rlc elc myopathy loop actin-binding loop helix- loop- helix motor domain lever-arm 50 kd cleft...

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