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Dynamics of a globular protein as studied by neutron scattering and solid-state NMR
ELSEVIER
Physica B 234-236 (1997) 228-230
Dynamics of a globular protein as studied by neutron scattering and solid-state NMR J.-M. Z a n o t t i a'*, M , - C . B e l l i s s e n t - F u n e l a, J. P a r e l l o b aLaboratoire LOon Brillouin (CEA-CNRS), CEA-Saclay, 91191 Gif-sur-Yvette Cedex, France b UPRES-A 5074 CNRS, Facultk de Pharmacie, Universitks Montpellier Iet II, 15 avenue Ch. Flahault, 34060 Montpellier Cedex 01, France
Abstract
Effect of hydration on the dynamics of parvalbumin, a 11.5 kDa Ca2+/Mg2+-binding globular protein has been studied, at room temperature, by incoherent quasi-elastic neutron scattering and 13C solid-state NMR. Samples were protein powders hydrated at different hydration levels. The increase of the quasi-elastic signal observed in neutron scattering upon hydration is interpreted as an increase of the local mobility of charged side-chain protons (Asp, Glu, Lys) and is in agreement with a parallel study of parvalbumin by solid-state natural abundance 13C NMR under cross-polarization and magic angle spinning (CP MAS) conditions. Keywords: Diffusion; Incoherent scattering; NMR; Proteins
We report here on the influence of hydration on protein dynamics using a typical calciprotein, parvalbumin (Pa). Powders of the fully Ca-loaded form (PaCa2) at different hydration levels (h in g of water/9 of dry protein) were investigated in parallel by incoherent quasi-elastic neutron scattering (IQNS) and by solid-state natural abundance 13C NMR. Lyophilized samples of parvalbumin (isoform pI 5.0 from pike muscle) were hydrated by vapor-phase adsorption of D20 for neutron experiments and of H20 for NMR experiments. The IQNS experiments were performed on the Mibrmol time-of-flight spectrometer of the Reactor Orphre of the Laboratoire Lron Brillouin using 6 A neutrons wavelength with a resolution of 96 laeV. The covered wave vector Q range was 0.32-1.93 A-1. Solid-state 13C NMR measurements were performed under CP MAS conditions at 6.5 kHz on a Bruker CXP 400 spectrometer operating at 100 MHz for 13C. * Corresponding author.
Fitting of the elastic and quasi-elastic part of the spectra is based on the model described in Ref. [1] using a delta function and a single Lorentzian line L(Q, co) with half-width at half maximum F, which leads to Sinc(Q, co) = e-Q2(u2)/3[(p + (1 - p)Ao(Q))6(co)
+ ( 1 - p)(1 - A o ( Q ) ) L ( Q , 09)],
(1)
where the exponential term is the Debye-Waller factor. We introduce a fraction p of immobile protons at the resolution of the spectrometer, that contribute to the elastic part of the spectra 6(o)). The ( l - p ) mobile protons undergo diffusive motions responsible for the quasi-elastic line L(Q, ~ ) and contribute to the elastic intensity via the elastic incoherent structure factor Ao(Q). In Fig. l(a) and l(b), F is plotted as a function of Q2 at h = 0.31 and 0.65. To identify selective effects of hydration on the protein dynamics, we
0921-4526/97/$17.00 © 1997 Elsevier ScienceB.V. All rights reserved PII S0921-4526(96)00921-0
J.-M. Zanotti et al. / Physica B 234 236 (1997) 228 230 I
I"
I
(a)
T = 298 K
Pa Ca2 h = 0.31
o.lo ~-
0,~ o
L I I
] 2
I
t
3
4
Q2 (~;2)
I
I
(b)
GI
E
T = 298 K
0.10
PaCa2
it
h = 0.65
00,
HALF
L
O0
RESOLUTION
I
1
'~
3
Q2(R2
Fig. 1. Variation of the half-width at half-maximum F of the Lorenztian line versus Q2 for the fully Ca-loaded form of parvalbumin, at T = 298 K, at hydration levels of (a) h = 0.31, (b) h = 0.65.
T=
,
200
,
,
,
t
150
.
.
.
.
295K
i
100
.
.
.
.
i
.
.
.
.
50
ppm Fig. 2. Proton-decoupled CP MAS 13C NMR spectra of PaCa2 at 295 K, at hydration levels of (a) dry sample, (b) h = 0.21 and (c) h = 0.38. Spinning side bands (labeled *) not suppressed.
investigated powdered samples at different hydration levels by solid state 13C NMR. As shown in Fig. 2, the CP MAS 13C NMR spectra ofparvalbumin at two h values, 0.21 and 0.38, differ significantly from the spectrum of the lyophilized powder in the dry state.
