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Motional freedom of the acetylcholinesterase-active-site-bound spin labels
JOIJRNAL
OF MAGNETIC
RESONANCE
41, 207-212 (1980)
Motional Freedom of the Acetylcholinesterase-Active-Site-Bound Spin Labels* M. J. Stefan
Institute,
PAL&c
E. Kardelj
AND University
M.
SCHARAt
of Ljubyana,
Ljubljana,
Yugoslavia
Received January 16, 1980; revised March 24, 1980 The computer simulations of EPR spectra of spin labels, selectively bound to the marmorata electric-organ-membrane-bound acetylcholinesterase active site, were used to select between isotropic Brownian diffusion and anisotropic fast libration of the piperidine ring. For both representative models a better fit was obtained for the libration of the piperidine ring of the label as reflected by the EPR spectrum. Torpedo
INTRODUCTION
The selectively bound active serine fluorphosphate spin labels are located in a pocket-shaped environment of the membrane-bound acetylcholinesterase surface (I). The enzyme is bound to the membrane as well as suspended between the pre- and the postsynaptic membrane. Because of the large molecular weight one cannot expect the motional freedom of the whole enzyme to be significant, but there might be parts of the enzyme where the conformation is changed, corresponding to reorientation in space of some enzyme sections. These modulations of the enzyme shape and orientation could influence in a random fashion the teorientation of the piperidine ring of the covalently bound spin label. Assuming that these reorientations are slow in terms of the EPR experiment (correlation times rR > lo-’ set) we must consider other possibilities of the spin-label motion. Though the rigid attachment of the label is confirmed at the phosphate group, thlere are chances for the piperidine ring to exhibit a restricted motion. There are now basically two possibilities: either there is some kind of Brownian rotation of the piperidine ring with respect to a combined mobility about several bonds, thus producing a nearly isotropic slow reorientation in space with all orientations equally probable, or the motion is restricted so as to allow only a librational motion of the piperidine ring between the extreme deflections. The electron paramagnetic resonance spectra were calculated for both the fast libration and the isotropic Brownian diffusion of the piperidine ring. We show that the first model is more probable since a better fit could be obtained between the experimental and the calculated spectra. Comparison of the spectra of labels of different size favors the librational model, and by this we corroborate the model suggested previously (2). * This work was supported by the Research Community of Slovenia. t Author to whom correspondence should be addressed. 207 0022-2364/80/140207-06$02.00/O Copyright B 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.
208
PAVSIC AND SCHARA
-
OCSL lexpcmentol I
FIG. 1. The EPR spectrum of the selectively bound spin label OcSL (I-oxyl-2,2,6,6-tetramethyl-4-piperidinyloctylphosphorofluoridate) to the active serine of the electric-organ-bound acetylcholinesterase (2) with the corresponding best-fit spectrum calculated from the librational model (T = 20°C).
MATERIALS
AND METHODS
We tried to simulate the EPR spectra and to select the more probable model. The Hamiltonian is X = /3I-I*g.S + S-A.1, [II where /3 is the Bohr magneton, II is the magnetic field, S and I are the electron and nuclear spins, while g and A are the Zeeman and hyperfine coupling tensors. Neglecting the terms which do not contribute significantly to the calculated energy levels we can write in the laboratory system X, Y, 2 with H parallel to Z X = g,zPHSz + A,,S,Z.z + A,S.zZx
PI
+ AYZSZZY.
Using the derivation of Griffith and Jost (3), and assuming libration of the piperidine ring about the molecular x axis, we can write the average values of the components of the A tensor in the molecular coordinate system xyz (A,,)
= Am
(A,,)
= A,, + (A,, - &,)‘/iU
- 0,
(A,)
= A,, - (A,, - 4,,)W
- PI.
