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Nanosecond study of fluorescently labeled troponin C
8i¢~'himica ,'t Biophysica A,'ta. 1121 (1992) 16-22 '.~'~ Ig92 Ei.~vicr Science Publishers B.V. All rights reserved 0167-4~38/92/$05.00
16
BBAPRO M I ~
Nanosecond study of fluorescently labeled troponin C Chien-Kao Wang ~'*, Ronglih Liao b'** and Herbert C. Cheung :' a Depurtmem of Biochemist~'. Unic,'rsiO' of Alabama at Birmingham. Birmingham. AL (USA) and i, (;raduate Program in Biophysical Sriem'es. Unirersity of Ah, bama at Birmingham, Birminghum. AL (USA)
(Received g September Iq91)
Key words: Exlrinsic fluorescence: Trol~min C: (Rabbit skclelai)
The time-resolved extrinsic fluorescence of rabbit skeletal troponin C v,as studied with the protein labcled at Cys-98 with N-(iodoacetyl)-N'-(5-sulfi~-l-naphthyl)cthylenediamine. Both the intensity and anisotropy decays fi)llowed a biexponcntial decay law, regardless of the ionic condition, pH. viscosity or temperaturc. The lifetimcs and their fractional amplitudes were insensitive to Mg -~+, and the lifetimes were also insensitive to Ca 2'. in response to Ca -~ binding to all four sites, the fractional amplitude (a I) associated with the short lifetime (r~)decreased by a factor of two, thus increasing the ratio of the two amplitudes a 2 / a ~ from !.6 to 4.3. These amplitude changcs suggest the existence of two conformaiional states of TnC-IAEDANS, with the conformation associated with the long-decay component (T~) being promoted by saturation of the two Ca-'+-specific sites. At pH 5.2 the ratio a~/a~ lot the apo-protein was 3.5 indicating different relative populations of the two decay compoi~ents when compared with pH 7.2. In the presence of Ca ~+ at the lower pH, a 2 / a I decreased to 2.1, suggesting a shift of the conformations in favor of the short-decay component. Thus Ca ~-~ elicited different conformational changes in TnC at the two pH values. The recovered anisotropies suggest that there were fast molelcular motions that were not resolved in the present expcriments, and some of these motions wcre sensitive to Ca 2 ~ binding to the specific sites. These results support the notion of communication between the N-domain and the C-terminal end of ~hc central helix of troponin C.
Introduction
Troponin from striated muscle consists of three nonidentical, and structurally and functionally distinct subunits. Troponin T is the tropomyosin-binding subunit, troponin I is the subunit responsible for the inhibition of actomyosin ATPas¢ and troponin C is the calciumbinding subunit which is responsible for transmission of the signal of activator calcium to other proteins in activation of actomyosin ATPase. In vertebrate muscle, troponin is associated with tropomyosin in a 1 : I stoichiometry, and this complex is bound to the actin
* Present address: Department ,)f Physiology and Biophysics. University of Washington, Seattle. WA. USA. ** Present address: Division of Cardiology. Belh Israel Hospital. Boston. MA. USA. Abbreviations: TnC. troponin C: CTnl. cardiac troponin l: IAEDANS. N-(iodoacelyD-N'45-sulfo. I-naphthyl)ethylenediaminc: TnC-IAEDANS. TnC labeled at Cys-98 with IAEDANS: Gu-HCI. guanidine hydrochloridc: D'Fr. dithiolhreiiol; HWHM, full width a! half-maximum height. Correspondence: H.C. Cheung. Department of Biochemistry. University of Alabama at Birmingham. Birmingham. AL 35294. USA.
filament with a stoichiometry of 1 tropomyosintroponin to 7 actin monomers. The three-dimensional crystal structures of T n C from the skeletal muscle of chicken [1] and turkey [2] have been reported and show the protein to have an unusual dumbbell shape, with the amino and c a r b o ~ l terminal segments folded into two globular domains. The two domains are connected by a 9-turn a-helix the middle one-third of which is totally exposed to solvent in the crystal structure. The structure has four E F hands, each of which consists of a helix-loop-helix motif, and the four loops are the sites at which Ca 2+ binds. The two sites located in the N-terminal half (sites ! and II) bind Ca 2÷ specifically with a low affinity ( K , , - - 2 - 10 ~ M - i ) , and the other two sites located in the C-terminal half (sites I!I and IV) bind Ca 2+ with a high affinity (K~,--2" 10 7 M - I ) and also bind Mg 2+ competitively. Becasuc of the relatively high intracellular Mg 2+ concentration, sites 111 and IV are likely saturated with Mg 2+ in relaxed muscle, and these sites do not appear to have a major role in calcium regulation. In contrast, sites ! and Ii (regulatory sites) are unoccupied until the arrival of activator Ca 2+ in the concentration range of 10/zM. The mechanism by which calcium triggers contraction is not fully understood. Early studies have sug-
17 gested that the interaction between TnC and Tnl is relatively weak in relaxed muscle and that the binding of calcium to the specific sites of TnC enhances this interaction and concomitantly weakens the interaction between Tnl and actin [3-5]. The latter event allows Tnl detachment from actin and a lateral movement of tropomyosin on the actin helix. It is now generally agreed that the binding of activator calcium induces structural changes not only in TnC itself, but also in the other proteins associated with the thin filament, namely, the other two troponin subunits, tropomyosin and actin. Many lines of evidence have suggested that these structural changes and the accompanying alterations of the affinities of these proteins provide a molecular basis for calcium regulation [6-8]. On the basis of energetic and other considerations, Cheung et al. [9] and Tap et al. [10] have suggested that troponin 1 and the T n i - T n C linkage may play a fundamental role in signal tansduction in skeletal muscle. in the present work, we report a time-resolved study of the extrinsic fluorescence of TnC. Since the crystals of TnC used in structure determination were obtained at pH 5.0-5.1, the present studies were carried out at two different pH values to investigate the effect of acidic pH on the global conformation of the protein. The results indicate that the binding of calcium to the specific sites can induce a spectrum of conformational changes and fast molecular motions. Some of these fast motions are sensitive to the binding of activator calcium. The studies provide further evidence of structural communication be,ween the N-domain and the C-terminal region of the long central helix connecting the two globular ends of the protein.
Materials and Methods
Protehz preparation Troponin was extracted from rabbit skeletal muscle as described in our previous work [! 1]. The protein was dissolved in 6 M urea and TnC was separated from the other two subunits by DEAE-cellulose (DE-52) column chromatography in the presence of urea [6]. The purity of the isolated TnC was monitored by SDS-PAGE, and its concentration was estimated from an absorbance of 0.23 g-~ c m - ' at 276 nm and a molecular weight of 18000. TnC was labeled at Cys-98 with IAEDANS as in our previous work [6]. The extent of labeling was determined to be about 0.95 tool of probe/tool of protein by absorbance measurements using an absorption coefficient of 6100 M - t c m -t at 337 nm [12]. Suifhydryl content was determined with 5,5'-dithiobis2-nitrobenzoic acid (DTNB) in 50 mM Tris, 90 mM KCI and 2 mM EGTA at pH 8.0.
