A measure of conformational entropy change during thermal protein unfolding using neutron spectroscopy

A measure of conformational entropy change during thermal protein unfolding using neutron spectroscopy

Fitter, Jorg

ABSTRACT Thermal unfolding of proteins at high temperatures is caused by a strong increase of the entropy change which lowers Gibbs free energy change of the unfolding transition ([Delta]G^sub unf^ = [Delta]H – T[Delta]S). The main contributions to entropy are the conformational entropy of the polypeptide chain itself and ordering of water molecules around hydrophobic side chains of the protein. To elucidate the role of conformational entropy upon thermal unfolding in more detail, conformational dynamics in the time regime of picoseconds was investigated with neutron spectroscopy. Confined internal structural fluctuations were analyzed for [alpha]-amylase in the folded and the unfolded state as a function of temperature. A strong difference in structural fluctuations between the folded and the unfolded state was observed at 30[degrees]C, which increased even more with rising temperatures. A simple analytical model was used to quantify the differences of the conformational space explored by the observed protein dynamics for the folded and unfolded state. Conformational entropy changes, calculated on the basis of the applied model, show a significant increase upon heating. In contrast to indirect estimates, which proposed a temperature independent conformational entropy change, the measurements presented here, demonstrated that the conformational entropy change increases with rising temperature and therefore contributes to thermal unfolding.


The stability of the folded state of a protein, which is the native and functional state under physiological conditions, is operated by a subtle balance of enthalpic and entropie contributions. Both contributions consist of opposing fractions which either stabilize or destabilize the folded state. The conformational entropy of the polypeptide chain is larger for unfolded state compared to the more compact folded state characterized by much more restricted conformational space. Therefore, this contribution stabilizes the unfolded state ([Delta]S^sub conf^ > 0). The interaction of solvent water with nonpolar side chains of the protein stabilizes the folded state, because solvation of these side groups induces ordering of water which is unfavorable. Since nonpolar groups are exposed to the solvent mainly in the unfolded state and not in the folded state, this contribution stabilizes the folded state ([Delta]S^sub hyd^

As an approach to elucidate more details about the role of the conformational entropy during thermal unfolding, neutron spectroscopy was applied to measure structural fluctuations of a well characterized protein, the [alpha]-amylase from B. licheniformis (BLA, 58,550 Da). This amylolytic enzyme is rather heat stable and well characterized with respect to thermal and thermodynamic stability (Feller et al., 1999, Fitter et al., 2001a) (Fig. 1). BLA has a monomeric structure and consists of 483 residues. In the time window of neutron spectroscopy, the observed picosecond motions are dominated by side-chain reorientations and segmental movements of flexible polypeptide backbone regions (Kneller and Smith, 1994; Tarek and Tobias, 2002). To obtain a measure of the conformational entropy change contribution, conformational fluctuations for the folded and the unfolded state of [alpha]-amylase were measured as a function of temperature. Although folded proteins exhibit a certain

In accordance with a vast body of experimental evidence and with principles of the famous “protein folding funnel” (Onuchic et al., 1997), the unfolded state is characterized by a larger degree of freedom for structural relaxations. Therefore, proteins in this state are more flexible (Receveur et al., 1997; Dyson and Wright, 1998; Bu et al., 2000; Fitter et al., 2001 b), with less defined and heterogeneous structures, leading to a higher conformational entropy as compared to the folded state.

The temperature dependence of dynamics in both states

For a meaningful statement on the role of protein dynamics for entropy changes during thermal unfolding, data of protein dynamics as a function of temperature are required. The evolution of internal dynamics for both states as a function of temperature is already apparent in the raw data. A comparison of spectra measured with proteins in the folded and in the unfolded state is shown for three different temperatures (Fig. 3). In all spectra the strong contribution of solvent scattering (dashed line) is visible. As already shown in the previous section, we find a smaller intensity in the elastic region and more pronounced quasielastic scattering for the unfolded stated as compared to the folded state at T = 30[degrees]C (Fig. 3 a). Qualitatively, this behavior is also recognizable for the other temperatures. Interestingly, the increase of quasielastic scattering with rising temperatures is much more pronounced for the unfolded state as compared to the folded state. This important feature is analyzed in more detail by fits applied to the corresponding difference spectra. For all temperatures we obtained reasonable fits with a Lorentzian width of H^sub 1^ = 150 + or – 10 [mu]eV. Within the limit of experimental errors, in the applied fits only the structure factors are affected significantly by protein unfolding or increasing the temperature. In Fig. 4 the elastic incoherent structure factors (A^sub 0^) are shown for the folded (a) and the unfolded state (b). The Q dependence of the structure factors, as measured for both states at different temperatures, yields the following features: i), With increasing temperature A^sub 0^ decreases more strongly with Q (i.e., more quasielastic intensity at high Q values). ii), The decrease of A^sub 0^ with Q is more distinctive for the unfolded state as compared to the folded state. This result was quantified by fitting the data with the diffusion inside a sphere model. The resulting radius parameters are given in Table 1. In terms of this model we obtain larger radii, and therefore a larger part of the conformational space explored by confined motions, with increasing temperature. The important result with respect to thermodynamics is given by the fact, that the difference in conformational dynamics between the folded state and the unfolded state increases with rising temperature. Due to lower energy barriers between adjacent conformational subslalcs in the unfolded stale, a certain increase in temperature results in more pronounced thermal fluctuations as compared to the folded state.

Conformational entropy calculation from protein dynamics and the impact on protein stability

Proper entropy calculations are difficult, because these calculations require knowledge of the complete conforma-

during unfolding with a radius of confinement for motions in the folded state r^sub f^ and in the unfolded state r^sub u^ is given by


With respect to experimental techniques, mainly NMR spectroscopy is used to calculate conformational entropies (Yang and Kay, 1996) from bond vector fluctuations in the backbone (Stone, 2001) and in side chains (Lee et al., 2002). So far, temperature-dependent studies have been performed only with proteins in the folded state (Stone, 2001). As shown here and within the above given limitations, neutron spectroscopy allows a direct measure of contributions to the conformational entropy change arising from conformational picosecond fluctuations, mainly related to diffusive side group reorientations. The presented results of the dynamical behavior of [alpha]a-amylase as a function of temperature are indicative for the fact that the conformational entropy change during unfolding was underestimated in the past (Privalov and Makhatadze, 1993; Sturtevant, 1977; Baldwin, 1986; Privalov, 1997) and seems to play a more important role in thermal protein unfolding.

Further studies with other proteins are required to state, whether the increasing conformational entropy change during thermal unfolding is a general phenomenon in proteins. Beyond this, further studies with other proteins give valuable information about strategies how in different proteins thermostability is determined. In particular, these findings are elucidative to understand mechanisms of thermal adaptation of thermophiles (Beadle et al., 1999; Colombo and Merz, 1999; Fitter and Heberle, 2000), and for developing thermostable enzymes for biotechnological applications (Stone, 2001; Arnold et al, 2001).

The Institute Laue-Langevin (Grenoble) and J. Ollivier are acknowledged for providing neutron beam facilities and for help during neutron scattering experiments. The author thanks G. Buldt for valuable discussions and for long standing and continuous support in his Institute.


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Jorg Fitter

Research Center Julich, IBI-2: Structural Biology, D-52425 Julich, Germany

Submitted October 10, 2002, and accepted for publication January 28, 2003.

Address reprint requests to Jorg Fitter, Research Center Julich, IBI-2: Structural Biology, D-52425 Julich, Germany. Tel.: +49-2461-612036; Fax: +49-2461-612020; E-mail: j.fitter@fz-juelich.de.

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