[%(l - P) = (sin2 cp)], [31
Here the x axis coincides with the C-O bond connecting the piperidine ring with the phosphate group of the spin-label molecule and P = L sin cpocos cpo,
[41
CPO
with cpothe librational angle. The average values of the component calculated analogously to Eqs. [3].
of g were
SPIN LABELS
-38 40
-2813
BOUND TO ACETYLCHOLINESTERASE
3785
-758
__
EtSLkxperimenlol)
----
calculated
2m I6OUSSI
123E
23 25
a.53
209
43a3
FIG. 2. The EPR spectrum of the selectively bound spin label EtSL (l-oxyl-2,2,6,6-tetramethyl4-piperidinylethylfluorophosphate) to the active serine of the electric-organ-bound acetylcholinesterase (2) with the corresponding best-fit spectrum calculated from the librational model
(T = 20°C).
‘These average values of the g and A tensors for fast lib&ion were used in the starting Hamiltonian of the stochastic Liouville equation-of-motion approach to calculate the EPR lineshape for slow motion. Here we used the rigid-limit spectrum which represents the random orientation in space of the membrane fragments to obtain the tensor values. The computer program of Bruno (4) and Freed (5) was used. The same program was used for the slow Brownian tumbling calculation of the spectra. The best tensor components were evaluated from the best fit on both the long and the short spin-label molecule spectra. The rigid~
HxSL kxperlmenloli 1:.2O'C cakuloted r,=ll lC6s
----
-3850
-28 13
-1785
-758
210 1Gauss I
1298
2325
3353
4380
FIG. 3. The spectrum of HxSL (l-oxyl-2,2,6,6-tetraethyl-4-piperidiny~hexylphosphorofluo~date) taken at -20°C with the corresponding fit of the original spectrum as evaluated by this calculation with A, = 7.50G, A, = 5.91 G, A,, = 33.3 G, and g, = 2.00901, g,, = 2.00619, g,, = 2.00283. The same values of the original A and g tensors were taken also in the calculations with libmtional averaging in Figs. 1 and 2.
210
PAVSIC AND SCHARA
0.015
FIG.
4. The fitting
parameter,
0.03
0.045
0.075
0.06
OD9 tsdg)
defined
as x = 100.95 & [(f’(H,)/Af’(Hi))T,,,, - (f’(H# -f’(H&,, f’(H) is the first derivative of the EPR of intervals), is shown as a function of the librational
Af’WJLp,r.11i2 (where AJYHI) = f’WJ,,, spectrum, and N is the number angle ‘pOvariation.
limit original spectrum limit spectrum.
was then compared
RESULTS
with the low-temperature
rigid-
AND DISCUSSION
Figures 1 and 2 show the experimental spectra (2) for two representative spin labels with a short and a long hydrocarbon chain, respectively, attached across the piperidine ring about the phosphate group which is irreversibly
X 10 91
7654i 3
t_,
/ 111A ‘1
10'0
16'
1
I
acm,c 1
1ti6
r,lsl
FIG. 5. The same parameter x as defined in Fig. 4 for the variation of the isotropic rotational correlation time ra with the corresponding tensor components given in the text to Fig. 3.