Time-resoh'ed fluorescence measurements Fluorescence intensity decay and fluorescence anisotropoy decay were measured with a PRA (Photochemical Research Asmciatcs) single-photon counting nanosecond fluorometer. The spark lamp was thyratron-triggered, H2-filled (14 mm Hg vaccum) and run at 32 kHz. The pulses had a typical FWHM of 2.0 ns. A Ditric 3-cavity 340-rim interference filter (FWHM 8.0 nm) was used to isolate the excitation light and the emission of IAEDANS was isolated with a Ditric 3-cavity 490-nm interference filter {FWHM 7.0 nm). Lifetimes were determined using rotation free optical conditions (excitation light polarized vertically and emitted light polarized at the magic angle of 54.7 ° from the vertical). Lamp profiles were measured at the same emission wavelengths with a pair of Ditric 3-cavity interference filters using Ludox as the scattering agent. To optimize the signal to noise ratio, a minimum of I0000 photon counts were collected in the peak channel. The data stored in a multichannel analyzer were transferred to either a DEC 11/03 microcomputer or an IBM P C / A T personal computer for analysis. Intensity decay curves were fitted to a sum of exponential terms:
F(t)
=
~ a , exp(-t/~'i)
(I)
i
where tr, are the amplitudes associated with the i decay components, each having a lifetime 7-,. The decay parameters were recovered using nonlinear leastsquares iterative procedures with the Marquardt algorithm [ 131. Emission anisotropy decay was determined by using vertically polarized excitation light and measuring alternately the emission polarized in the vertical [FIl(t)] and horizontal [Fj. (t)] directions. This was achieved by changing the orientation of the emission polarizer at 20-min intervals. Photon counts were collected until at least 20000 counts were reached in the peak channel for the Fu(t) component. The F±(t) component was collected for the .same period of time as for Ftt(t). The anisotropy decay r(t) was calculated from
r(t) =
Ftl - F l (t) Fll(t ) + 2 F j . ( t )
(2)
and the deca~' data were fitted to a sum of exponential terms,
r( t) = rt,~gi exp(-ttd~,)
(3)
i
where g, are the fractional amplitudes of the components associated with the correlation times d'i. For a
18 biexponentiai decay law the total anisotorpy r. is given by r o = glro + g2ro with ~g, -- 1.0. The goodness of a fit between a given set of observed data and the chosen function was evaluated by the weighted residuals, the autocorrelation function of the weighted residuals, the reduced chi-square ratio (~C~), the Durbin-Watson paremeter (D-W)[14,15] and the runs test (Z,~.) [16]. A fit was considered acceptable when plots of the weighted residuals and the autocorrelation function showed random deviation about zero with a ,¢~ value of about 1 and a value of the D-W parameter that satisfied theoretical estimates. The log-likelihood ratio test [17] was also used to discriminate between a two-exponential and a oneexponential model.
TABLE I Fluorescence intensity decays o f T n C - I A E D A N S at p H 7 2 Measurements were carried out with TnC-IAEDANS ( = 2. l0 -s M) in 0.1 M KCI, 50 mM Tris, 1 mM DTT. I mM EGTA (pH 7.2). When present, [Ca-" + ]= 1.5 raM, [Mg -~+ I = 2 raM, s u c r o ~ = 25% ( W / V ) . [Gu-HCI] = 6 M. The decay was resolvvd into two cor~ponenls, with ¢1 referring to the short lifetime and ~'z to the long lifetime, a I and a , refer to the fractional amplitudes associated with the two respeclive lifetimes. The weighted average lifetime is given by ( v ) = r,,,,,T~/F,a, Ti. D - W is the Durbin-Watson parameter. Medium
T(°C)
r, (ns)
ai
EGTA
20
9.27 16.43 10.21 17.99
0.39 0.61 0.41 0.59
9.52 16.13 9.36 17.08
0.43 0.57 0.33 0.67
9.q6 16.60 8.82 17.81
0. ! 9 0.81 0.14 0.86
7.36 15.89 7.05 17.08
0.26 0.74 0.28 0.72
6.23 11.84 5.60 12.35
0.14 0.86 0.18 0.82
4 Magnesium
Chemicals and reagents
IAEDANS was purchased from Molecular Probes (Eugene, OR) and used without further purification. Ultra-pure urea and ammonium sulfate were obtained from S. S. Biochemicals and other chemicals were of reagent grade.
4 Calcium
20 4
Sucrose
Results
20
20 4
Emission properties o f troponin C labeled with I A E D A N S
The emission decay of TnC labeled with IAEDANS was biexponential, regardless of the conditions under which the measurements were made, including both pH 7.2 and 5.2. A typical intensity decay curve is shown
F -0
!
| Z
'r . . . . . . .
o 0
'
t5 4
-
" -IP Ww-'-",~r-',e,"
30 8
4e 2
~
v
r
~
'
61 6
w
~
77 0
TIME (all Fig. I. Fluorescence intensity decay of T n C - I A E D A N S ( 2 . 1 0 5 M ) in 0.1 M KCI, 50 mM Tris, I m M E G T A , 0.5 mM D T T ( p H 7.2), 20°C. The sharp peak on the left is lamp profile, The data were fitted to a two-exponential function: ~'i = 9.27 ns, r 2 = 16.43 ns; a~ = 0.39, "2 = 0.61. The panel across the figure is the weighted residual p%t and the inset in the upper right-hand corner is the autocorrelation function of the weighted residuals. X~ = 1.3. D . W = 1.8 and Zru n = 1.1. A log-likelihood ratio test indicates that the two-exponential fit is justified over a one-exponential fit.
Gu-HCI
20 4
(~') (ns)
~
D-W
14.54
1.3
i.7
15.79
1. I
2.0
14.11
1.2
1.7
15.42
1.2
1.8
15.79
1.4
1.7
17.16
I.I
1.9
14.71
1.0
1.9
15.70
1.4
1.4
11.41
1.2
1.7
11.76
1.2
1.9
in Fig. 1. The observation of two fluorescence lifetimes in the absence of bound cations is in agreement with our early study [11]. Upon binding either Mg 2+ or Ca 2+, the emission decay remained biexponential. This result differed from the early report that the decay became monoexponential in the presence of Mg 2+ or Ca 2+. The reduced chi-square ratio in the previous study was in the range of 1.9-2.0 and higher than in the present study. The present biexponential fit was additionally supported by other statistical paremeters, and the log-likelihood ratio test ruled out a one-exponential model. Since the loss of sulfhydryl content after labeling agreed with the degree of labeling, the biexponential decay characteristic was not due to lab,cling heterogeneity. The best fitted parameters recovered for different conditions at pH 7.2 are given in Table I. In the absence of cations at 20°C, the fractional amplitude (a I) associated with the short decay component (9.27 ns) was 0.39. Addition of Mg 2+ to saturate the two high-affinity sites did not affect either the lifetimes or the amplitudes. When Ca 2+ was added to saturate both the two high-affinity and the two low-affinity sites, the two decay times were little affected, but
19 T A B L E !1
o~2eE
Fluorescence intensity decays of TnC-IAEDANS at pH 5.2 All conditions are the same as shown in Table I. Medium
T (°C)
"r,(ns)
a i
EGTA
20
10.09 18.34 10.96 19.27
0.22 0.78 0.23 0.77
9.66 18.02 10.01 18.92
0.27 0.73 0.31 0.69
12.24 19.55 8.27 19.10
0.32 0.68 0.14 0.86
7.89 18.13 9.07 19.28
0.13 0.87 0.14 0.86
10.58 14.85 10.92 16.04
0.80 0.20 0.78 0.22
4 Magnesium
20 4
Calcium
20 4
Sucrose
20 4
Gu-HCI
20 4
(I") (ns)
X~
D-W Lm
17.26
1.4
1.4
18.05
I. I
2.2
16.64
1.2
1.8
17.21
i.3
1.8
17.89
i.7
1.3
18.36
!.1
1.7
17.16
i.2
1.8
18.01
I. I
1.7
11.80
1.4
1.7
12.40
i.3
1.8
i.., I
a, decreased by a factor of two to 0.19. This result indicated a shift of the two decay components in favor of the long component (a2). This shift was also observed at 4°C. in the presence of sucrose, both lifetimes were slightly decreased, but the decay was shifted toward to long component with a 33% reduction in a~ and no change in the weighted mean lifetimes. Denaturation of the protein in 6 M guanidine hydrochloride led to a significant decrease in both lifetimes and an increase of a2 by 41%. The intensity decay parameters recovered at pH 5.2 are listed in Table II. In general, the two decay components and the weighted mean lifetime were slightly higher than at pH 7.2. The amplitude a= was significantly smaller (0.22) than at neutral pH. While Mg 2+ had little effect on both the lifetimes and their amplitudes, Ca 2+ binding to all four sites increased the short lifetime by 2 ns and the long lifetime by 1 ns. When compared with pH 7.2, the decay observed at pH 5.2 was shifted by the addition of Ca z+ toward the short component with a mincreasing from 0.22 to 0.32. This Ca 2+ effect at pH 5.2 was in the opposite direction observed for pH 7.2.
