(Received for publication, October 21, 1994; and in revised form, December 12, 1994)
From the
Recoverin, a new member of the EF-hand protein superfamily,
serves as a Ca sensor in vision. A myristoyl or
related N-acyl group covalently attached to the amino terminus
of recoverin enables it to bind to disc membranes when the
Ca
level is elevated. Ca
-bound
recoverin prolongs the lifetime of photoexcited rhodopsin, most likely
by blocking its phosphorylation. We report here Ca
binding studies of myristoylated and unmyristoylated recombinant
recoverin using flow dialysis, fluorescence, and NMR spectroscopy.
Unmyristoylated recoverin exhibits heterogeneous and uncooperative
binding of two Ca
with dissociation constants of 0.11
and 6.9 µM. In contrast, two Ca
bind
cooperatively to myristoylated recoverin with a Hill coefficient of
1.75 and an apparent dissociation constant of 17 µM. Thus,
the attached myristoyl group lowers the calcium affinity of the protein
and induces cooperativity in Ca
binding.
One-dimensional
H and two-dimensional
N-
H shift correlation NMR spectra of
myristoylated recoverin measured as a function of Ca
concentration show that a concerted conformational change occurs when
two Ca
are bound. The Ca
binding
and NMR data can be fit to a concerted allosteric model in which the
two Ca
binding sites have different affinities in
both the T and R states. The T and R conformational states are defined
in terms of the Ca
-myristoyl switch; in the T state,
the myristoyl group is sequestered inside the protein, whereas in the R
state, the myristoyl group is extruded. Ca
binds to
the R state at least 10,000-fold more tightly than to T. In this model,
the dissociation constants of the two sites in the R state of the
myristoylated protein are 0.11 and 6.9 µM, as in
unmyristoylated recoverin. The ratio of the unliganded form of T to
that of R is estimated to be 400 for myristoylated and <0.05 for
unmyristoylated recoverin. Thus, the attached myristoyl group has two
related roles: it shifts the T/R ratio of the unliganded protein more
than 8000-fold, and serves as a membrane anchor for the fully liganded
protein.
Recoverin is a 23-kDa calcium-binding protein in retinal rod and
cone cells. The Ca-bound form of recoverin prolongs
the photoresponse (Gray-Keller et al., 1993), most likely by
blocking the phosphorylation of photoexcited rhodopsin (Kawamura,
1993). The light-induced lowering of the Ca
level in
rod cells is thought to reverse this inhibition, thereby promoting
recovery of the dark state. The shortening of the lifetime of
photoexcited rhodopsin at low Ca
concentration may
also contribute to adaptation to background light. Calcium ion is known
to play a critical role in adaptation (Koch, 1994; Koch and Stryer,
1988; Matthews et al., 1988).
Retinal recoverin contains a
covalently attached N-terminal myristoyl or related fatty acyl group
(Dizhoor et al., 1992). Ca binding to
recoverin is thought to induce the extrusion of the myristoyl group of
the protein, enabling it to interact with the disc membranes (Dizhoor et al., 1993; Zozulya and Stryer, 1992). This
Ca
-induced membrane interaction, referred to as a
``Ca
-myristoyl switch,'' may be important
in regulating the phosphorylation of photoexcited rhodopsin.
Sequence comparison of recoverin with other members of the EF-hand
protein superfamily has revealed the existence of four EF-hand
Ca binding motifs (Ray et al., 1992). Fig. 1shows the amino acid sequence of the four EF-hands of
recoverin. The Ca
-coordinating residues at positions
5, 9, and 12 of the 12-residue loop are well preserved for all of the
four motifs. However, EF-1 and EF-4 lack essential acidic residues at
positions 1 and 3. Thus, EF-2 and EF-3, but not EF-1 and EF-4, are
predicted to have high affinity for Ca
.
Figure 1:
Primary sequence of the four EF-hand
motifs found in recoverin. The EF-hand consensus sequence is shown at
the top with Ca ligating residues denoted as X, Y, and Z; n is any nonpolar
residue; and a dash is any residue. Conserved amino acid
residues in the Ca
binding loop and three tryptophans
are in boldface.
The
crystal structure of recoverin shows that it in fact contains four
EF-hands (Flaherty et al., 1993). However, the structure of
recoverin is significantly different from that of calmodulin (Babu et al., 1988) and troponin C (Herzberg and James, 1988). In
calmodulin and troponin C, two EF-hand pairs are separated by a long
central helix, forming a dumbbell-shaped structure in the crystal.
