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Cardiovascular Program, Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472 John P. Sumida, Eleanor Wu, Knut Langsetmo, David Hayes, Shu Qin Jiang, John Stewart, Zenon Grabarek, and Sherwin S. Lehrer Conserved Destabilization of Tropomyosin at Asp 137: Functional Significance of Flexibility. This work was supported by the NIH NIH grant HL 022461
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Platform Presentation at 2008 Biophysical Meeting
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Cardiovascular Program, Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472
John P. Sumida, Eleanor Wu, Knut Langsetmo, David Hayes, Shu Qin Jiang, John Stewart, Zenon Grabarek, and Sherwin S. Lehrer
Conserved Destabilization of Tropomyosin at Asp 137: Functional Significance of Flexibility.
This work was supported by the NIH
NIH grant HL 022461
Outline
1. Conserved Asp 137 destabilizes the middle of the Tropomyosin
2. Asp 137 is necessary to maintain local conformational dynamics, and imparts a flexible region to the middle of Tropomyosin
3. This locally flexible region in the middle of Tropomyosin is important to properly tune the regulation of the thin filament.
Conserved Destabilization of Tropomyosin at Asp 137: Functional Significance of Flexibility.
Cardiovascular Program, Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472
John P. Sumida, Eleanor Wu, Knut Langsetmo, David Hayes, Shu Qin Jiang, John Stewart, Zenon Grabarek, and Sherwin S. Lehrer
The Thin Filament Proteins
The 3 State ModelLehrer, et. al. JBC, 257, 1982
Vibert, et. al, JMB, 266, 1997
residue position
0 50 100 150 200 250
Pe
rcen
t Fre
que
ncy
0
20
40
60
80
100 Lys 15 Asp 137Glu 218
Lys 29
• Charged residues are highly conserved at four positions
• Asp at position 137 is 100% conserved
Helical wheel
Occurrence of charged residues in “a” and “d” positions of 84 long form isoforms of Tm
Hydrophobic ridge
Recombinant chicken tropomyosin used in this study
Controls
Leu Mutant
Temp20 25 30 35 40 45 50 55 60
Kca
l/mol
0
5
10
15
20
25
30
35
Asp to Leu mutation increases tropomyosin’s thermal stability
Sumida et al, Biophys J 2006, 90
Pre-transition
Co
Main transition
Co
WT 41.5 46.6
C190A 40.5 46.2
D137LC190A 40.6 47.2
The naturally occurring Asp at 137 destabilizes tropomyosin
WT Leu Mutant Differential
Scanning Calorimetry
Trypsin Cleaves preferentially at Arg 133
Tryptic digestion of tropomyosin quenched at different time points with soybean trypsin inhibitor, STI
The Leu Mutant remains uncleaved over the course of the experiment compared to both control tropomyosins.
Change in intensity of the Tm band over time
Comparison of Asp 137 with Leu 207 at equivalent “d” positions in tropomyosin.
Asp 137 Leu 207
“knobs into holes” packing
Charged side chains are extended out of the core.
Brown, J. H., Zhou, Z., Reshetnikova, L., Robinson, H., Yammani, R. D., Tobacman, L. S., and Cohen, C. (2005) Proc Natl Acad Sci U S A 102(52), 18878-18883
cleavagefold
unfobs k
k
kk
Tropomyosins with the naturally occurring Asp 137 exhibit a much greater degree of flexibility in the middle of the molecule
Under conditions of limited proteolysis observed rates provide a qualitative measure of the rate of local conformational fluctuation at Asp 137
log[Ca2+]
-9.0 -8.0 -7.0 -6.0 -5.0 -4.0
M A
TP
sec
-1
0.00
0.05
0.10
0.15
0.20
0.25
0.30
Sumida et al, JBC 2008
The Leu Mutant regulated thin filament exibits more activity than control in the open state at high Ca2+
Leu Mutant
Control
No change in calcium sensitivity is observed
Blocked
Closed
TmTm
log[Ca2+]
-9.0 -8.0 -7.0 -6.0 -5.0 -4.0
M A
TP
sec
-1
0.00
0.05
0.10
0.15
0.20
0.25
0.30
Sumida et al, JBC 2008
The Leu Mutant regulated thin filament exibits more activity than control in the open state at high Ca2+
Leu Mutant
Control
No change in calcium sensitivity is observed
Closed
Open
TmTm
M S10 4 8 12 16
M A
TP
sec
-1
0.0
0.2
0.4
0.6
(+Tn/+Ca2+)
(+Tn/-Ca+2
)
(-Tn/-Ca+2
)
Sumida et al, JBC 2008
Greater myosin activation of Actin-S1 ATPase for the Leu mutant
Squares = Leu MutantCircles = ControlBlocked
Closed
Open
TmTm
Conclusion: the conserved Asp at position 137…
1. imparts flexibility to tropomyosin by creating a conformationally dynamic region in the middle of the molecule
2. is important in the myosin activation of the thin filament by regulating the closed to open equilibrium.
Zenon Grabarek & Sam Lehrer
Eleanor Wu
Knut Langsetmo & David Hayes
Shu Qin Jiang & John Stewart,
Proteolysis experiments
Preparation of recombinant and native proteins
Stability measurments
Technical advice, and direction
Danuta Szczena-CordaryJose PintoUniversity of Miami