O0
1
I
i
3120
~
48 0
I
.
64.0
t
B0 o
Fig. 2. A representative fluorescence anisotropy decay curve af TnC-IAEDANS (pH 5.2), 20°C. Conditions are the same as for Fig. I, except pH 5.2 (50 mM Mes). The decay was titled to a double-exponential function: 6j = 1.34 ns, dPz = 13.59 ns; glro = 0.1a.5, g 2 r , = 0.136, r o = 0.281. A'~ = !.8, D - W = 1.7.
7.2 are given in Table !II, and the parameter~ recovered for pH 5.2 are given in Table IV. The short rotational correlation times (d)~) at pH 7.2 were in the range of 1 to 3 ns, and the long correlation times (d)2) were in the range of 11-41 ns. At 20°C and in the absence of added cations, d)~ = 1.17 ns and 4~2- 11.3 ns. The short correlation time could be attributed to probe motion, and the long correlation time may reflect the overall rotational motion of the protein. The rotational correlation time of an equivalent T A B L E 11!
Anisotropy decays of TnC-IAEDANS at pH Z 2 Conditions are the .same as for Table I. The anisotropy decay was resolved into two components, with 0 t referring :o the short correlation time and 0 z referring to the long correlation lime. The amplitudes of the decays associated with the short and long correlation times are g=r o and g2ro, respectively. The zero-lime anisotropy is r 0 , = g l r o + g2ro. The angular range of the probe motion 0 was calculated according In Eqn. 4. Medium
T(°C)
d)i(ns)
gtro
EGTA
20
1.17 11.33 1.76 16.42
0.222 0.060 0.145 0.085
1.17 11.35 1.57 15.88
0.184 0.088 0.130 0.118
2.00 13.90 2.02 19.50
0.120 0.110 0.106 0.145
1.78 24.57 3.13 41.45
0.147 0.085 0.115 0300
4 Magnesium
20 4
Calcium
20 4
Anisotropy decay of troponin C labeled with IAEDANS The anisotropy decays of TnC labeled with IAEDANS were fitted to both a monoexponential and a double-exponential function. The best fits were obtained in all cases with a two-component model (Fig. 2). The anisotropy decay parameters recovered for pH
~
|60
Sucrose
20 4
ro
X2
D-W
0(deg.)
0.282
1.4
2.0
54.4
0.230
1.1
2.0
44.7
0.272
1.4
2.0
41.0
0.248
1.3
2.3
33.0
0.230
1.5
2.2
41.0
0.751
1.3
2.0
33.9
0.232
1.4
2.0
44.9
0.215
1.6
1.9
39.6
20
r,
~C~ D-W 0(deg.)
tion time of apo-TnC was 13.59 ns at pH 5.2 as compared to I 1.33 ns at pH 7.2. In sucrose the long correlation time increased by 28% from 24.57 to 31.58 ns when the pH was dropped from neutral to 5.2. The general effect of pH 5.2 was to increase the long rotational correlation time.
0.281
1.8
1.7
38.6
Discussion
0.271
1.3
1.9
31.1
0.328
1.9
1.8
43.6
0.243
1.4
1.8
3(I.8
1.6
1.9
35.6
2.2
2.(i
35.2
1.3
2.0
28.3
!.4
2.1
28.3
TABLE IV
Anisotropg" decays of TnC-IAEDANS at pH 5.2 All conditions are the same as shown in Table Ill, exccpl pH = 5.2 (50 mM Mes). Medium
T(°C)
~(ns)
g, ra
EGTA
20
1.34 13.59 1.24 19.38
0.145 0.136 0.100 0.171
1.00 12.84 1.91 16.57
0.200 0.128 0.088 0.155
1.77 14.88 1.36 2(I.68
0.120 0.143 (I.263 (1.134 0.165 0.299
5.67 31.58 4.36 53.79
0.061 0.133 0.194 0.(]68 0.148 0.216
4 Magnesium
20 4
Calcium
20 4
Sucrose
20 4
hydrated sphere (&0)with a partial specific volume (T) of 0.74 cma/g and a hydration (h) of 0.2 g w a t e r / g protein is 7.0 ns from the relationship 6 , = V,7/kT = M('~ + h ) T I / k T , where V is the molecular volume, T/ the medium viscosity, k the Boltzmann constant, and T the absolute temperature. The ratio &2/&0 is 1.61, indicating that TnC is not highly symmetric in neutral solution. Mg 2+ had no effect on both correlation times, but Ca 2+ increased d~t and (b2 by l and 2 ns, respectively. The two correlation times increased significantly upon lowering the temperature to 4°C. When the medium viscosity was increased by the addition of sucrose, 4~t increased by a factor of 1.5 and &2 by a factor of 2.2. If the probe motion was confined within a cone of semiangle 0, its angular range (0) is related to the amplitudes of the two rotational modes by r2
cos20( 1 + cos 0 ) 2
r t +r 2
4
(4)
The results of this calculation are shown in the last column in Tables Ill and IV and indicated that the attached probe had considerable rotational freedom. The angular displacement decreased significantly when the protein was saturated with either Mg 2+ o r C a 2+. With increasing viscosity, the rotational freedom of the probe was also significantly reduced. As expected, the motion of the probe was more restricted when the temperature was lowered. The anisotropy decay pattern at pH 5.2 was similar to that observed at pH 7.2. At 20°C the long correla-
The probe attached to Cys-98 of TnC is near one of the two high-affinity C a / M g sites, but far removed from the two low-affinity, Ca 2*-specific regulatory sites on the basis of either the amino acid sequence or the reported crystal structure. At neutral pH, the ratio of the two amplitudes a 2 / a I for apo-protein is 1.6. Saturation of the two high-affinity sites with Mg 2÷ has little or no effect on the two lifetimes and reduces the amplitude ratio slightly to 1.3, in spite of the close proximity of Cys-98 to one of the Mg 2÷ sites. While both ~-m and r 2 are also relatively unchanged when M g 2* in the solution is replaced by sufficient C a 2+ t o saturate both the high-affinity and the low-affinity sites, a 2 / a ~ increases to 4.3. This change indicates a shift of the two decay components in favor of the long component and suggests the presence of two conformational states. We have no decay data for the system in which only the two high-affinity sites are saturated with C a 2 + and do not know the precise effect of Ca 2. binding to these sites on the decay properties of TnC-IAEDANS. However, we [l 1] previously showed that M g 2÷ elicited the same increase in the quantum yield of TnCIAEDANS as an stoichiometric amount of Ca 2÷ for the two high-affinity sites. These previous results suggest that the decay properties of the labeled protein are very similar regardless of whether the high-affinity sites are occupied by Mg 2+ or Ca 2+. Thus, the large increase in a 2 / a j induced by Ca 2+ binding to all four sites may be taken as evidence for a significant effect of Ca 2 * binding to the low-affinity sites on the environment of Cys-98. The conformation associated with the long-decay component is promoted by Ca 2+ binding to its specific sites. This result adds to the current knowledge that there is possible molecular communication between the two ends of the dumbbell-shaped STnC. Since the two specific sites located in the N-domain are separated from Cys-98 by a long central helix, it is difficult to visualize how the structural changes occurring in the N-domain may be sensed at a distal site. It has been suggested in various reports that the central helix may not be rigid because of the presence of a glycine in posistion 92 in the middle of the D / E linker. A recent 2-D NMR study from this laboratory has shown that a peptide with a sequence corresponding to the middle of the central helix of TnC can exist in a bent structure with a bend around Gly-92 (unpublished data). We have recently shown that the half-width of
