Exploring the Mutated Troponin Subunit to better understand Distal Arthrogryposis

Minjung Kim, Alice Ward Racca, Michael Bamshad, Michael Regnier

Dept. of Bioengineering, University of Washington, Seattle, WA
Motivation

Abstract

IVM Data Acquisition and Analysis

To develop a protocol for the expression of the human troponin subunit and determine the impact of DA mutations myosin activity

Distal Arthrogryposis (DA) is an umbrella term for non

progressive congenital skeletal muscle defect characterized
by abnormal contractures of the limbs and the face. It occurs
in 1 in every 3,000 live births and is inherited in an
autosomal dominant manner. Currently, there are no
treatments for the disease, and the patients usually go
through regular physical therapy throughout their lifetime.
Severe cases require surgical intervention and amputation.
One of the more common syndromes is DA 2B, or Sheldon
Hall syndrome, which is caused by mutations in two genes,
TNNI2 and TNNT3. This research focuses on studying the
R63H mutation that impacts the fast skeletal troponin T
(TNNT3) subunit. The troponin subunits are critical in
regulating the interaction between actin and myosin
myofilaments. Mutations in the TnT can alter the way the
troponin subunit interacts with the tropomyosin and result
in large additive impacts on overall muscle mechanics. From
the phenotype of the disease, we hypothesize that this
mutation increases the contractility and tension of muscle
mechanics. Wild type will be compared to the mutated
version of TNNT3 by putting a tag on the proteins.
Differences in calcium sensitivity, ATPase, and maximum
filamented actin velocity in a sliding filament assay known as
the in vitro motility assay (IVM), will be observed to piece
together information about how the mutation impacts the
overall muscle mechanics. Making connections between the
genotype and the phenotype of the disease to better
understand it on a molecular level will hopefully open up
doors for therapies.

Background
Muscle is a highly ordered system of proteins
Each muscle cell contains myofibrils
Myofibrils contain sarcomeres
Sarcomeres are the contractile units

Creating Recombinant Human Troponin

Left: Video data is acquired via

IVM data acquisition program.

Troponin Complex Formation

Vector Construction

Right: Individual filaments (red)

are identified along with their
paths (grey) and velocities.

Protein Verification
human TnT
TnI

Starting
sequence

TnC

Human skeletal
muscle mRNA

Rabbit TNNC2

TnT

original unbound
pellet fractions

Human skeletal
muscle mRNA

peak
fractions

Fractions around absorbance

peak were verified for subunit
expression via Western blot.

30 kDa

1. Mutagenesis
Starting sequence is modified using short genetic sequences
(primers) and enzyme TAQ that create a copy of the starting
sequence containing a small change
Rabbit TnC (TNNC gene) is very similar to human TNNC (below)
Human TNNT is mutated to R63H
Human TNNT is mutated to add a flag (DYKKDDDDK in the C
terminus)

R63H
+flag

R63H

WT
+flag WT

30 kDa

Rabbit HMM
Rigor Solution (0 mM ATP)

The TnT proteins with flag are

slightly heavier than the ones
without flag. Therefore the
proteins with flag are found
further up in the gel.

The rigor solution (top) is the negative

control and the 2mM ATP (bottom) is the
positive control.

Formation of the Troponin Complex

99.4% Homology in amino acid sequence between

TNNC2 Rabbit and TNNC2 Human (160 residues)

1. Dialysis of each subunit to

have the proteins in the
right buffer concentration.

2mM ATP Solution

2. Determine concentration of
subunits.

New Constructs:
Human TnC;
Human TnT + flag;
Human TnT R63H;
Human TnT R63H+flag

As expected, the rigor solution has

negligible mean speed for all filaments
while the 2mM ATP has much higher
mean speed of ~4.5 um/s.

3. Add subunits drop by drop

in the right ratio.
4. Determine concentration of
whole troponin complex.

2. Anneal construct into vector

(right)

5. Dialysis of whole complex to

IVM working buffer.

3. Sequencing Verification
confirms construct is correct

In Vitro Motility (IVM)

(4)
Rigor Solution (0 mM ATP)

Making Heavy Mero-Myosin(HMM)

(3)

Protein expression

Myosin in glycerol

In rigor solution, the mean speed of the

filaments depend heavily on the set
cutoff speed SD/mean, which means
that the mean speed of the individual
non-erratic filaments is inconsistent.

4. Insert vector construct into BL21 E. coli cells

(1)

Sarcomeres contain:
Thin filament (TF)
Regulatory Troponin complex (TnT, TnI, &TnC)
Actin backbone binds myosin
Thick Filament
Myosin protein heavy and light chains
Acts as the motor, pulls against the TF

5. Addition of IPTG encourages production of protein of interest

BED (+), centrifuge, and re-suspend to pull

myosin out of glycerol

2mM ATP Solution

6. Centrifuge

+ 12.5 uL chymotripsin digest per 1 mL myosin

to cut myosin tail

Cell Pellet
Protein Purification

In 2mM ATP solution, the mean speed

of the filaments are generally
independent from the cutoff speed
SD/mean. Therefore the filaments have
similar mean speed no matter how
much they deviate from the mean
speed.

7. Sonicate to release the

protein from the cell

Thin
Filament

Centrifuge to separate tail and head of myosin

Thick
Filament
(1)

8. Use (NH4)2SO4 to
precipitate protein of
interest

On going work
In the first test of the troponin subunit, the actin filaments did not fully stop at
pCa of 4.0. Therefore we can try three additional steps for a better outcome:
1. Increase the concentration of the complex.
2. Use excess TnC subunit to ensure that all TnT and TnI can bind to
TnC and form the whole complex.
3. Dialyze the complex into the IVM working buffer more gradually
to decrease the likelihood of protein unfolding.

Myosin

Takeda, 2003 (2)

10. Collect fractions at

and around peak
absorbance

Purified Protein
Elution buffer of
various salt
concentrations

1)
2)
3)
4)
5)

Absorbance of fractions

The troponin complex

Troponin C (TnC):
- Binds Ca2+ to initiate muscle contraction
Troponin I (TnI):
- Inhibitory protein
- Keeps the complex on the myosin binding site
of actin when calcium is not present
Troponin T (TnT):
- Structural protein

9. Use Anion Exchange

column (DE52) to filter
protein by charge (below)

Protein
sample

(4)

Apply adhesive solutions to glass

Apply myosin and let it bind to glass
Apply photo-labeled actin
Add tropomyosin and tropnoin complex of interest
Apply buffer with appropriate Ca2+ concentration
and ATP concetration.

IVM will be carried out with the mutant complex to see the full impact of mutant
TnT on molecular muscle mechanics.

Reference
1. http://www.rtmsd.org/page/1790)
2. Takeda S, Yamashita A, et al (2003). Structure of the core domain of human cardiac troponin in the
a(2+) saturated form. Nature. 2003 Jul 3;424(6944):35-41.
3. http://en.wikipedia.org/wiki/File:Recombinant_formation_of_plasmids.svgc
4. Biochemistry Voet and Voet 4th Edition

Contact
Fractions

Undergraduate student and first author: Minjung Kim (mjkim25@uw.edu)

Senior author: Michael Regnier (mregnier@uw.edu)