RLC ELC Myopathy Loop Actin-Binding Loop Helix- 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, and3) have excellent temporal resolution. Fluorescence Spectroscopy

Skel. YLRKSPFDAKSSVFVVHPKES / EKM.FLWMVIRIN / KKEGIEWEFID Card. EAQTRPFDLKKDVFVPDDKQE / ERM.FNWMVTRIN / KKEGIEWTFID Dcty KLTVSDKRYIWYNPDPKERDS / GRL.FLWLVKKIN / LKEKINWTFID Smooth LAQA.DWSAKKLVWVPSEKHG / ERL.FRWILTRVN / QREGIEWNFID     F F F F 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 Normal R413Q mutation

MAX (nm)  Steady-State Fluorescence Properties

K SV (M -1 ) k q (M -1 ·ns -1 ) Acrylamide Quenching F 0 /F = 1 + K SV [Q]

Relative Fluorescence of 625 MDE Bound to Actin

Actin-Induced Conformational Changes MAX (nm) 333 Trp625 - UnchangedTrp546 -adopts a more buried conformation

Actin Bound Fluorescence: ADP-Bound vs. Rigor MAX

DHNBS Quenching: ADP-Bound vs. Rigor

Fluorescence from F425W-MDE in the absence of actin

Steady-State Fluorescence of F425W-MDE Nucleotide Bound Peak Intensity MAX K SV (M -1 ·ns -1 ) None 100% ± 0.02 MgADP 97% ± 0.02 MgATP 80% ± 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