21 the distribution of the distances between Met-25 and Cys-98 as determined by fluorescence resonance energy transfer is relatively insensitive to Mg 2+, but considerably reduced by the presence of Ca 2+ [18]. This altered conformational dynamics induced by specific Ca z+ binding may be the basis for the transfer of structural information between the N-domain and the region of Cys-98 as demonstrated here. It is of interest to examine the origin of the short correlation time observed with TnC-IAEDANS. A conjugate of cysteine-lAEDANS has a rotational correlation time of 0.092 ns at 20°C and 0.15 ns at 4°C [19]. if the probe attached to the Cys-98 of TnC is assumed to have a completely free rotation independent of protein structure, its rotational correlation time would be in the range of 0.092-0.5 ns at 20°(: and 0.15-0.9 ns at 4°C. The two lower limits correspond to probe motion without orientational restriction and the upper limits to motion confined to a single axis. The values of &~ observed at both temperatures are at least one order of magnitude larger than the correlation time expected of free rotation. Clearly, the probe motion is far from being free and independent of protein structure. The dependence of &~ on medium viscosity provides a means to examine the possibility that the short correlation time may arise from a hindered motion of the attached probe [18]. Such a correlation time would depend upon the rate of transition between a set of preferred positions corresponding to encrgy minima. This transition in turn is a function of medium viscosity. From the ratio of the two &n values determined in water and in sucrose and the ratio of the viscosities of the two solutions, a correlation time (&) of the protein can be estimated for the hindered rotation model. The estimated values are a factor of 5-10 smaller than the observed long correlation time. Although these estimates are only qualitative, the results suggest that the observed short correlation time is unlikely due to restricted probe motion. Instead, they may suggest internal motion of the protein involving a segment of the polypeptide chain adjacent to Cys-98. This motion may be related to the putative segmental motion of the central helix. In the absence of bound ligand, 79% of the total anisotropy of TnC-IAEDANS decays at 2ff'C with the short component and the recovered total anisotropy r o is close to the expected value of the probe in the absence of molecular motion. The angular range of the motion associated with the short component is quite large, and this suggests that the probe is likely on the surface of the protein, in the presence of Mg z+, this contribution decreases to 68% without much effect on the recovered anisotropy. However, the contributions of the two components become about equal with a 20% decrease in r 0 when all four sites are occupied by Ca 2+. This loss in anisotropy suggests the existence of
some fast motion in the Ca 2+ complex that is outside the time window of the experiment. Coupled with the Iower anisotropy is a more restricted angular motion as 0 is now reduced from 54 to 41 °. Since we have no information on the magnitude of the Ca-' +-induced fast motion (except that it is subnanosecond), its origin remains unclear. At 4°C r o is significantly lower than at 20°C, but both Mg z+ and Ca :'+ have little or no effect on this value. If anything, these cations seem to recover some of the lost anisotropy at the lower temperature. This observation is to be contrasted with the Ca-" + effect at 20°C. These 4°C results are complicated by both thermal and viscosity effects on localized motions of the attached probe or a segment of the protein linked to the probe. An increase in bulk viscosity at 20°C induces isothermal depolarization effects as reflected by a lower r, value in the presence of sucrose. These viscosity-dependent depolarization effects are enhanced by lowering of the temperature. The present data do not allow delineation of viscosity effect from thermal effect on the depolarization of the probe. Importantly, at a sufficiently high temperature (i.e., 20°C) Ca 2 +, not Mg z+, induces a significant depolarization of the attached probe and this depolarization can be interpreted in terms of enhanced localized motions occurring in the subnanosecond regime. At pH 5.2 the emission decay parameters of TnCIAEDANS are different from those observed at neutral pH in two aspects. The ratio of a 2 / a ~ is factor of two larger than at pH 7.2. This suggests an alteration of the relative proportions of the two conformations when compared with the pH 7.2 system. Acidic pH induces a 20% increase ( > 2 ns) in the long correlation time. This result is compatible with the previous report that TnC has a slightly larger sedimentation coefficient and is a dimer at pH 5.2 [20]. In summary, the time-resolved fluorescence results show that the conformation of skeletal TnC is more sensitive to the binding of activator calcium to its specific sites than the binding of calcium or magnesium to the high-affinity sites. Subnanosecond motions originating from a region of the protein far removed from the Ca2+-specifie sites are influenced by saturation of these sites. These results support the notion of a molecular communication between the N-domain and a distal site along the central helix, a feature that may have relevance in signal transduction in skeletal muscle.
Acknowledgement This work was supported in part by a grant from the U.S. National Institutes of Health, AR25193.