Recoverin adopts a compact globular shape that can be divided into
amino- and carboxy-terminal domains. The amino-terminal domain contains
EF-1 and EF-2, and the carboxyl-terminal domain contains EF-3 and EF-4.
Ca was found to bind to EF-3 in this crystal form,
and Sm
, used as an isomorphous heavy atom derivative,
was found to bind to both EF-2 and EF-3. EF-1 is distorted from a
favorable Ca
-binding geometry by Cys-39 and Pro-40 at
positions 3 and 4 of the 12-residue loop. EF-4 contains a salt bridge
between Lys-161 and Glu-171 at positions 2 and 12. The crystal
contained no metal ion in EF-1 and EF-4.
Extensive Ca binding studies have been carried out on the EF-hand proteins
calmodulin (Haiech et al., 1981; Minowa and Yagi, 1984; Klee,
1988; Linse et al., 1991; Porumb, 1994), troponin C (Iida,
1988), parvalbumin (Ogawa and Tanokura, 1986), and calbindin-D
(Akke et al., 1991). In calmodulin, two high affinity
Ca
binding sites are present in the carboxyl-
terminal domain, and two low affinity sites are present in the
amino-terminal domain. Cooperativity was observed for the pair of sites
in each domain, but not between the amino- and carboxyl-terminal
domains (Minowa and Yagi, 1984). However, little is known about calcium
binding to recoverin. To understand the molecular mechanism of the
Ca
-myristoyl switch, a thorough knowledge of the
calcium binding properties of recoverin is necessary. We report here
flow dialysis, fluorescence, and NMR studies of myristoylated and
unmyristoylated recoverin that were carried out to delineate the
mechanism of Ca
binding and gain insight into the
mechanism of the calcium-myristoyl switch.
Tryptophan fluorescence experiments were
performed with 1-2 µM myristoylated or
unmyristoylated recoverin in 2 ml of 0.1 M KCl, 1 mM dithiothreitol, 50 mM HEPES (pH 7.4) at 25 °C. The
free calcium concentration (50 nM to 2 µM) was
set using an EGTA buffer system. The protein samples initially
contained an equal amount of total Ca and EGTA (2
mM); the free Ca
concentration was adjusted
by adding aliquots of 0.1 M EGTA. The free Ca
concentration was calculated based on the total amount of
Ca
and EGTA present using the program
Bound-and-Determined (Brooks and Storey, 1992). The calculated free
calcium concentrations agreed closely with measured Ca
concentrations using fluorescent indicator dyes fluo-3 and rhod-2
(Molecular Probes, Eugene, OR) with K
of 0.4 and
1.0 µM, respectively (Tsien and Pozzan, 1989). Calcium
concentrations above 1 µM were measured using a
Ca
-selective electrode (Orion, Boston, MA).
Samples for NMR experiments were prepared by dissolving lyophilized
protein (1 mM) in 95% HO, 5%
H
O or 99.9%
H
O (total
volume, 500 µl each) containing 0.1 M KCl and 10 mM [
H
]dithiothreitol. The pH was
adjusted to 6.8-7.0 using 4% NaO
H or 4%
HCl without correction for isotope effects. The total
calcium concentration of the NMR samples was adjusted by successively
adding 1.0-1.5-µl aliquots of 0.1 M CaCl
.
Two-dimensional NMR experiments were performed on a
UNITY-plus 500 spectrometer equipped with a triple resonance probe with
an actively shielded z gradient together with a pulse field
gradient accessory. N-
H HSQC
(Bodenhausen and Ruben, 1980; Bax et al., 1990) spectra as a
function of Ca
were recorded using the enhanced
sensitivity method of Kay et al.(1992). The spectra were
recorded with the following numbers of complex points and acquisition
times:
N (F
) 128, 64 ms,
H
(F
) 512, 77 ms. The HSQC spectrum of the
Ca
-free state was recorded with 64 transients.
Spectra with 0.75, 1.5, 2.25, 3.0, 4.0, and 5.0 equivalents of
Ca
were recorded with eight transients.
All data
sets were processed on a Sun Sparc10 workstation using commercial
software (NMRZ, New Methods Research Inc., Syracuse, NY; and FELIX,
Biosym, San Diego, CA). The programs Capp and Pipp (Garrett et
al., 1991) were used for peak selection and spectral analysis.
