of FRET measurements may be attained from
the expression below, which takes into account the fluorescence intensities
of both the donor and acceptor.
5
Principal Objective: It was our aim to identify structural abnormalities
between HcTnI and HcTnC in the presence of disease mutation HcTnT R92L as well
as Del 160E and its associated links to decreased availability at sites 23, 24 of HcTnI
for phosphorylation by PKA. The inability of PKA to phosphorylate sites 23 and 24
of HcTnI is significant in that the phosphorylation at those sites via PKA allow for a
“decrease in myofilament Ca2+ sensitivity” (4) allowing for increased relaxation of
cardiac muscle. However, the ability to relax may be compromised in the absence of
normal PKA activity. As such, the inability of PKA to phosphorylate at sites 23 and
24 of HcTnI may be explained through distance alterations between HcTnI and
HcTnC in the presence of HcTnT disease mutation R92L.
Materials and Methods
Mutagenesis:
To yield the desired troponin complex proteins, pET-3d vectors that consisted of HcTnI,
HcTnT, HcTnC and TM insert were over expressed in E.Coli BL21 DE3cells. FRET
based analysis requires the conjugation of a site-specific probe to a Cys. Therefore the
endogenous Cys in cTnC and cTnI were mutated to either Ser or Ile. The endogenous Cys
of cTnI were mutated to C80S/C97I. The endogenous Cys in cTnC were mutated to
C35S/C84S. Therefore 3 mutations were performed to yield cTnI (C80S/C97I/S17C),
cTnI (C80S/C97I/S24C), cTnI (C80S/C97I/S28C). Since there are no endogenous Cys in
TnT the R92L and del 160 clones were made with ease. The Agilent Technologies primer
design application was used to derive the primers, which were used for mutagenesis. A
Quick Change XL II kit was used to incorporate the site specific mutations whereby
modified plasmids were sent to the University of Arizona Genomics Core Facility for
sequencing to confirm the accuracy of the site specific mutagenesis.
Protein Purification
6
Purification of HcTnC: Protein Over Expression and Purification
pET 3d- HcTnC were transformed into BL21 bacterial cells, plated onto agar plates with
ampicillin (100mg/ml) and grown overnight at 37deg C. A single colony was grown in
5ml of pre-culture in the presence of ampicillin. .5ml of the pre-culture was loaded in to a
1L Terrific Broth (TB) growth medium with ampicillin (100mg/ml) after growing for 7
hours at 37degC and a gyration of 250RPm. The growth medium with single colony was
shaken overnight at 37 degC.
Spun down Culture grown previously was shook at 4000 revolutions per minute for 20
minutes at 4 degC using centrifugation. The aqueous layer was decanted and the pellets
suspended in S sepharous Buffer (20 ml/ 1 pellet). Following re-suspension sonication
took place for eight to ten times for a total of ten minutes at setting 8 with 30-second
intervals per two-minute pauses. Centrifugation of our samples was done at 14k RPM for
45 minutes at 4 degC and decanted immediately afterward. Both the supernatant and
pellet were run onto a 15% agarose gel to identify overexpression. Once protein
expression has been confirmed using gel based analysis protein was transferred into a
Pre-equilibrated Q-Seph Column (1200 ml Q sepharous buffer-6M Urea, 20mM Tris,
1mM EDTA, 0.3mM DTT) to be used for the purification of our newly grown HcTnC
followed by a Q seph Buffer wash overnight at 1.3ml/min.
HcTnC protein was eluted using 600mM KCL in Q-Seph Buffer with a standard elution
profile. Eluted samples were taken in order to identify protein in intervals of 3 using a
profile of 45 minutes at 200V. Elution fractions were chosen which correspond to HcTnC
expression (18kDa) and dialyzed in Phenyl Sepharous Buffer.
Pooled fractions of HcTnC were dialyzed in 4X 2L Phenyl Sepharous (50mM Tris, 1mM
CaCL2, 1mM MgCl2, 50 mM NaCl, 1mM DTT). Phenyl Seph Column was equilibrated
with Phenyl Sepharous buffer +0.5 Ammonium Sulfate overnight. Expressed HcTnC was
run into the Phenyl Seph column using a desired program followed by a Phenyl Seph
wash overnight. The elution of HcTnC was performed using Phenyl Seph plus 0.5
7
Ammonium Sulfate elution buffer. Agarose gel analysis was performed and the fractions,
which corresponded to protein expression, were pooled and stored in the -80deg C fridge.