22 References 1 Sundaralingam, M., Bergstrom, B., Strasbury, G., Rao, S,T., Roychowdhury, P., Greaser, M. and Wang. B.C. (1985) Science 227, 945-948. 2 Herzberg, O. and James, M.N.G. (1985) Nature 313. 653-659. 3 Hitchcock, S.E., Huxley, H. and Szent-Gyorgyi, A.G. (1973) J. Mol. Biol. 80, 825-836. 4 Margossian, S.S. and Cohen, C. (1973) J. Mol. Biol. 81,409-413. 5 Potter, J.D. and Gergely, J. (1974) Biochemistry 13, 2697-2703. 6 Wang, C.-K. and Cheung, H.C. (1985) Biophys. J. 48, 727-739. 7 Wang, C,-K, and Cheung, H.C. (1986)J. Mol. Biol. 191,727-739. 8 Tao, T., Gowell, E., Strasburg, G.M., Gergely, J. and Leavis, P.C. (1989) Biochemistry 28, 5902-5908. 9 Cheung, H.C., Wang, C.-K. and Malik, N.A. (1987) Biochemistry 26, 5904-5907. 10 Tao, T., Gong, B.-J. and Leavis, P.C. (1990) Science 247, 13391341. 11 Cheung, H.C., Wang, C.-K. and Garland, F. (1982) Biochemistry 21, 5135-5142,
12 Hudson, E.N. ann Weber, G. (1973) Biochemistry 12, 4154-4161. 13 Grinvald, A. and Steinberg, I.Z. (1974) Anal. Biocbem. 59, 583593. 14 Durbin, J. and Watson, G.S. (1951) Biometrika 38, 159-t78. 15 Lamp~rt, R.A., Chewter, L.A., Phillips, D., O'Conner, D.V., Roberts, A.J. and Meech, S.R. (1983) Anal. Chem. 55, 68-73. 16 Gunst, R.F. and Mason, R.L. (1980) Regression Analysis and Its Application, Marcel Dckker, New York, pp. 231-141. 17 Gross, A.J. and Clark, V.A. (1975) Survival Distributions: Reliabilily Application in the Biomedical Sciences, Wiley, New York, pp. 228-233. 18 Cheung, H.C., Wang, C.-K, Gryczynski, I.o Wiczk, W., Laczko, G., Johnson, M.L. and Lakowicz, J.R. (19ql) Biochemistry 30, 5238-5247. 19 Onto. J., Bucci, E., Steiner, R.F,, Fronticelli. C., Franchi, D., Montemarano, J. and Martinez, A. (1981) J. Biol. Chem. 256. 7248-7256. 20 Wang, C.-K., Lebowitz, J. and Cheung, H.C. (1989) Proteins: Structure, Function and Genetics 6. 424-430.
16
BBAPRO M I ~
Nanosecond study of fluorescently labeled troponin C Chien-Kao Wang ~'*, Ronglih Liao b'** and Herbert C. Cheung :' a Depurtmem of Biochemist~'. Unic,'rsiO' of Alabama at Birmingham. Birmingham. AL (USA) and i, (;raduate Program in Biophysical Sriem'es. Unirersity of Ah, bama at Birmingham, Birminghum. AL (USA)
(Received g September Iq91)
Key words: Exlrinsic fluorescence: Trol~min C: (Rabbit skclelai)
The time-resolved extrinsic fluorescence of rabbit skeletal troponin C v,as studied with the protein labcled at Cys-98 with N-(iodoacetyl)-N'-(5-sulfi~-l-naphthyl)cthylenediamine. Both the intensity and anisotropy decays fi)llowed a biexponcntial decay law, regardless of the ionic condition, pH. viscosity or temperaturc. The lifetimcs and their fractional amplitudes were insensitive to Mg -~+, and the lifetimes were also insensitive to Ca 2'. in response to Ca -~ binding to all four sites, the fractional amplitude (a I) associated with the short lifetime (r~)decreased by a factor of two, thus increasing the ratio of the two amplitudes a 2 / a ~ from !.6 to 4.3. These amplitude changcs suggest the existence of two conformaiional states of TnC-IAEDANS, with the conformation associated with the long-decay component (T~) being promoted by saturation of the two Ca-'+-specific sites. At pH 5.2 the ratio a~/a~ lot the apo-protein was 3.5 indicating different relative populations of the two decay compoi~ents when compared with pH 7.2. In the presence of Ca ~+ at the lower pH, a 2 / a I decreased to 2.1, suggesting a shift of the conformations in favor of the short-decay component. Thus Ca ~-~ elicited different conformational changes in TnC at the two pH values. The recovered anisotropies suggest that there were fast molelcular motions that were not resolved in the present expcriments, and some of these motions wcre sensitive to Ca 2 ~ binding to the specific sites. These results support the notion of communication between the N-domain and the C-terminal end of ~hc central helix of troponin C.
Introduction
Troponin from striated muscle consists of three nonidentical, and structurally and functionally distinct subunits. Troponin T is the tropomyosin-binding subunit, troponin I is the subunit responsible for the inhibition of actomyosin ATPas¢ and troponin C is the calciumbinding subunit which is responsible for transmission of the signal of activator calcium to other proteins in activation of actomyosin ATPase. In vertebrate muscle, troponin is associated with tropomyosin in a 1 : I stoichiometry, and this complex is bound to the actin
* Present address: Department ,)f Physiology and Biophysics. University of Washington, Seattle. WA. USA. ** Present address: Division of Cardiology. Belh Israel Hospital. Boston. MA. USA. Abbreviations: TnC. troponin C: CTnl. cardiac troponin l: IAEDANS. N-(iodoacelyD-N'45-sulfo. I-naphthyl)ethylenediaminc: TnC-IAEDANS. TnC labeled at Cys-98 with IAEDANS: Gu-HCI. guanidine hydrochloridc: D'Fr. dithiolhreiiol; HWHM, full width a! half-maximum height. Correspondence: H.C. Cheung. Department of Biochemistry. University of Alabama at Birmingham. Birmingham. AL 35294. USA.
filament with a stoichiometry of 1 tropomyosintroponin to 7 actin monomers. The three-dimensional crystal structures of T n C from the skeletal muscle of chicken [1] and turkey [2] have been reported and show the protein to have an unusual dumbbell shape, with the amino and c a r b o ~ l terminal segments folded into two globular domains. The two domains are connected by a 9-turn a-helix the middle one-third of which is totally exposed to solvent in the crystal structure. The structure has four E F hands, each of which consists of a helix-loop-helix motif, and the four loops are the sites at which Ca 2+ binds. The two sites located in the N-terminal half (sites ! and II) bind Ca 2÷ specifically with a low affinity ( K , , - - 2 - 10 ~ M - i ) , and the other two sites located in the C-terminal half (sites I!I and IV) bind Ca 2+ with a high affinity (K~,--2" 10 7 M - I ) and also bind Mg 2+ competitively. Becasuc of the relatively high intracellular Mg 2+ concentration, sites 111 and IV are likely saturated with Mg 2+ in relaxed muscle, and these sites do not appear to have a major role in calcium regulation. In contrast, sites ! and Ii (regulatory sites) are unoccupied until the arrival of activator Ca 2+ in the concentration range of 10/zM. The mechanism by which calcium triggers contraction is not fully understood. Early studies have sug-
17 gested that the interaction between TnC and Tnl is relatively weak in relaxed muscle and that the binding of calcium to the specific sites of TnC enhances this interaction and concomitantly weakens the interaction between Tnl and actin [3-5]. The latter event allows Tnl detachment from actin and a lateral movement of tropomyosin on the actin helix. It is now generally agreed that the binding of activator calcium induces structural changes not only in TnC itself, but also in the other proteins associated with the thin filament, namely, the other two troponin subunits, tropomyosin and actin. Many lines of evidence have suggested that these structural changes and the accompanying alterations of the affinities of these proteins provide a molecular basis for calcium regulation [6-8]. On the basis of energetic and other considerations, Cheung et al. [9] and Tap et al. [10] have suggested that troponin 1 and the T n i - T n C linkage may play a fundamental role in signal tansduction in skeletal muscle. in the present work, we report a time-resolved study of the extrinsic fluorescence of TnC. Since the crystals of TnC used in structure determination were obtained at pH 5.0-5.1, the present studies were carried out at two different pH values to investigate the effect of acidic pH on the global conformation of the protein. The results indicate that the binding of calcium to the specific sites can induce a spectrum of conformational changes and fast molecular motions. Some of these fast motions are sensitive to the binding of activator calcium. The studies provide further evidence of structural communication be,ween the N-domain and the C-terminal region of the long central helix connecting the two globular ends of the protein.