Residual water signal in N-
H HSQC
spectra was removed using a time-domain deconvolution approach reported
by Marion et al.(1989).
Figure 2:
A, Ca binding curves of
myristoylated (filledsquares) and unmyristoylated (opencircles) recoverin obtained by flow dialysis.
The solidlines represent the best fit to the
allosteric model presented in Fig. 4(see Table 1for
refined parameters). The crossedpoints show the
relative change in fluorescence intensity of myristoylated recoverin as
a function of Ca
concentration. B, Scatchard
plots of Ca
binding data of myristoylated (lefty axis) and unmyristoylated (righty axis) recoverin. The calcium binding curves measured by flow
dialysis appear to saturate at about two Ca
bound per
protein (2.3 for myristoylated and 1.6 for unmyristoylated). The NMR
data presented below clearly demonstrate that in fact 2 ± 0.15
Ca
bind to recoverin. Thus, a Ca
stoichiometry of two was used to normalize the Ca
binding curves in Fig. 2A.
Figure 4:
Allosteric model for the binding of
Ca to recoverin. The equilibrium expressions and
parameters are defined in the text.
The binding equilibria for two different
Ca-binding sites are
where [Ca] is the free calcium
concentration, P
denotes the occupancy of sites i and j, and the K
values are
the dissociation constants. Both sites are empty in P
, one is filled in P
and P
, and both are filled in P
. The fractional occupancy Y of the
calcium binding sites is given by
If the sites are independent (K = K
and K
= K
), the binding expression simplifies
to
The binding data for unmyristoylated recoverin are well fit by
this equation with K = 0.11
µM and K
= 6.9
µM. As discussed below, it seems likely that EF-3 is the
higher affinity site and EF-2 is the lower affinity site (Flaherty et al., 1993). P
, then, denotes
occupancy of EF-3, and P
denotes occupancy of
EF-2.
The binding curve for myristoylated recoverin, in contrast
with that of the unmyristoylated protein, exhibits a steep
Ca dependence. The data for the myristoylated protein
can be fit to the Hill equation
with a Hill coefficient () of 1.75 and an apparent
dissociation constant (K
) of 17.4 µM (Table 1). The observed Hill coefficient of 1.75 requires
that two or more Ca
bind cooperatively. Cooperativity
is also demonstrated by the downward curvature seen in the Scatchard
plot (Fig. 2B). The binding curve of myristoylated
recoverin was also fit by the two-site model described above, yielding K
= 100 µM, K
= 2.4 µM, K
= 2.4 mM, and K
= 0.1 µM.
The
dependence of the fraction f of P, P
, P
, and P
as a function of [Ca]
was
calculated using the best-fit parameters of the two-site model and
plotted in Fig. 3. For unmyristoylated recoverin (Fig. 3A), f
builds up to a
maximum of about 0.79 as [Ca]
is raised
to 0.85 µM and then falls as the Ca
concentration is raised. The fraction of the other one-calcium
species, f
, is always less than 0.014. Thus,
Ca
binds almost exclusively to the first site of the
unmyristoylated protein before binding to the second. By contrast, for
myristoylated recoverin (Fig. 3B), the maximum value of f
is less than 0.08. In fact, the fraction of
molecules in the P
state closely parallels Y, showing that the binding of Ca
to
myristoylated recoverin is highly cooperative.
Figure 3:
Concentration profiles of protein states
of (A) unmyristoylated and (B) myristoylated
recoverin with zero, one, and two Ca bound for a
two-site model. The fractional concentrations of the protein states are
defined as f
, where the
subscripts i and j denote whether or not
Ca
is bound to the low affinity (EF-2) and high
affinity (EF-3) sites, respectively. The data points represent the
fractional saturation of myristoylated recoverin measured by flow
dialysis. The solidlines represent concentration
profiles calculated for a two-site model (see
text).
The concerted
allosteric model (Monod-Wyman-Changeux model by Monod et
al.(1965)) can be adapted to quantitatively account for both the
uncooperative binding of Ca by unmyristoylated
recoverin and the cooperative binding by myristoylated recoverin. In
the model depicted in Fig. 4, recoverin has two intrinsically different binding sites for calcium and two
global protein conformational states, T and R. Let K
and K
denote the dissociation constants of
the two sites (most likely EF-3 and EF-2, respectively) in the R state.
