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Figure 2.
Profilin binding causes a moderate opening of the nucleotide
cleft in actin. (A) Superimposition of the structures of
profilin–Dictyostelium-actin (blue and cyan) and uncomplexed
monomeric actin (28) (blue and magenta). Two orientations are
shown, rotated by 90°. The latter structure was obtained by
mutagenesis in subdomain 4 and is thought to be free of
perturbations resulting from the binding of an ABP or chemical
cross-linking. For clarity, profilin is not shown in this figure
(see Figs. S5 and S6 for a full view of the profilin–actin
structure). Subdomains 3 and 4 of the structures were
superimposed (blue) to highlight the relative movement of
subdomains 1 and 2 (magenta or cyan). Using the classical view
of actin as a reference (left view), the 4.7° rotation
(calculated with the program DynDom,
http://www.sys.uea.ac.uk/dyndom/) between the two major domains
of actin can be visualized as two perpendicular rotations of
≈3.3°. The center of this rotation approximately coincides
with the junctions between domains, consisting of residue
Lys-336 and the helix between residues Ile-136 and Gly-146.
Comparison of the profilin–actin structures with any other
structure of actin, except for the wide-open structure of
profilin–β-actin (36), results in a similar motion of the two
major domains (see also Movies S2 and S3). This movement appears
less dramatic than previously anticipated (36, 37), but it is
probably sufficient to explain the stimulation of nucleotide
exchange by profilin. (B) Quenching of tryptophan fluorescence
on profilin binding (the results of two identical experiments,
with different preparations of both actins, are shown). Profilin
binds pY53-actin and unphosphorylated actin with similar
affinities (K[d] = 0.090 and 0.057 μM, respectively), but the
quenching of tryptophan fluorescence is significantly less for
profilin–pY53-actin.
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