Materials and Methods
Protehz preparation Troponin was extracted from rabbit skeletal muscle as described in our previous work [! 1]. The protein was dissolved in 6 M urea and TnC was separated from the other two subunits by DEAE-cellulose (DE-52) column chromatography in the presence of urea [6]. The purity of the isolated TnC was monitored by SDS-PAGE, and its concentration was estimated from an absorbance of 0.23 g-~ c m - ' at 276 nm and a molecular weight of 18000. TnC was labeled at Cys-98 with IAEDANS as in our previous work [6]. The extent of labeling was determined to be about 0.95 tool of probe/tool of protein by absorbance measurements using an absorption coefficient of 6100 M - t c m -t at 337 nm [12]. Suifhydryl content was determined with 5,5'-dithiobis2-nitrobenzoic acid (DTNB) in 50 mM Tris, 90 mM KCI and 2 mM EGTA at pH 8.0.
Time-resoh'ed fluorescence measurements Fluorescence intensity decay and fluorescence anisotropoy decay were measured with a PRA (Photochemical Research Asmciatcs) single-photon counting nanosecond fluorometer. The spark lamp was thyratron-triggered, H2-filled (14 mm Hg vaccum) and run at 32 kHz. The pulses had a typical FWHM of 2.0 ns. A Ditric 3-cavity 340-rim interference filter (FWHM 8.0 nm) was used to isolate the excitation light and the emission of IAEDANS was isolated with a Ditric 3-cavity 490-nm interference filter {FWHM 7.0 nm). Lifetimes were determined using rotation free optical conditions (excitation light polarized vertically and emitted light polarized at the magic angle of 54.7 ° from the vertical). Lamp profiles were measured at the same emission wavelengths with a pair of Ditric 3-cavity interference filters using Ludox as the scattering agent. To optimize the signal to noise ratio, a minimum of I0000 photon counts were collected in the peak channel. The data stored in a multichannel analyzer were transferred to either a DEC 11/03 microcomputer or an IBM P C / A T personal computer for analysis. Intensity decay curves were fitted to a sum of exponential terms:
F(t)
=
~ a , exp(-t/~'i)
(I)
i
where tr, are the amplitudes associated with the i decay components, each having a lifetime 7-,. The decay parameters were recovered using nonlinear leastsquares iterative procedures with the Marquardt algorithm [ 131. Emission anisotropy decay was determined by using vertically polarized excitation light and measuring alternately the emission polarized in the vertical [FIl(t)] and horizontal [Fj. (t)] directions. This was achieved by changing the orientation of the emission polarizer at 20-min intervals. Photon counts were collected until at least 20000 counts were reached in the peak channel for the Fu(t) component. The F±(t) component was collected for the .same period of time as for Ftt(t). The anisotropy decay r(t) was calculated from
r(t) =
Ftl - F l (t) Fll(t ) + 2 F j . ( t )
(2)
and the deca~' data were fitted to a sum of exponential terms,
r( t) = rt,~gi exp(-ttd~,)
(3)
i
where g, are the fractional amplitudes of the components associated with the correlation times d'i. For a
18 biexponentiai decay law the total anisotorpy r. is given by r o = glro + g2ro with ~g, -- 1.0. The goodness of a fit between a given set of observed data and the chosen function was evaluated by the weighted residuals, the autocorrelation function of the weighted residuals, the reduced chi-square ratio (~C~), the Durbin-Watson paremeter (D-W)[14,15] and the runs test (Z,~.) [16]. A fit was considered acceptable when plots of the weighted residuals and the autocorrelation function showed random deviation about zero with a ,¢~ value of about 1 and a value of the D-W parameter that satisfied theoretical estimates. The log-likelihood ratio test [17] was also used to discriminate between a two-exponential and a oneexponential model.
TABLE I Fluorescence intensity decays o f T n C - I A E D A N S at p H 7 2 Measurements were carried out with TnC-IAEDANS ( = 2. l0 -s M) in 0.1 M KCI, 50 mM Tris, 1 mM DTT. I mM EGTA (pH 7.2). When present, [Ca-" + ]= 1.5 raM, [Mg -~+ I = 2 raM, s u c r o ~ = 25% ( W / V ) . [Gu-HCI] = 6 M. The decay was resolvvd into two cor~ponenls, with ¢1 referring to the short lifetime and ~'z to the long lifetime, a I and a , refer to the fractional amplitudes associated with the two respeclive lifetimes. The weighted average lifetime is given by ( v ) = r,,,,,T~/F,a, Ti. D - W is the Durbin-Watson parameter. Medium
T(°C)
r, (ns)
ai
EGTA
20
9.27 16.43 10.21 17.99
0.39 0.61 0.41 0.59
9.52 16.13 9.36 17.08
0.43 0.57 0.33 0.67
9.q6 16.60 8.82 17.81
0. ! 9 0.81 0.14 0.86
7.36 15.89 7.05 17.08
0.26 0.74 0.28 0.72
6.23 11.84 5.60 12.35
0.14 0.86 0.18 0.82
4 Magnesium
Chemicals and reagents
IAEDANS was purchased from Molecular Probes (Eugene, OR) and used without further purification. Ultra-pure urea and ammonium sulfate were obtained from S. S. Biochemicals and other chemicals were of reagent grade.
4 Calcium
20 4
Sucrose
Results
20
20 4
Emission properties o f troponin C labeled with I A E D A N S
The emission decay of TnC labeled with IAEDANS was biexponential, regardless of the conditions under which the measurements were made, including both pH 7.2 and 5.2. A typical intensity decay curve is shown
F -0
!
| Z
'r . . . . . . .
o 0
'
t5 4
-
" -IP Ww-'-",~r-',e,"
30 8
4e 2
~
v
r
~
'
61 6
w
~
77 0
TIME (all Fig. I. Fluorescence intensity decay of T n C - I A E D A N S ( 2 . 1 0 5 M ) in 0.1 M KCI, 50 mM Tris, I m M E G T A , 0.5 mM D T T ( p H 7.2), 20°C. The sharp peak on the left is lamp profile, The data were fitted to a two-exponential function: ~'i = 9.27 ns, r 2 = 16.43 ns; a~ = 0.39, "2 = 0.61. The panel across the figure is the weighted residual p%t and the inset in the upper right-hand corner is the autocorrelation function of the weighted residuals. X~ = 1.3. D . W = 1.8 and Zru n = 1.1. A log-likelihood ratio test indicates that the two-exponential fit is justified over a one-exponential fit.