We assume that the global conformational transition from R to T changes
the binding constant of both sites by the same factor c. L is
the T/R ratio in the absence of Ca
. The equilibria
for this model are given by
where R and T
represent recoverin in the R and T states, and the subscripts i and j denote whether or not Ca
is
bound to the low affinity (EF-2) and high affinity (EF-3) sites,
respectively. The fractional saturation is then given by
where = c + L
and
= c
+ L
. The
dissociation constants of the R state (K
and K
) are taken to be equal to K
and K
obtained by the independent sites model for unmyristoylated
recoverin. Using K
= 0.11 µM and K
= 6.9 µM, L is calculated to be 400 for myristoylated and
0.05 for
unmyristoylated recoverin. The parameter c is estimated from
the data for the myristoylated form to be less than
10
, indicating that Ca
binds
almost exclusively to the R state.
Both cooperative and
uncooperative Ca binding can be modeled by varying L while keeping K
, K
, and c fixed (Fig. 2A).
Positive cooperativity is produced when T is thermodynamically favored
over R (L > 1) and Ca
binds more tightly
to R than to T (c = 10
). In the
absence of the myristoyl group, the R state is much more stable. The
myristoyl group induces cooperative Ca
binding by
markedly favoring T. The myristoyl group changes the T/R equilibrium by
a factor of at least 8000 (400/0.05). Thus, the myristoyl group serves
as a covalently attached allosteric effector.
Figure 5:
1D H NMR spectra of (A)
unmyristoylated and (B) myristoylated recoverin at various equivalents
of added Ca
. Intensities of highlighted peaks are
plotted in Fig. 6.
Figure 6:
Intensities of 1D H NMR peaks
of (A) unmyristoylated recoverin and (B) myristoylated recoverin as a
function of Ca
. It should be noted that the x axis represents Ca
added/recoverin and not free
calcium concentration as in Fig. 2A. Since the
concentration of recoverin (1 mM) used in the NMR experiments
is
100-fold greater than the Ca
dissociation
constant (K
= 17 µM),
all Ca
added, up to two equivalents, was bound to the
protein.
Fig. 6B shows selected
Ca-dependent peak intensities from 1D
H
NMR spectra of myristoylated recoverin. The peak intensities changed
monotonically on the addition of up to two equivalents of
Ca
(in going from P
to P
). No further change was detected beyond 2 bound
Ca
/recoverin. This dependence shows that 2 ±
0.15 calcium ions bind per protein, in agreement with the flow dialysis
measurements. None of the resolved peak intensities saturated at 1
equivalent of Ca
. The absence of detectable
intermediate Ca
binding states supports a highly
cooperative Ca
binding mechanism for myristoylated
recoverin ( Fig. 2and Fig. 3B). A similar
phenomenon has been observed for the high affinity sites of calmodulin
(Ikura et al., 1983; Klevit et al., 1984; Thulin et al., 1984) and troponin C (Tsuda et al., 1988).
Two-dimensional N-
H HSQC spectra of
uniformly
N-labeled myristoylated recoverin were obtained
as a function of Ca
. Complete sequence-specific
backbone assignments have been obtained for myristoylated recoverin in
the Ca
-free state (Ames et al., 1994). This
enables one to monitor specific residues that undergo structural
changes upon Ca
binding. The
N-
H HSQC spectral changes are so
substantial upon the addition of Ca
that very few
peaks coincide in the spectra of the Ca
-free and
Ca
-bound states (Fig. 7).
N-
H cross-peak intensities of backbone
amide and tryptophan imide protons of specific residues in the
Ca
-free state are plotted as a function of bound
Ca
(Fig. 8). Cross-peaks from residues in the
amino-terminal domain and also in the carboxyl-terminal domain decrease
monotonically to zero when the number of bound Ca
increases from 0 to 2. These data indicate that
Ca
-induced structural changes occur throughout the
protein in a concerted fashion.
Figure 7:
Two-dimensional N-
H HSQC NMR spectra of (A)
Ca
-free and (B) Ca
-bound
myristoylated recoverin uniformly labeled with
N
(>95%). CHAPS detergent (20 mM) was added to prevent
aggregation of the Ca
-bound state (Kataoka et
al., 1993). The addition of CHAPS decreased the
linewidths (Anglister et al., 1993) but did not change the
peak positions of the cross-peaks in
H-
N
HSQC spectrum. Therefore, the addition of CHAPS most likely has little
effect on the structure of recoverin.