SPIN
LABELS
BOUND
TO ACETYLCHOLINESTERASE TABLE
A COMPAFU~~N MODELS
@I OCSL EtSL
1
OF THE LIBRATIONAL AND BROWNIAN FOR THE ACETYLCHOLINESTERASE-ACTIVESITE-BOUND SPIN LABELS Librational model (TH = 5.5 x lo-’ set)
21.2” 27.8
211
Brownian
DIFFUSION
diffusion
Xllli”
nt (-1
Xmin
4.0 4.7
2 x 10-1 2 x 10-a
4.5 5.6
bound to the serine. The corresponding calculated spectra are shown for comparison. We have had to get the best fit to the OcSL spectrum (Fig. 1) by changing the hyperfine tensor components as well as the g tensor components. Using these tensors we tried to apply a larger librational angle to provide the best fit for the experimental spectrum of EtSL (Fig. 2). So finally we were able to evaluate the original tensor for the immobilized OcSL and EtSL, when the calculated values cpO= 21.2”andcp, = 27.8”,r espectively, were used. Figure 3 shows the comparison with the low-temperature rigid-limit spectrum. We took this agreement as another proof that the system is characterized by the same original tensor but with a fast libration about the x axis. Figures 4 and 5 show the best fit represented by the minimum in the x(ra) and x(q,-J functions. Here the linewidth parameter Tzk = 4.5 x 10’ set-’ has been used for the best fit, assuming T& = 0 for all calculated spectra. It is possible to see that for EtSL we get a better fit for the librational model. So we believe that the fast librational model, where the selectively bound spin label of the fluorphosphate type librates rapidly between the obstructions of the active serine pocket of acetylcholinesterase, interprets satisfactorily the observed EPR data. Table 1 summarizes our findings. We also tried to use approximate methods to determine the correlation times from the high- and low-field EPR spectra component linewidths (5). Unfortunately these could not be made to fit our experimental results. CONCLUSION
We have shown by computer simulation of EPR spectra for both the librational and the Brownian diffusion model that the first, by which the nitroxide group is librating fast in the environment where it is bound, reproduces better the experimental spectra.
212
PAVSIC: AND SCHARA REFERENCES
1. M. SENTJURC, A. STALC, 2. S. PEEAR, M. SCHARA,
AND A. 0. ZUPANW, Biochim. Biophys. Acta 438, 131 (1976). M. SENTJURC, A. STALC, AND A. 0. %JPANEIE, Stud. Biophys.
77,
33 (1979). 3. 0. H. GRIFFITH AND P. C. JOST, in “Spin Labeling” (L. J. Berliner, Ed.), p. 453, Academic Press, New York/San Francisco/London, 1976. 4. G. BRUNO, Dissertation, Cornell University, 1973. 5. J. H. FREED, in “Spin Labeling” (L. J. Berliner, Ed.), p. 53, Academic Press, New York/San Francisco/London, 1976.
OF MAGNETIC
RESONANCE
41, 207-212 (1980)
Motional Freedom of the Acetylcholinesterase-Active-Site-Bound Spin Labels* M. J. Stefan
Institute,
PAL&c
E. Kardelj
AND University
M.
SCHARAt
of Ljubyana,
Ljubljana,
Yugoslavia
Received January 16, 1980; revised March 24, 1980 The computer simulations of EPR spectra of spin labels, selectively bound to the marmorata electric-organ-membrane-bound acetylcholinesterase active site, were used to select between isotropic Brownian diffusion and anisotropic fast libration of the piperidine ring. For both representative models a better fit was obtained for the libration of the piperidine ring of the label as reflected by the EPR spectrum. Torpedo
INTRODUCTION
The selectively bound active serine fluorphosphate spin labels are located in a pocket-shaped environment of the membrane-bound acetylcholinesterase surface (I). The enzyme is bound to the membrane as well as suspended between the pre- and the postsynaptic membrane. Because of the large molecular weight one cannot expect the motional freedom of the whole enzyme to be significant, but there might be parts of the enzyme where the conformation is changed, corresponding to reorientation in space of some enzyme sections. These modulations of the enzyme shape and orientation could influence in a random fashion the teorientation of the piperidine ring of the covalently bound spin label. Assuming that these reorientations are slow in terms of the EPR experiment (correlation times rR > lo-’ set) we must consider other possibilities of the spin-label motion. Though the rigid attachment of the label is confirmed at the phosphate group, thlere are chances for the piperidine ring to exhibit a restricted motion. There are now basically two possibilities: either there is some kind of Brownian rotation of the piperidine ring with respect to a combined mobility about several bonds, thus producing a nearly isotropic slow reorientation in space with all orientations equally probable, or the motion is restricted so as to allow only a librational motion of the piperidine ring between the extreme deflections. The electron paramagnetic resonance spectra were calculated for both the fast libration and the isotropic Brownian diffusion of the piperidine ring. We show that the first model is more probable since a better fit could be obtained between the experimental and the calculated spectra. Comparison of the spectra of labels of different size favors the librational model, and by this we corroborate the model suggested previously (2). * This work was supported by the Research Community of Slovenia. t Author to whom correspondence should be addressed. 207 0022-2364/80/140207-06$02.00/O Copyright B 1980 by Academic Press. Inc. All rights of reproduction in any form reserved.