Gu-HCI
20 4
(~') (ns)
~
D-W
14.54
1.3
i.7
15.79
1. I
2.0
14.11
1.2
1.7
15.42
1.2
1.8
15.79
1.4
1.7
17.16
I.I
1.9
14.71
1.0
1.9
15.70
1.4
1.4
11.41
1.2
1.7
11.76
1.2
1.9
in Fig. 1. The observation of two fluorescence lifetimes in the absence of bound cations is in agreement with our early study [11]. Upon binding either Mg 2+ or Ca 2+, the emission decay remained biexponential. This result differed from the early report that the decay became monoexponential in the presence of Mg 2+ or Ca 2+. The reduced chi-square ratio in the previous study was in the range of 1.9-2.0 and higher than in the present study. The present biexponential fit was additionally supported by other statistical paremeters, and the log-likelihood ratio test ruled out a one-exponential model. Since the loss of sulfhydryl content after labeling agreed with the degree of labeling, the biexponential decay characteristic was not due to lab,cling heterogeneity. The best fitted parameters recovered for different conditions at pH 7.2 are given in Table I. In the absence of cations at 20°C, the fractional amplitude (a I) associated with the short decay component (9.27 ns) was 0.39. Addition of Mg 2+ to saturate the two high-affinity sites did not affect either the lifetimes or the amplitudes. When Ca 2+ was added to saturate both the two high-affinity and the two low-affinity sites, the two decay times were little affected, but
19 T A B L E !1
o~2eE
Fluorescence intensity decays of TnC-IAEDANS at pH 5.2 All conditions are the same as shown in Table I. Medium
T (°C)
"r,(ns)
a i
EGTA
20
10.09 18.34 10.96 19.27
0.22 0.78 0.23 0.77
9.66 18.02 10.01 18.92
0.27 0.73 0.31 0.69
12.24 19.55 8.27 19.10
0.32 0.68 0.14 0.86
7.89 18.13 9.07 19.28
0.13 0.87 0.14 0.86
10.58 14.85 10.92 16.04
0.80 0.20 0.78 0.22
4 Magnesium
20 4
Calcium
20 4
Sucrose
20 4
Gu-HCI
20 4
(I") (ns)
X~
D-W Lm
17.26
1.4
1.4
18.05
I. I
2.2
16.64
1.2
1.8
17.21
i.3
1.8
17.89
i.7
1.3
18.36
!.1
1.7
17.16
i.2
1.8
18.01
I. I
1.7
11.80
1.4
1.7
12.40
i.3
1.8
i.., I
a, decreased by a factor of two to 0.19. This result indicated a shift of the two decay components in favor of the long component (a2). This shift was also observed at 4°C. in the presence of sucrose, both lifetimes were slightly decreased, but the decay was shifted toward to long component with a 33% reduction in a~ and no change in the weighted mean lifetimes. Denaturation of the protein in 6 M guanidine hydrochloride led to a significant decrease in both lifetimes and an increase of a2 by 41%. The intensity decay parameters recovered at pH 5.2 are listed in Table II. In general, the two decay components and the weighted mean lifetime were slightly higher than at pH 7.2. The amplitude a= was significantly smaller (0.22) than at neutral pH. While Mg 2+ had little effect on both the lifetimes and their amplitudes, Ca 2+ binding to all four sites increased the short lifetime by 2 ns and the long lifetime by 1 ns. When compared with pH 7.2, the decay observed at pH 5.2 was shifted by the addition of Ca z+ toward the short component with a mincreasing from 0.22 to 0.32. This Ca 2+ effect at pH 5.2 was in the opposite direction observed for pH 7.2.
O0
1
I
i
3120
~
48 0
I
.
64.0
t
B0 o
Fig. 2. A representative fluorescence anisotropy decay curve af TnC-IAEDANS (pH 5.2), 20°C. Conditions are the same as for Fig. I, except pH 5.2 (50 mM Mes). The decay was titled to a double-exponential function: 6j = 1.34 ns, dPz = 13.59 ns; glro = 0.1a.5, g 2 r , = 0.136, r o = 0.281. A'~ = !.8, D - W = 1.7.
7.2 are given in Table !II, and the parameter~ recovered for pH 5.2 are given in Table IV. The short rotational correlation times (d)~) at pH 7.2 were in the range of 1 to 3 ns, and the long correlation times (d)2) were in the range of 11-41 ns. At 20°C and in the absence of added cations, d)~ = 1.17 ns and 4~2- 11.3 ns. The short correlation time could be attributed to probe motion, and the long correlation time may reflect the overall rotational motion of the protein. The rotational correlation time of an equivalent T A B L E 11!
Anisotropy decays of TnC-IAEDANS at pH Z 2 Conditions are the .same as for Table I. The anisotropy decay was resolved into two components, with 0 t referring :o the short correlation time and 0 z referring to the long correlation lime. The amplitudes of the decays associated with the short and long correlation times are g=r o and g2ro, respectively. The zero-lime anisotropy is r 0 , = g l r o + g2ro. The angular range of the probe motion 0 was calculated according In Eqn. 4. Medium
T(°C)
d)i(ns)
gtro
EGTA
20
1.17 11.33 1.76 16.42
0.222 0.060 0.145 0.085
1.17 11.35 1.57 15.88
0.184 0.088 0.130 0.118
2.00 13.90 2.02 19.50
0.120 0.110 0.106 0.145
1.78 24.57 3.13 41.45
0.147 0.085 0.115 0300
4 Magnesium
20 4
Calcium
20 4
Anisotropy decay of troponin C labeled with IAEDANS The anisotropy decays of TnC labeled with IAEDANS were fitted to both a monoexponential and a double-exponential function. The best fits were obtained in all cases with a two-component model (Fig. 2). The anisotropy decay parameters recovered for pH
~
|60
Sucrose
20 4
ro
X2
D-W
0(deg.)
0.282
1.4
2.0
54.4
0.230
1.1
2.0
44.7
0.272
1.4
2.0
41.0
0.248
1.3
2.3
33.0
0.230
1.5
2.2
41.0
0.751
1.3
2.0
33.9
0.232
1.4
2.0
44.9
0.215
1.6
1.9
39.6
20
r,
~C~ D-W 0(deg.)
tion time of apo-TnC was 13.59 ns at pH 5.2 as compared to I 1.33 ns at pH 7.2. In sucrose the long correlation time increased by 28% from 24.57 to 31.58 ns when the pH was dropped from neutral to 5.2. The general effect of pH 5.2 was to increase the long rotational correlation time.
