Florida State University, National High Magnetic Fields Laboratory. Conformational Changes Associated with Muscle Activation and Force Generation by Pulsed EPR Methods. Piotr Fajer. myosin head. Motor proteins. Ca activation. actin. troponin C. myosin. Force generation.
Florida State University, National High Magnetic Fields Laboratory
function demands large conformational changes;
Labeling Cysteine Scanning
nitroxide - nitroxide
Rabenstein & Shin, PNAS, 92 (1995)
sensitivity: 8-20 Å
Sensitivity: 10–50 Å
Long Distance: 18 –50 Å
Sensitive to distance distribution
myosin cleft closure
myosin head interactions in smooth muscle
opening of K+ - channel
Site specific spin labelling
structure of troponin I
A. Málnási-Csizmadia, C. Bagshaw, P. Connibear
Cleft closure associated with lever swing
Wendt et al. (1999)
Wahlstrom et al. (2003)
Vassylyev et al. PNAS, 1998
Tung et. al, Protein Sci, 2000
Ca switch mechanism shown in isolated TnC but NOT in ternary complex of TnI, TnC and TnT
what is the structure of TnC in ICT complex ?
what are the Ca induced conformational changes in ICT ?
Spin labels: 12, 51, 89, 94
Gd3+: sites III & IV
Excellent agreement with X-ray and NMR
TnC in solution is extended
N- to C-domain distance decreases by 9 Ǻ central helix bends in a complex
15-94 15-136 12-136
(assume no changes in the N-domain which senses Ca)
All distances are in (Ǻ)
TnI N-terminal helix moves v. little (2Å) with respect to TnC C-domain on Ca2+ binding.
TnC is more compact in ternary complex than isolated TnC.
Calcium switch might well be same in troponin complex as in isolated TnC.
3. N-domain of TnI remains in proximity of C-domain of TnC.
N domain movement
central helix bending
Tn (+ Ca)
Tn (- Ca)
Y. Li, E. Perozo
Homology model is wrong.
Scatter = 6 Å
Modelling the spin label decreases scatter = 3 Å
“cysteine scanning” from 130-146
P1/2= 60 mW
P1/2= 20 mW
power ½ (mW) ½
TnI inhibitory region
X-ray CS data, homology model
Vassylyev et al PNAS 95:4847 ‘98
-hairpin loop (nmr)
Tung et al Prot.Sci. 9:1312 ‘00
Binary/ternary “difference” map
Ternary: TnI mutants
Dipolar EPR excellent for 10-20 A
Pulsed EPR extends the range to 20-50 A
“Easy” protein chemistry
Large macromolecular complexes
Determination of secondary structure.