Figure 8:
Two-dimensional N-
H HSQC cross-peak intensities of
selected residues in the amino- and carboxyl-terminal domains of
myristoylated recoverin at various equivalents of added
Ca
.
The calcium binding properties of recoverin were determined
to help understand how the Ca-myristoyl switch
operates. Many differences between the Ca
binding
properties of the myristoylated and unmyristoylated protein are
evident. Myristoylated recoverin cooperatively binds two Ca
with a Hill coefficient of 1.75 and an apparent dissociation
constant of 17 µM. Unmyristoylated recoverin, by contrast,
binds two Ca
noncooperatively with markedly different
affinities (0.11 and 6.9 µM). The covalently attached
myristoyl group lowers the calcium affinity of the protein and induces
cooperativity.
Our NMR data indicate that two Ca bind per recoverin molecule and that a global conformational
change occurs when two Ca
bind to the myristoylated
protein. Many of the proton and
N resonances of recoverin
exhibit chemical shift changes when up to two Ca
bind, but no marked changes were detected when more than two
Ca
equivalents were added. Sequence-specific
assignments have been obtained for the backbone amide (Ames et
al., 1994) and tryptophan indole resonances. (
)These
assignments identify the residues that undergo structural change upon
Ca
binding. Almost all backbone amide resonances from
residues in both the amino- and carboxyl-terminal domains change
chemical shift simultaneously when up to two Ca
bind (Fig. 8). The Ca
-induced conformational change
also affects the side chain indole resonances of Trp-31, Trp-104, and
Trp-156. These results suggest that Ca
-induced
structural changes are occurring throughout the protein in a concerted
fashion.
From the amino acid sequence and x-ray structure it seems
clear that only EF-2 and EF-3 can bind Ca. Which is
the high affinity site? A large data base of amino acid sequences of
the EF-hand protein family suggests that aspartic acid is highly
conserved at the third position of the 12-residue Ca
binding loop, whereas the fifth position can be more frequently
substituted by other amino acids such as asparagine (Wylie and Vanaman,
1988). Sequence comparison of the Ca
binding loops of
EF-2 and EF-3 (Fig. 1) indicates that the most favorable residue
(aspartic acid) at the third position is present in EF-3, whereas it is
substituted with asparagine in EF-2. This suggests that EF-3 has a
higher affinity for Ca
than EF-2. Furthermore, the
x-ray structure shows Ca
bound to EF-3 and not EF-2
(Flaherty et al., 1993). Sequence-specific NMR assignments for
H resonances at 5.61 and -0.47 ppm (Fig. 6A), which monitor Ca
binding
to the high affinity site, will determine whether EF-3 is in fact the
high affinity site.
The myristoyl group promotes cooperative binding
of Ca to EF-2 and EF-3 sites. What is the structural
basis of this cooperativity? The x-ray crystal structure of
unmyristoylated recoverin with one Ca
bound (Flaherty et al., 1993) showed that the helices of EF-3 pack closely
against the amino-terminal helix of EF-2, resulting in a compact
globular fold. In addition, the myristoyl group is thought to bind to a
hydrophobic cleft located between EF-1 and EF-2. The present study
indicates that Ca
binding to EF-2 and EF-3 is
independent when the myristoyl group is absent. The myristoyl group
may, therefore, alter EF-2 to allow communication between EF-2 and
EF-3. Specifically, the amino-terminal helix of EF-2 is unstable in the
myristoylated protein (Ames et al., 1994), whereas it is
stable in the unmyristoylated form (Flaherty et al., 1993).
The effect of the myristoyl group on the Ca binding cooperativity is somewhat analogous to the effect of a
target peptide on Ca
binding to calmodulin. In the
absence of a target, there is little cooperativity between the amino-
and carboxyl-terminal domains of calmodulin (Minowa and Yagi, 1984;
Klee, 1988). In the presence of a target such as a 26-residue peptide
derived from myosin light-chain kinase and mastoparan, the two domains
interact with each other, resulting in highly cooperative
Ca
binding (Yazawa et al., 1987). This
target-induced interaction between the two domains of calmodulin has
been accounted for by the compact structures of calmodulin complexed
with myosin light chain kinase peptides determined by NMR spectroscopy
(Ikura et al., 1992) and x-ray crystallography (Meador et
al., 1992). For recoverin, the myristoyl group may similarly
induce an interaction between the amino- and carboxyl-terminal domains,
perhaps by binding to the interface of the two domains. The crystal
structure of Ca
-bound unmyristoylated recoverin
(Flaherty et al., 1993) shows a compact shape. Recent NMR
studies (Ames et al., 1994) and small-angle x-ray scattering
studies (Kataoka et al., 1993) on myristoylated recoverin also
suggest a compact shape even in the absence of Ca
.