208
PAVSIC AND SCHARA
-
OCSL lexpcmentol I
FIG. 1. The EPR spectrum of the selectively bound spin label OcSL (I-oxyl-2,2,6,6-tetramethyl-4-piperidinyloctylphosphorofluoridate) to the active serine of the electric-organ-bound acetylcholinesterase (2) with the corresponding best-fit spectrum calculated from the librational model (T = 20°C).
MATERIALS
AND METHODS
We tried to simulate the EPR spectra and to select the more probable model. The Hamiltonian is X = /3I-I*g.S + S-A.1, [II where /3 is the Bohr magneton, II is the magnetic field, S and I are the electron and nuclear spins, while g and A are the Zeeman and hyperfine coupling tensors. Neglecting the terms which do not contribute significantly to the calculated energy levels we can write in the laboratory system X, Y, 2 with H parallel to Z X = g,zPHSz + A,,S,Z.z + A,S.zZx
PI
+ AYZSZZY.
Using the derivation of Griffith and Jost (3), and assuming libration of the piperidine ring about the molecular x axis, we can write the average values of the components of the A tensor in the molecular coordinate system xyz (A,,)
= Am
(A,,)
= A,, + (A,, - &,)‘/iU
- 0,
(A,)
= A,, - (A,, - 4,,)W
- PI.
[%(l - P) = (sin2 cp)], [31
Here the x axis coincides with the C-O bond connecting the piperidine ring with the phosphate group of the spin-label molecule and P = L sin cpocos cpo,
[41
CPO
with cpothe librational angle. The average values of the component calculated analogously to Eqs. [3].
of g were
SPIN LABELS
-38 40
-2813
BOUND TO ACETYLCHOLINESTERASE
3785
-758
__
EtSLkxperimenlol)
----
calculated
2m I6OUSSI
123E
23 25
a.53
209
43a3
FIG. 2. The EPR spectrum of the selectively bound spin label EtSL (l-oxyl-2,2,6,6-tetramethyl4-piperidinylethylfluorophosphate) to the active serine of the electric-organ-bound acetylcholinesterase (2) with the corresponding best-fit spectrum calculated from the librational model
(T = 20°C).
‘These average values of the g and A tensors for fast lib&ion were used in the starting Hamiltonian of the stochastic Liouville equation-of-motion approach to calculate the EPR lineshape for slow motion. Here we used the rigid-limit spectrum which represents the random orientation in space of the membrane fragments to obtain the tensor values. The computer program of Bruno (4) and Freed (5) was used. The same program was used for the slow Brownian tumbling calculation of the spectra. The best tensor components were evaluated from the best fit on both the long and the short spin-label molecule spectra. The rigid~
HxSL kxperlmenloli 1:.2O'C cakuloted r,=ll lC6s
----
-3850
-28 13
-1785
-758
210 1Gauss I
1298
2325
3353
4380
FIG. 3. The spectrum of HxSL (l-oxyl-2,2,6,6-tetraethyl-4-piperidiny~hexylphosphorofluo~date) taken at -20°C with the corresponding fit of the original spectrum as evaluated by this calculation with A, = 7.50G, A, = 5.91 G, A,, = 33.3 G, and g, = 2.00901, g,, = 2.00619, g,, = 2.00283. The same values of the original A and g tensors were taken also in the calculations with libmtional averaging in Figs. 1 and 2.
210
PAVSIC AND SCHARA
0.015
FIG.
