Ca2+-induced conformational changes of scallop myosin regulatory domain (RD
) were studied using intrinsic fluorescence. Both the intensity and anisotr
opy of tryptophan fluorescence decreased significantly upon removal of Ca2. By making a mutant RD we found that the Ca2+-induced fluorescence change
is due mainly to Trp21 of the essential light chain which is located at the
unusual Ca2+-binding EF-hand motif of the first domain. This result sugges
ts that Trp21 is in a less hydrophobic and more flexible environment in the
Ca2+-free state, supporting a model for regulation based on the 2 Angstrom
resolution structure of scallop RD with bound Ca2+ [Houdusse A. and Cohen
C. (1996) Structure 4, 21-32]. Binding of the fluorescent probe, 8-anilinon
aphthalene-1-sulphonate (ANS) to the RD senses the dissociation of the regu
latory light chain (RLC) in the presence of EDTA, by energy transfer from a
tryptophan cluster (Trp818, 824, 826, 827) on the heavy chain (HC). We ide
ntified a hydrophobic pentapeptide (Leu836-Ala840) at the head-rod junction
which is required for the effective energy transfer and conceivably is par
t of the ANS-binding site. Extension of the HC component of RD towards the
rod region results in a larger ANS response, presumably indicating changes
in HC-RLC interactions, which might be crucial for the regulatory function
of scallop myosin.