Hence, cooperative Ca
binding by myristoylated
recoverin may be the consequence of rather subtle structural
differences brought about by the myristoyl group.
A concerted
allosteric mechanism is proposed to explain how the myristoyl group
induces cooperative Ca binding to recoverin. The
model shown in Fig. 4is like the Monod-Changeux-Wyman model
(Monod et al., 1965), except that the Ca
binding sites are taken to be intrinsically different and hence
have different affinities. The existence of two protein conformations T
and R, defined by the Ca
-myristoyl switch (Zozulya
and Stryer, 1992), is supported by the fluorescence and NMR data
presented in this study. The T state can be pictured to have the
myristoyl group buried inside the protein core, allowing recoverin to
be stable in solution. The R state places the myristoyl group outside
so that it can interact with disc membranes or another protein. The
intrinsic dissociation constants of the R state for Ca
are K
and K
, and those
of the T state are K
/c and K
/c, where c is estimated to be less than
10
. Thus, Ca
binds at least
10,000-fold as tightly to R than to T. Unlike the Monod-Wyman-Changeux
model, K
and K
are allowed to
be different and are assumed to be 0.11 and 6.9 µM in the
R state, the same as measured for unmyristoylated recoverin. Based on
these values of K
and K
, the
ratio (L) of the unliganded form of T to that of R is
estimated to be 400 for myristoylated and <0.05 for unmyristoylated
recoverin. Using the obtained values of L and c (Table 1), myristoylated recoverin is calculated to be
almost exclusively in the T state in the absence of Ca
(99.8% T
for L = 400). The
binding of each Ca
changes the equilibrium between T
and R by a factor of c in the direction of R. When both sites
are occupied, the ratio of T to R
([T
]/[R
]) is 4
10
. Therefore, about 5 kcal/mol of the binding
energy of each Ca
is used to drive the conformational
equilibrium to the R state so that recoverin is almost entirely in the
R state when two Ca
bind.
The apparent low
Ca affinity of myristoylated recoverin in solution is
linked to an unfavorable extrusion of the myristoyl group during the T
to R transition (L = 400,
G° =
+3.5 kcal/mol). The T
R
transition is opposed in solution because the myristoyl group is
then forced to interact with H
O. A membrane environment
should stabilize the extruded myristoyl group in the R state and hence
cause L to decrease. Fig. 2A shows the
calculated effect of lowering L on the Ca
binding curve. The apparent Ca
affinity of
myristoylated recoverin is brought into the physiological range when L is lowered. An L value of 40 predicts a binding
curve with apparent dissociation constant of about 4 µM.
This calculated curve is similar to the curve describing the binding of
myristoylated recoverin to rod outer segment membranes in which
half-maximal binding occurs at
4 µM Ca
(Zozulya and Stryer, 1992). S-Modulin, a close relative
of recoverin found in frog rods, was found to associate with frog rod
outer segment membranes and inhibit phosphorylation of rhodopsin with a
Ca
dissociaton constant of
1 µM (Kawamura, 1993).
The allosteric model in Fig. 4has
implications concerning the kinetics of the Ca myristoyl switch. Upper limits on the rate constants for binding
and release of Ca
from the R and T states can be
inferred. For both states, the rate constant for binding Ca
is limited by diffusion to be less than about 10
M
s
. The
dissociation rates from the high affinity (EF-3) and low affinity
(EF-2) sites can be calculated from the dissociation constant for each
site. The calculated dissociation rate of Ca
from the
low affinity site (EF-2) in the R state is less than 7000
s
and that from the high affinity site (EF-3) in the
R state is less than 100 s
. Similarly, the
dissociation rates of Ca
from the high and low
affinity sites in the T state are less than 10
s
and 7
10
s
, respectively. The rate constants for the
allosteric transitions between R and T are not known. Stopped-flow
kinetic measurements of the Ca
-induced tryptophan
fluorescence changes of myristoylated recoverin and of Ca
uptake by fluorescent buffers are currently in progress to
determine the kinetics of Ca
binding and the
allosteric transition.