Purification of HcTnI: Protein Overexpression and Purification
pET 3d- HcTnC were transformed into Rosetta bacterial cells, plated onto agar plates
with ampicillin (100mg/ml) and grown overnight at 37deg C. A single colony was grown
in 5ml of pre-culture in the presence of ampicillin. 1ml of the pre-culture was loaded in to
a 1L Terrific Broth (TB) growth medium with ampicillin (100mg/ml) after growing for 7
hours at 37degC and a gyration of 250RPm. The growth medium with single colony was
shaken overnight at 37 degC.
Spun down Culture grown previously was shook at 4000 revolutions per minute for 20
minutes at 4 degC using centrifugation. The aqueous layer was decanted and the pellets
suspended in S sepharous Buffer (20 ml/ 1 pellet). Following re-suspension sonication
took place for eight to ten times for a total of ten minutes at setting 8 with 30-second
intervals per two-minute pauses. Centrifugation of our samples was done at 14k RPM for
45 minutes at 4 degC and decanted immediately afterward. Both the supernatant and
pellet were run onto a 15% agarose gel to identify overexpression. Once protein
expression had been confirmed using gel based analysis protein was transferred into a
Pre-equilibrated S-Seph Column (1200 ml S sepharous buffer-6M Urea, 50mM Tris,
2mM EDTA, 1mM DTT) to be used for the purification of newly grown protein (TnI)
followed by an S seph Buffer wash overnight at 1.3ml/min.
Elution of our desired protein was performed using 600mM KCL in S-Seph Buffer using
a standard elution profile. Eluted samples were run on a 15% agarose gel in order to
identify whether our protein was present in intervals of 3 using an operating time of 45
minutes at 200V. Elution fractions corresponding to HcTnI expression (24kDa) were
chosen and dialyzed in TnC Affinity buffer 2x overnight.
8
A TnC Affinity column was equilibrated (50mM Tris, 2mM CaCl2, 1M NaCl, 1mM
DTT) overnight using 200 ml TnC Buffer A. Selected HcTnI was run into the TnC
Affinity column at 1.3ml/min and eluted using 300mL Buffer A (50mM Tris, 2mM
CaCl2, 1M NaCl, 1mM DTT) followed by 300mL buffer B (50mM Tris, 3mM EDTA,
1M NaCl, 1mM DTT, 6M Urea). Eluted samples were run on a 15% agarose gel in order
to identify whether our protein was present in intervals of 3 using an operating time of 45
minutes at 200V. Elution fractions were chosen which corresponded to HcTnI expression
(24kDa) and stored in the -80degC fridge.
Purification of HcTnT: Protein Over Expression and Purification
pET 3d- HcTnC were transformed into Rosetta bacterial cells, plated onto agar plates
with ampicillin (100mg/ml) and grown overnight at 37deg C. A single colony was grown
in 5ml of pre-culture in the presence of ampicillin. 1ml of the pre-culture was loaded in to
a 1L ZYP growth medium with ampicillin (100mg/ml) after growing for 7 hours at
37degC and a gyration of 250RPm. The growth medium with single colony was shaken
overnight at 37 degC.
Spun down Culture grown previously was shook at 4000 revolutions per minute for 20
minutes at 4 degC using centrifugation. The aqueous layer was decanted and the pellets
suspended in S sepharous Buffer (20 ml/ 1 pellet). Following, re-suspension sonication
took place for eight to ten times for a total of ten minutes at setting 8 with 30-second
intervals per two-minute pauses. Centrifugation of our samples was done at 14k RPM for
45 minutes at 4 degC and decanted immediately afterward. Both the supernatant and
pellet were run onto a 15% agarose gel to identify overexpression. Once protein
expression had been confirmed using gel based analysis protein was transferred into a
Pre-equilibrated S-Seph Column (1200 ml S sepharous buffer-6M Urea, 50mM Tris,
2mM EDTA, 1mM DTT) to be used for the purification of newly grown protein (TnT)
followed by an S seph Buffer wash overnight at 1.3ml/min.
9
Elution of our desired protein was performed using 600mM KCL in S-Seph Buffer using
a standard elution profile. Eluted samples were run on a 15% agarose gel in order to
identify whether our protein was present in intervals of 3 using an operating time of 45
minutes at 200V. Elution fractions corresponding to HcTnT expression (36kDa) were
chosen and dialyzed in 2L of Q-Sepharous.