229
A relatively sharp signal at 41.9ppm progressively emerges from a 13C resonance envelope by increasing the hydration level of the protein. From assignments of the 13C resonances of pike 5.0Pa Ca2 in solution [2], this signal is to correspond to the 13C~ resonances of the lysyl residues. The prominent signal at 18.04ppm that is clearly apparent at h = 0.38 is due to the 13C/~ resonances of Ala. The invariance of iv as a function of Q2, at h = 0.31 (Fig. l(a)), is to be interpreted as the result of reorientational motions of the protons in the protein at the observation time scale of 10 ps. At higher hydration (h = 0.65), F shows a plateau in the range 0.2 to 1.2 i -2 and becomes clearly dependent on Q2 at higher Q2 values (Fig. l(b)). This indicates that diffusive motions of protein protons occur within a confined volume at this relatively high hydration value. The volume of diffusion as deduced from Ao(Q) accounts for a sphere of radius 1.7 A [3]. The use of Eq. (1), then leads to a fraction p = 0.72 of immobile protons which indicates that about 30% of the protons in the protein are involved in 10 ps short time diffusive motions. Those mobile protons at a time scale of 10-20ps are thought to correspond to the abundant protons from Lys, Asp and Glu side chains at the surface of the protein. The NMR data suggest that only the Nz-terminal region of the Lys polymethylene side chain is sufficiently unconstrained in the hydrated state (h > 0.2) to allow a selective sharpening of both 13C~: and 13C6 (29.11 ppm)resonances. The time-dependent reorientation of the Ala 13C~SH vectors, at the origin of the sharpening of the Ala 13C~ signal at 18.04ppm, involves a dipolar coupling with correlation times in the ns range that are probably associated with collective dynamics of the protein backbone. These motions are too slow to be detected in the present IQNS signal. The uniaxial rotational diffusion of Ala methyl groups which have very short correlation times might contribute to our IQNS signal as a fiat background. In closing, we have observed in this work that hydration has a major influence on the dynamics of a globular protein, such as parvalbumin. Both experimental approaches, IQNS and solid-state 13C NMR, indicate the occurrence of selective dynamical events upon progressive hydration of the dry protein. It is not only the polar
230
J. -M. Zanotti et al. / Physica B 234-236 (1997) 228-230
side chains, as expected, at the surface of the protein which are primarily affected during the early steps of the hydration process, but also non-polar side chains thus suggesting that hydration acts on the dynamics of the protein at both local and global levels.
References [1] M.-C. Bellissent-Funel, J. Teixeira, K.F. Bradley and S.H. Chert, J. Phys. I 2 (1992) 995. [2] T. Alattia, A. Padilla and A. Cave, Eur. J. Biochem. 237 (1996) 561. [3] F. Volino and A.J. Dianoux, Mol. Phys. 41 (1980) 271.
Physica B 234-236 (1997) 228-230
Dynamics of a globular protein as studied by neutron scattering and solid-state NMR J.-M. Z a n o t t i a'*, M , - C . B e l l i s s e n t - F u n e l a, J. P a r e l l o b aLaboratoire LOon Brillouin (CEA-CNRS), CEA-Saclay, 91191 Gif-sur-Yvette Cedex, France b UPRES-A 5074 CNRS, Facultk de Pharmacie, Universitks Montpellier Iet II, 15 avenue Ch. Flahault, 34060 Montpellier Cedex 01, France
Abstract
Effect of hydration on the dynamics of parvalbumin, a 11.5 kDa Ca2+/Mg2+-binding globular protein has been studied, at room temperature, by incoherent quasi-elastic neutron scattering and 13C solid-state NMR. Samples were protein powders hydrated at different hydration levels. The increase of the quasi-elastic signal observed in neutron scattering upon hydration is interpreted as an increase of the local mobility of charged side-chain protons (Asp, Glu, Lys) and is in agreement with a parallel study of parvalbumin by solid-state natural abundance 13C NMR under cross-polarization and magic angle spinning (CP MAS) conditions. Keywords: Diffusion; Incoherent scattering; NMR; Proteins
We report here on the influence of hydration on protein dynamics using a typical calciprotein, parvalbumin (Pa). Powders of the fully Ca-loaded form (PaCa2) at different hydration levels (h in g of water/9 of dry protein) were investigated in parallel by incoherent quasi-elastic neutron scattering (IQNS) and by solid-state natural abundance 13C NMR. Lyophilized samples of parvalbumin (isoform pI 5.0 from pike muscle) were hydrated by vapor-phase adsorption of D20 for neutron experiments and of H20 for NMR experiments. The IQNS experiments were performed on the Mibrmol time-of-flight spectrometer of the Reactor Orphre of the Laboratoire Lron Brillouin using 6 A neutrons wavelength with a resolution of 96 laeV. The covered wave vector Q range was 0.32-1.93 A-1. Solid-state 13C NMR measurements were performed under CP MAS conditions at 6.5 kHz on a Bruker CXP 400 spectrometer operating at 100 MHz for 13C. * Corresponding author.