4. The fitting
parameter,
0.03
0.045
0.075
0.06
OD9 tsdg)
defined
as x = 100.95 & [(f’(H,)/Af’(Hi))T,,,, - (f’(H# -f’(H&,, f’(H) is the first derivative of the EPR of intervals), is shown as a function of the librational
Af’WJLp,r.11i2 (where AJYHI) = f’WJ,,, spectrum, and N is the number angle ‘pOvariation.
limit original spectrum limit spectrum.
was then compared
RESULTS
with the low-temperature
rigid-
AND DISCUSSION
Figures 1 and 2 show the experimental spectra (2) for two representative spin labels with a short and a long hydrocarbon chain, respectively, attached across the piperidine ring about the phosphate group which is irreversibly
X 10 91
7654i 3
t_,
/ 111A ‘1
10'0
16'
1
I
acm,c 1
1ti6
r,lsl
FIG. 5. The same parameter x as defined in Fig. 4 for the variation of the isotropic rotational correlation time ra with the corresponding tensor components given in the text to Fig. 3.
SPIN
LABELS
BOUND
TO ACETYLCHOLINESTERASE TABLE
A COMPAFU~~N MODELS
@I OCSL EtSL
1
OF THE LIBRATIONAL AND BROWNIAN FOR THE ACETYLCHOLINESTERASE-ACTIVESITE-BOUND SPIN LABELS Librational model (TH = 5.5 x lo-’ set)
21.2” 27.8
211
Brownian
DIFFUSION
diffusion
Xllli”
nt (-1
Xmin
4.0 4.7
2 x 10-1 2 x 10-a
4.5 5.6
bound to the serine. The corresponding calculated spectra are shown for comparison. We have had to get the best fit to the OcSL spectrum (Fig. 1) by changing the hyperfine tensor components as well as the g tensor components. Using these tensors we tried to apply a larger librational angle to provide the best fit for the experimental spectrum of EtSL (Fig. 2). So finally we were able to evaluate the original tensor for the immobilized OcSL and EtSL, when the calculated values cpO= 21.2”andcp, = 27.8”,r espectively, were used. Figure 3 shows the comparison with the low-temperature rigid-limit spectrum. We took this agreement as another proof that the system is characterized by the same original tensor but with a fast libration about the x axis. Figures 4 and 5 show the best fit represented by the minimum in the x(ra) and x(q,-J functions. Here the linewidth parameter Tzk = 4.5 x 10’ set-’ has been used for the best fit, assuming T& = 0 for all calculated spectra. It is possible to see that for EtSL we get a better fit for the librational model. So we believe that the fast librational model, where the selectively bound spin label of the fluorphosphate type librates rapidly between the obstructions of the active serine pocket of acetylcholinesterase, interprets satisfactorily the observed EPR data. Table 1 summarizes our findings. We also tried to use approximate methods to determine the correlation times from the high- and low-field EPR spectra component linewidths (5). Unfortunately these could not be made to fit our experimental results. CONCLUSION
We have shown by computer simulation of EPR spectra for both the librational and the Brownian diffusion model that the first, by which the nitroxide group is librating fast in the environment where it is bound, reproduces better the experimental spectra.
212
PAVSIC: AND SCHARA REFERENCES
1. M. SENTJURC, A. STALC, 2. S. PEEAR, M. SCHARA,
AND A. 0. ZUPANW, Biochim. Biophys. Acta 438, 131 (1976). M. SENTJURC, A. STALC, AND A. 0. %JPANEIE, Stud. Biophys.
77,
33 (1979). 3. 0. H. GRIFFITH AND P. C. JOST, in “Spin Labeling” (L. J. Berliner, Ed.), p. 453, Academic Press, New York/San Francisco/London, 1976. 4. G. BRUNO, Dissertation, Cornell University, 1973. 5. J. H. FREED, in “Spin Labeling” (L. J. Berliner, Ed.), p. 53, Academic Press, New York/San Francisco/London, 1976.