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RLC
ELC
Myopathy Loop
Actin-Binding LoopHelix-
Loop-Helix
Motor Domain
Lever-Arm
50 kD Cleft
Active
Site
Crystal Structure of Myosin S1
Lever Arm
Pi, ADP
ATP
Hydrolysis
Structure/Function Relationships in Myosin
•Hydrolyze ATP
•Bind Actin
•Generate Powerstroke
•Coordinate Functions
Myosin: “A Whale of a Protein”
Mouth
Tail
Blow Hole
Active Site -
Actin-binding Cleft -
Rigid Relay Loop -
Lever-arm -
along with converter domain coordinates active site and lever arm.
generates powerstroke.
Structure/Function Relationships in Myosin
hydrolyzes ATP.
mediates affinity for actin.
How Can the Details of Changes Be Established?
Crystallograhy can provide some information about changes since myosin can be crystallized in different nucleotide states.
However, to address DYNAMIC changes in the protein other methods are required which are
1) compatible with biological reaction conditions,2) sensitive to structural changes,
and 3) have excellent temporal resolution.
Fluorescence Spectroscopy
29 36 441 512Skel. YLRKSPFDAKSSVFVVHPKES / EKM.FLWMVIRIN / KKEGIEWEFID Card. EAQTRPFDLKKDVFVPDDKQE / ERM.FNWMVTRIN / KKEGIEWTFID Dcty KLTVSDKRYIWYNPDPKERDS / GRL.FLWLVKKIN / LKEKINWTFID Smooth LAQA.DWSAKKLVWVPSEKHG / ERL.FRWILTRVN / QREGIEWNFID
F F F F
546 597 625
Skel. / ILEEECMFPKATD / DYNISGWLEKNK / KTLALLFATY... Card. / ILEEECMFPKATD / DYNIIGWLQKNK / KLLSTLFANY...Dicty / LLDEQSVFPNATD / MYEIQDWLEKNK / NVVTKLFND....Smooth / LLDEECWFPKATD / TYNASAWLTKNM / KFVADLWKDVDRI
M F W
W546M
V413W
ELC
Upper 50 kDa Subdomain
Actin-Binding Cleft
Lower 50 kDa Subdomain
F425W
FHC – Familial Hypertrophic Cardiomyopathy
NormalR413Q mutation
Wavelength (nm)300 320 340 360 380 400 420 440
Rel
ati
ve
Flu
ores
cen
ce625-MDE546-MDEL-Tryptophan
MAX(nm) 333 0.36 344 0.20 351 0.14
Steady-State Fluorescence Properties
Acrylamide (mM)0 30 60 90 120 150 180
F0/
F
1
2
3
4
5546-MDE625-MDE
KSV (M-1) kq (M
-1·ns-1)
11.5 6.4 5.3 1.7
Acrylamide QuenchingF0/F = 1 + KSV[Q]
Relative Fluorescence of 625 MDE
Bound to Actin
0
0.2
0.4
0.6
0.8
1
1.2
300 325 350 375 400
Wavelength (nm)
Rel
ativ
e Fl
uore
scen
ce
625 MDE:Actin
Actin
Null MDE:Actin
625 MDE
Wavelength (nm)300 320 340 360 380 400
Nor
mal
ized
Flu
ores
cenc
e
625-MDE625-MDE:Actin
Actin-Induced Conformational Changes
Wavelength (nm)300 320 340 360 380 400
Nor
mal
ized
Flu
ores
cenc
e
546-MDE546-MDE:Actin
334 344
MAX (nm)MAX (nm)
333
Trp625 - Unchanged Trp546 -adopts a more buried conformation
Wavelength(nm)
300 320 340 360 380 400
Nor
mal
ized
Flu
ores
cenc
e
W546-MDE RigorW546-MDE ADP-Bound
Wavelength (nm)300 320 340 360 380 400
Nor
mal
ized
Flu
ores
cenc
e
V413W-MDE Rigor 1V413W-MDE Rigor 2V413W-MDE ADP-Bound
Actin Bound Fluorescence: ADP-Bound vs. Rigor
336337
347341338
MAX
MAX
Wavelength (nm)300 320 340 360 380 400
Fluo
resc
ence
(arb
ritr
ary
units
)
0
5000
10000
15000
20000
25000
W546-MDE ADP-Bound DHNBSW546-MDE Rigor DHNBS
Wavelength (nm)300 320 340 360 380 400
Fluo
resc
ence
(arb
ritr
ary
units
)
0
5000
10000
15000
20000
V413W ADP-Bound DHNBSV413W Rigor DHNBS
DHNBS Quenching: ADP-Bound vs. Rigor
Fluorescence from F425W-MDE in the absence of actin
Wavelength (nm)
300 320 340 360 380 400
Fluo
resc
ence
(au)
RigorMgADPMgATP
A
[Acrylamide] mM
0 20 40 60 80 100 120 140 160 180
Fluo
resc
ence
(au)
RigorMgADPMgATP
B
Steady-State Fluorescence of F425W-MDE
Nucleotide Bound Peak Intensity MAX KSV (M-1·ns-1)
None 100% 338 4.1 ± 0.02
MgADP 97% 339 4.1 ± 0.02
MgATP 80% 345 5.7 ± 0.06
Cleft Conformational
Changes
A:M-ATP A:M-ADP A:M
Weak Binding Cleft Closure Motor Domain Rotation
Structural Model of Acto-Myosin Interactions
W546 V413W
F425W
Actin