Volume 23 June 1, 2012 Structure of Shroom domain 2 | 2141
Cy3 or Cy5 maleimide (GE Healthcare) in cysteine labeling buffer
(20 mM HEPES, pH 7.6, 100 mM NaCl, 8% glycerol). Small (821–
938) dRock was labeled at C862 with Cy3 maleimide as described.
Large dRock (724–938) was labeled at the N-terminus with Cy5 suc-
cinimidyl ester (GE Healthcare) in amino labeling buffer. All labeling
reactions included 10× molar excess of ﬂuorophore at room tem-
perature for 2 h. Excess ﬂuorophore was removed from the samples
through extensive dialysis with labeling buffer. The labeling efﬁ-
ciency was quantiﬁed using the extinction coefﬁcient of the dye
compared with the protein concentration determined from a stan-
dard curve using a Bradford assay and found to be essentially 1:1.
FRET binding experiments
FRET titrations were performed in dShrm reaction buffer, using a
50 nM of Cy3-labeled dShrm or dRock and increasing concentra-
tions of Cy5-labeled dRock or dShrm. Cy3 was excited at 552 nM,
and the donor emission maximum (563 nm) was corrected for dilu-
tion, normalized, and plotted as a function of protein concentration
as the average of three independent experiments. Fluorescence
) titrations were ﬁtted to a single binding equation:
is the normalized change in donor ﬂuorescence intensity
is the dissociation constant.
We thank Jeff Brodsky and Karen Arndt for critical comments on the
manuscript. Operations at the National Synchrotron Light Source
are supported by the Department of Energy, Ofﬁce of Basic Energy
Research, and by the National Institutes of Health. Data collection at
the National Synchrotron Light Source was funded by the National
Center for Research Resources. This work was supported by Na-
tional Institutes of Health Grant GM097204.
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