0.281
1.8
1.7
38.6
Discussion
0.271
1.3
1.9
31.1
0.328
1.9
1.8
43.6
0.243
1.4
1.8
3(I.8
1.6
1.9
35.6
2.2
2.(i
35.2
1.3
2.0
28.3
!.4
2.1
28.3
TABLE IV
Anisotropg" decays of TnC-IAEDANS at pH 5.2 All conditions are the same as shown in Table Ill, exccpl pH = 5.2 (50 mM Mes). Medium
T(°C)
~(ns)
g, ra
EGTA
20
1.34 13.59 1.24 19.38
0.145 0.136 0.100 0.171
1.00 12.84 1.91 16.57
0.200 0.128 0.088 0.155
1.77 14.88 1.36 2(I.68
0.120 0.143 (I.263 (1.134 0.165 0.299
5.67 31.58 4.36 53.79
0.061 0.133 0.194 0.(]68 0.148 0.216
4 Magnesium
20 4
Calcium
20 4
Sucrose
20 4
hydrated sphere (&0)with a partial specific volume (T) of 0.74 cma/g and a hydration (h) of 0.2 g w a t e r / g protein is 7.0 ns from the relationship 6 , = V,7/kT = M('~ + h ) T I / k T , where V is the molecular volume, T/ the medium viscosity, k the Boltzmann constant, and T the absolute temperature. The ratio &2/&0 is 1.61, indicating that TnC is not highly symmetric in neutral solution. Mg 2+ had no effect on both correlation times, but Ca 2+ increased d~t and (b2 by l and 2 ns, respectively. The two correlation times increased significantly upon lowering the temperature to 4°C. When the medium viscosity was increased by the addition of sucrose, 4~t increased by a factor of 1.5 and &2 by a factor of 2.2. If the probe motion was confined within a cone of semiangle 0, its angular range (0) is related to the amplitudes of the two rotational modes by r2
cos20( 1 + cos 0 ) 2
r t +r 2
4
(4)
The results of this calculation are shown in the last column in Tables Ill and IV and indicated that the attached probe had considerable rotational freedom. The angular displacement decreased significantly when the protein was saturated with either Mg 2+ o r C a 2+. With increasing viscosity, the rotational freedom of the probe was also significantly reduced. As expected, the motion of the probe was more restricted when the temperature was lowered. The anisotropy decay pattern at pH 5.2 was similar to that observed at pH 7.2. At 20°C the long correla-
The probe attached to Cys-98 of TnC is near one of the two high-affinity C a / M g sites, but far removed from the two low-affinity, Ca 2*-specific regulatory sites on the basis of either the amino acid sequence or the reported crystal structure. At neutral pH, the ratio of the two amplitudes a 2 / a I for apo-protein is 1.6. Saturation of the two high-affinity sites with Mg 2÷ has little or no effect on the two lifetimes and reduces the amplitude ratio slightly to 1.3, in spite of the close proximity of Cys-98 to one of the Mg 2÷ sites. While both ~-m and r 2 are also relatively unchanged when M g 2* in the solution is replaced by sufficient C a 2+ t o saturate both the high-affinity and the low-affinity sites, a 2 / a ~ increases to 4.3. This change indicates a shift of the two decay components in favor of the long component and suggests the presence of two conformational states. We have no decay data for the system in which only the two high-affinity sites are saturated with C a 2 + and do not know the precise effect of Ca 2. binding to these sites on the decay properties of TnC-IAEDANS. However, we [l 1] previously showed that M g 2÷ elicited the same increase in the quantum yield of TnCIAEDANS as an stoichiometric amount of Ca 2÷ for the two high-affinity sites. These previous results suggest that the decay properties of the labeled protein are very similar regardless of whether the high-affinity sites are occupied by Mg 2+ or Ca 2+. Thus, the large increase in a 2 / a j induced by Ca 2+ binding to all four sites may be taken as evidence for a significant effect of Ca 2 * binding to the low-affinity sites on the environment of Cys-98. The conformation associated with the long-decay component is promoted by Ca 2+ binding to its specific sites. This result adds to the current knowledge that there is possible molecular communication between the two ends of the dumbbell-shaped STnC. Since the two specific sites located in the N-domain are separated from Cys-98 by a long central helix, it is difficult to visualize how the structural changes occurring in the N-domain may be sensed at a distal site. It has been suggested in various reports that the central helix may not be rigid because of the presence of a glycine in posistion 92 in the middle of the D / E linker. A recent 2-D NMR study from this laboratory has shown that a peptide with a sequence corresponding to the middle of the central helix of TnC can exist in a bent structure with a bend around Gly-92 (unpublished data). We have recently shown that the half-width of
21 the distribution of the distances between Met-25 and Cys-98 as determined by fluorescence resonance energy transfer is relatively insensitive to Mg 2+, but considerably reduced by the presence of Ca 2+ [18]. This altered conformational dynamics induced by specific Ca z+ binding may be the basis for the transfer of structural information between the N-domain and the region of Cys-98 as demonstrated here. It is of interest to examine the origin of the short correlation time observed with TnC-IAEDANS. A conjugate of cysteine-lAEDANS has a rotational correlation time of 0.092 ns at 20°C and 0.15 ns at 4°C [19]. if the probe attached to the Cys-98 of TnC is assumed to have a completely free rotation independent of protein structure, its rotational correlation time would be in the range of 0.092-0.5 ns at 20°(: and 0.15-0.9 ns at 4°C. The two lower limits correspond to probe motion without orientational restriction and the upper limits to motion confined to a single axis. The values of &~ observed at both temperatures are at least one order of magnitude larger than the correlation time expected of free rotation. Clearly, the probe motion is far from being free and independent of protein structure. The dependence of &~ on medium viscosity provides a means to examine the possibility that the short correlation time may arise from a hindered motion of the attached probe [18]. Such a correlation time would depend upon the rate of transition between a set of preferred positions corresponding to encrgy minima. This transition in turn is a function of medium viscosity. From the ratio of the two &n values determined in water and in sucrose and the ratio of the viscosities of the two solutions, a correlation time (&) of the protein can be estimated for the hindered rotation model. The estimated values are a factor of 5-10 smaller than the observed long correlation time. Although these estimates are only qualitative, the results suggest that the observed short correlation time is unlikely due to restricted probe motion. Instead, they may suggest internal motion of the protein involving a segment of the polypeptide chain adjacent to Cys-98. This motion may be related to the putative segmental motion of the central helix. In the absence of bound ligand, 79% of the total anisotropy of TnC-IAEDANS decays at 2ff'C with the short component and the recovered total anisotropy r o is close to the expected value of the probe in the absence of molecular motion. The angular range of the motion associated with the short component is quite large, and this suggests that the probe is likely on the surface of the protein, in the presence of Mg z+, this contribution decreases to 68% without much effect on the recovered anisotropy. However, the contributions of the two components become about equal with a 20% decrease in r 0 when all four sites are occupied by Ca 2+. This loss in anisotropy suggests the existence of
some fast motion in the Ca 2+ complex that is outside the time window of the experiment. Coupled with the Iower anisotropy is a more restricted angular motion as 0 is now reduced from 54 to 41 °. Since we have no information on the magnitude of the Ca-' +-induced fast motion (except that it is subnanosecond), its origin remains unclear. At 4°C r o is significantly lower than at 20°C, but both Mg z+ and Ca :'+ have little or no effect on this value. If anything, these cations seem to recover some of the lost anisotropy at the lower temperature. This observation is to be contrasted with the Ca-" + effect at 20°C. These 4°C results are complicated by both thermal and viscosity effects on localized motions of the attached probe or a segment of the protein linked to the probe. An increase in bulk viscosity at 20°C induces isothermal depolarization effects as reflected by a lower r, value in the presence of sucrose. These viscosity-dependent depolarization effects are enhanced by lowering of the temperature. The present data do not allow delineation of viscosity effect from thermal effect on the depolarization of the probe. Importantly, at a sufficiently high temperature (i.e., 20°C) Ca 2 +, not Mg z+, induces a significant depolarization of the attached probe and this depolarization can be interpreted in terms of enhanced localized motions occurring in the subnanosecond regime. At pH 5.2 the emission decay parameters of TnCIAEDANS are different from those observed at neutral pH in two aspects. The ratio of a 2 / a ~ is factor of two larger than at pH 7.2. This suggests an alteration of the relative proportions of the two conformations when compared with the pH 7.2 system. Acidic pH induces a 20% increase ( > 2 ns) in the long correlation time. This result is compatible with the previous report that TnC has a slightly larger sedimentation coefficient and is a dimer at pH 5.2 [20]. In summary, the time-resolved fluorescence results show that the conformation of skeletal TnC is more sensitive to the binding of activator calcium to its specific sites than the binding of calcium or magnesium to the high-affinity sites. Subnanosecond motions originating from a region of the protein far removed from the Ca2+-specifie sites are influenced by saturation of these sites. These results support the notion of a molecular communication between the N-domain and a distal site along the central helix, a feature that may have relevance in signal transduction in skeletal muscle.
Acknowledgement This work was supported in part by a grant from the U.S. National Institutes of Health, AR25193.
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