A Q-Seph column was equilibrated using Q-Sepharose buffer (6M Urea, 20mM Tris
base, 1mM EDTA, 0.3mM DTT) overnight. Selected HcTnT was run into the Q-Seph
Column at 1.3ml/min and eluted using 500mM in NaCl using an elution specific
program. Eluted samples were run on a 15% agarose gel in order to identify whether our
protein was present in intervals of 3 using an operating time of 45 minutes at 200V.
Elution fractions were chosen which corresponded to HcTnT expression (36kDa) and
stored in the -80degC fridge.
Actin Purification
All experiments, which took place-using actin, were done so at 4deg C unless described
otherwise. 4 grams of acetone powder was extracted via pulverization using a coffee
grinder followed by dissolving in 80mL of buffer A (10mM Tris, 0.2 mM CaCl2, 0.2 mM
ATP, 0.2 mM DTT at PH=8.0) for 30 minutes. The solution was then filtered through a
Buchner Funnel with three layers of cheesecloth. The filtrate was then transferred to a
400mL beaker on ice. The residual filtrate was then re-dissolved using 20mL of buffer
and subsequently filtered through the Buchner Funnel, with the remaining solution being
added to the 400mL beaker described previously. The gathered filtrate was then placed
into designated centrifuge tubes of equivalent weight at 18,000 RPM for 30 minutes
followed by decanting of the supernatant. The slow addition of KCl (final concentration
0.05M) and MgCl2 (2mM) polymerized the gathered actin into F-actin. A stir bar was
added to the F-actin and was used to gently mix the solution for one hour at room
temperature. Following an hour of spinning at room temperature the F- actin was placed
into a 4-degree cold room and solid KCl was added for a final concentration of (0.6M)
and left to stir for 1.5 hours.
10
Spin down polymerized actin at 18,000 RPM for 6 hours followed by decanting of the
supernatant. We then rinsed the actin pellets with 1 mL of Actin buffer A followed by
soaking of the pellet in 0.5 mL buffer an overnight. The next morning we homogenized
the pellets for 5 minutes in 10 mL of buffer A to reconvert the F-actin to G-actin. Using
one dialysis buffer change per day the homogenized G-actin was dialyzed against 500 ml
of buffer A (10mM Tris, 0.2 mM CaCl2, 0.2 mM ATP, 0.2 mM DTT at PH=8.0).
Following centrifugation of G-actin at 15,000 RPM for 50 minutes the actin
concentration was measured using a UV spec. Given the absorption wavelength of
protein (280nm) we were primarily concerned with the absorbance level of actin at 280
nm.
Thin Filament Reconstitution:
Following the dialysis of Troponin complex proteins in 6M Urea (6M Urea, 30mM
Mops, 1.25 mM CaCl2, 1.25mM MgCl2, 0.5M Kcl, 1mM DTT) the concentrations of the
proteins were measured using a UV-VIS spectrophotometer. The E coefficients, which
were used in order to identify the absorbance of each protein, included HcTnT (16960 M1-cm-1), HcTnC (4470 M-1cm-1), HcTnI (9970M-1cm-1). Once the concentrations of
the proteins were identified they were reconstituted in a 1:1:1 ratio. Following
reconstitution both the Urea and KCL concentrations were lowered. The Urea
concentrations were lowered via 6M-4M-2M-0M decreases while the KCL concentration
was gradually lowered to physiological conditions at 150mM KCl. The Thin Filament
reconstitution (Troponin + Tropomyosin + Actin) took place within a 1:1:7 ratio at
350mM KCl and was decreased 200mM KCl. Following the final KCl concentration of
200mM being reached the Thin Filament was placed titration buffer at 150mM in
preparation for FRET. In preparation for FRET and Life Time analysis our reconstituted
thin filament was placed in working buffer (50mM Mops Ph: 7.0, 2mM EGTA, 5mM
NTA, 1mM DTT, 150mM KCL, 5mM MgCl2) and changed three times.
Fret: Measurements:
11
Fret measurements were performed using a Photon Technology International (PTI)
Quantamaster spectroflurometer with each sample measured at 1mM protein. The
FRET and Lifetime measurements were performed in both the EGTA and Ca2+
saturated state. Distance measurements in both the calcium and EGTA state were
measured and collected.
Life Time FRET based results
The figures below display Lifetime FRET based results, which uses intensity as a
function of time between a donor-acceptor pair to identify distance measurements.
1.
The first figure provides Life Time FRET results for cTnT (WT) using donoracceptor pairs Aedans and DDPM
2.
12
The second figure provides Life Time FRET results for cTnT (R92L) using donoracceptor pairs Aedans and DDPM.
