Can Fo
¨
rster Resonance Energy Transfer
Measurements Uniquely Position Troponin Residues
on the Actin Filament? A Case Study in Multiple-
acceptor FRET
John M. Robinson
*
, Wen-Ji Dong and Herbert C. Cheung
Department of Biochemistry
and Molecular Genetics
University of Alabama at
Birmingham, Birmingham, AL
35294-2041, USA
Straightforward interpretation of Fo
¨
rster resonance energy transfer
(FRET) data in terms of the distance from donor-labeled troponon-tropo-
myosin (TnTm) to acceptor-labeled actin is complicated by the potential
for energy transfer to acceptors on neighboring actin monomers (cross-
transfer). Calculations indicate that cross-transfer can account for a sub-
stantial percentage of the total transfer efficiency. In some cases, this
renders isolated FRET data uninterpretable. To overcome these limi-
tations, we have developed an analysis method that incorporates cross-
transfer and can, in principle, define the most probable (in the “least-
squares” sense) position of a TnTm residue on the actin filament. The
technique analyzes data from four or more FRET experiments using
acceptors attached to different residues on actin. We have used this
method to specify the coordinates of skeletal troponin I (sTnI) residue
133 relative to the actin filament under Mg
2þ
and Ca
2þ
-saturating con-
ditions. Ca
2þ
-activation causes the C terminus of the regulatory domain
of TnI to move away from the actin surface by 6.3 A
˚
, laterally along the
actin surface toward actin subdomain 3 by 22.0 A
˚
, and azimuthally
toward the actin inner domain by 13.2 A
˚
. This information is used to
construct a low-resolution structural model of thin filament activation.
q 2003 Elsevier Science Ltd. All rights reserved
Keywords: actin model; Ca
2þ
-regulation; cross-transfer; muscle activation;
macromolecular complex
*Corresponding author
Introduction
The striated muscle thin filament is organized
around a central filament of actin. The actin fila-
ment can be represented, as a double right-handed
helix comprised of two long-pitch strands spiraling
around the filament axis with each strand opposite
the other. Alternatively, the actin filament can
be viewed as a single left-handed genetic helix
of actin monomers staggered axially by 27.5 A
˚
(Lorentz model)
1
and each rotated from the next
by 193.848 in the plane normal to the filament axis
(transverse plane). We shall utilize the latter rep-
resentation and denote consecutive actin mono-
mers: actin
i21
, actin
i
, actin
iþ1.
Actin monomers can
be represented as oblate elipsoids of revolution
tilted with respect to all three filament principal
axes. Tilt in the transverse plane of the actin fila-
ment causes deviation from cylindrical symmetry
and creates a semi-flat surface for tropomyosin
(Tm) and troponin (Tn) to reside. The surface is
broken by a crevice that delineates the inner and
outer domains of actin. The actin outer surface has
an approximate 30 A
˚
radius. Muscle activation is
thought to involve first a Ca
2þ
-induced, then a
strong cross-bridge induced, repositioning (azi-
muthal rotation) of Tm across the actin filament
surface in the direction of the inner domain.
2,3
Fo
¨
r-
ster resonance energy transfer (FRET) has been
used to quantify activation-induced distance
changes in TnTm along the actin surface. The lit-
erature contains a collection of FRET measure-
ments between sites on TnI and sites on the actin
0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved
E-mail address of the corresponding author:
jmr@uab.edu
Abbreviations used: Acc
i
, FRET acceptor on actin
i
;
C-TnC, C-domain of TnC; FRET, Fo
¨
rster resonance
energy transfer; hc, human cardiac; N-TnC, N-domain of
TnC; R
o
,Fo
¨
rster distance; sk, fast skeletal; Tm, tropo-
myosin; Tn, troponin; TnI, troponin I; TnI-R, TnI
regulatory region; TnI-I, TnI inhibitory region.
doi:10.1016/S0022-2836(03)00424-8 J. Mol. Biol. (2003) 329, 371–380