Fitting of the elastic and quasi-elastic part of the spectra is based on the model described in Ref. [1] using a delta function and a single Lorentzian line L(Q, co) with half-width at half maximum F, which leads to Sinc(Q, co) = e-Q2(u2)/3[(p + (1 - p)Ao(Q))6(co)
+ ( 1 - p)(1 - A o ( Q ) ) L ( Q , 09)],
(1)
where the exponential term is the Debye-Waller factor. We introduce a fraction p of immobile protons at the resolution of the spectrometer, that contribute to the elastic part of the spectra 6(o)). The ( l - p ) mobile protons undergo diffusive motions responsible for the quasi-elastic line L(Q, ~ ) and contribute to the elastic intensity via the elastic incoherent structure factor Ao(Q). In Fig. l(a) and l(b), F is plotted as a function of Q2 at h = 0.31 and 0.65. To identify selective effects of hydration on the protein dynamics, we
0921-4526/97/$17.00 © 1997 Elsevier ScienceB.V. All rights reserved PII S0921-4526(96)00921-0
J.-M. Zanotti et al. / Physica B 234 236 (1997) 228 230 I
I"
I
(a)
T = 298 K
Pa Ca2 h = 0.31
o.lo ~-
0,~ o
L I I
] 2
I
t
3
4
Q2 (~;2)
I
I
(b)
GI
E
T = 298 K
0.10
PaCa2
it
h = 0.65
00,
HALF
L
O0
RESOLUTION
I
1
'~
3
Q2(R2
Fig. 1. Variation of the half-width at half-maximum F of the Lorenztian line versus Q2 for the fully Ca-loaded form of parvalbumin, at T = 298 K, at hydration levels of (a) h = 0.31, (b) h = 0.65.
T=
,
200
,
,
,
t
150
.
.
.
.
295K
i
100
.
.
.
.
i
.
.
.
.
50
ppm Fig. 2. Proton-decoupled CP MAS 13C NMR spectra of PaCa2 at 295 K, at hydration levels of (a) dry sample, (b) h = 0.21 and (c) h = 0.38. Spinning side bands (labeled *) not suppressed.
investigated powdered samples at different hydration levels by solid state 13C NMR. As shown in Fig. 2, the CP MAS 13C NMR spectra ofparvalbumin at two h values, 0.21 and 0.38, differ significantly from the spectrum of the lyophilized powder in the dry state.
229
A relatively sharp signal at 41.9ppm progressively emerges from a 13C resonance envelope by increasing the hydration level of the protein. From assignments of the 13C resonances of pike 5.0Pa Ca2 in solution [2], this signal is to correspond to the 13C~ resonances of the lysyl residues. The prominent signal at 18.04ppm that is clearly apparent at h = 0.38 is due to the 13C/~ resonances of Ala. The invariance of iv as a function of Q2, at h = 0.31 (Fig. l(a)), is to be interpreted as the result of reorientational motions of the protons in the protein at the observation time scale of 10 ps. At higher hydration (h = 0.65), F shows a plateau in the range 0.2 to 1.2 i -2 and becomes clearly dependent on Q2 at higher Q2 values (Fig. l(b)). This indicates that diffusive motions of protein protons occur within a confined volume at this relatively high hydration value. The volume of diffusion as deduced from Ao(Q) accounts for a sphere of radius 1.7 A [3]. The use of Eq. (1), then leads to a fraction p = 0.72 of immobile protons which indicates that about 30% of the protons in the protein are involved in 10 ps short time diffusive motions. Those mobile protons at a time scale of 10-20ps are thought to correspond to the abundant protons from Lys, Asp and Glu side chains at the surface of the protein. The NMR data suggest that only the Nz-terminal region of the Lys polymethylene side chain is sufficiently unconstrained in the hydrated state (h > 0.2) to allow a selective sharpening of both 13C~: and 13C6 (29.11 ppm)resonances. The time-dependent reorientation of the Ala 13C~SH vectors, at the origin of the sharpening of the Ala 13C~ signal at 18.04ppm, involves a dipolar coupling with correlation times in the ns range that are probably associated with collective dynamics of the protein backbone. These motions are too slow to be detected in the present IQNS signal. The uniaxial rotational diffusion of Ala methyl groups which have very short correlation times might contribute to our IQNS signal as a fiat background. In closing, we have observed in this work that hydration has a major influence on the dynamics of a globular protein, such as parvalbumin. Both experimental approaches, IQNS and solid-state 13C NMR, indicate the occurrence of selective dynamical events upon progressive hydration of the dry protein. It is not only the polar
230
J. -M. Zanotti et al. / Physica B 234-236 (1997) 228-230
side chains, as expected, at the surface of the protein which are primarily affected during the early steps of the hydration process, but also non-polar side chains thus suggesting that hydration acts on the dynamics of the protein at both local and global levels.
References [1] M.-C. Bellissent-Funel, J. Teixeira, K.F. Bradley and S.H. Chert, J. Phys. I 2 (1992) 995. [2] T. Alattia, A. Padilla and A. Cave, Eur. J. Biochem. 237 (1996) 561. [3] F. Volino and A.J. Dianoux, Mol. Phys. 41 (1980) 271.