Wavelength
480.
481.
482.
483.
484.
485.
486.
487.
488.
489.
490.
491.
492.
493.
494.
495.
496.
497.
498.
499.
500.
WT
27155.23
26820.23
26900.96
27178.79
27045.40
26584.19
26362.01
26837.57
26851.81
25785.46
26383.27
26509.52
25471.93
26268.62
26293.75
24938.68
26203.61
24981.32
25580.15
24853.30
24025.12
Del160E
26333.63
25957.84
26344.21
26325.06
27109.01
26024.75
26437.39
25798.03
26691.18
25778.18
25762.19
25499.34
25448.37
24896.63
24874.25
25218.08
24547.00
24418.56
24094.78
23280.71
23891.73
R92L
35770.76
36870.53
35954.25
35428.00
35699.16
35445.41
35585.72
35184.17
35159.18
34628.27
34344.60
33999.24
34614.18
33207.17
34122.72
33441.50
33445.50
33416.29
32776.59
31615.53
32964.53
Wavelength measurements for cTnT- (WT, Del 160E, R92L) using FRET based techniques.
13

In order to provide greater resolution as to the direction of movement
between the N-Terminal extension of cTnI and cTnC the donor acceptor pair
S24C Aedans (cTnI) and S89C (cTnC) DABMI were used which increased the
Ro value from 28A to 40A.
Experiment Part Two: Using cTnI S24C (Aedans) with S89C (DABMI)
3.
Figure number 3 describes the FRET Life Time measurements between S24C cTnI
(Aedans) in conjunction to S89C cTnC (DABMI) in the presence of cTnT (WT)
14
4.
Figure number 4 describes the FRET Life Time measurements between S24C cTnI
(Aedans) in conjunction to S89C cTnC (DABMI) in the presence of cTnT (Del 160E)
5.
Figure number 5 describes the FRET Life Time measurements between S24C cTnI
(Aedans) in conjunction to S89C cTnC (DABMI) in the presence of cTnT (R92L)

It is identifiable that there is significant FRET in figures 4,5 (Del 160E, R92L)
relative to figure number 3 (WT). The change in intensity for figures 4,5 is
15
suggestive of a decrease in in distance between the N-terminal extension of
cTnI and cTnC in the presence of cTnT mutation Del 160E as well as in R92L.
Results and Conclusion:
It was identified via FRET based analysis that in the presence of cTnT disease
mutation R92L as well as in the presence of cTnT mutation Del 160E the Nterminal extension of HcTnI experiences a decrease in distance with
relationship to cTnC. This suggests that there may be a relationship between
a decreased distance between cTnI and cTnC, which contributes to PKA not
being capable of phosphorylating sites 23, 24 of cTnI. Fret pairs, which
provided the most significant evidence for corroboration of our hypothesis,
include cTnC S89C (DABMI) and cTnI 24C (Aedans). This study additionally
verified the potential for in-silico (computer based predictions) methods to
identify outcomes prior to in-vitro experimentation.
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Acknowledgements:
I would like to thank Dr. Jil Tardiff as well as all other members of Dr. Jil Tardiff’s lab
for their mentorship and support through the development of this thesis.
The Physiological Sciences Undergraduate Program for the opportunity to
participate in Biophysical research.
Works Cited:
1. Tardiff, Jil C., and Jeffrey Robbins. "Developing an Integrative Approach to a
Complex Disorder. “American Heart Association, n.d. Web. 2 May 2014.
<http://circres.ahajournals.org/content/108/6/765.full>.
2. Farah, CS, and FC Reinach. "The troponin complex and regulation of muscle
contraction." The Journal of the Federation of American Societies for Experimental
Biology: n. pag. Web. 2 May 2014.
3. Heisler, Jennifer. "Blood Flow Through the Heart.” N.p., 3 Apr. 2014. Web. 2 May
2014. <http://surgery.about.com/od/beforesurgery/a/HeartBloodFlow.htm>.
4. Wijnker, Paul, D. Foster, Allison Tsao, Aisha Frazier, Cristobal Remedios, Anne
Murphy, Ger Stienen, and Jolanda Velden. "Abstract." National Center for
Biotechnology Information. U.S. National Library of Medicine, 9 Nov. 2012. Web. 2
May 2014. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3543672/>.
5. Moens, Pierre. "Fluorescence Resonance Energy Transfer spectroscopy." FRET
Page. N.p., n.d. Web. 2 May 2014.
<http://www.anatomy.usyd.edu.au/mru/fret/abot.html>.
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