Neutron Scattering in Biology - Fitter Gutberlet and Katsaras

thermosome. The SANS approach with trapping the reaction intermediates can be used in this case for stepping through the reaction.

The ATP-binding structure was observed by mimicking ATP with its nonhydrolysable analogue AMP-PNP, whereas the subsequent hydrolysis step was investigated by adding ADP together with an excess of phosphate to the thermosome. The phosphate-ADP condition drives the protein back to the state shortly after the hydrolysis reaction where the phosphate is no longer covalently bound, but still not ejected from the binding site. In order to probe the temperature dependence of the nucleotide-driven structural changes the experiments were performed by scanning di erent temperatures and nucleotides. Since the thermosome is found in an archea, which can stand high temperatures, a temperature range up to 50◦C was chosen. These extreme experimental parameters are easily accessible to small-angle scattering.

The scattering curves corresponding to the closed and open states of the thermosome are shown in Fig. 10.5. The di erence of the two states can be clearly distinguished by SANS. The experiments were performed on protonated thermosome in a bu er containing D2O in order to enhance the scattering signal-to-noise ratio. As can be seen in Fig. 10.1, the contrast of natural protein is highest in D2O, and at the same time, the incoherent scattering from D2O is significantly lower than from H2O.

The results listed in Table 10.2 show the transition of the open thermosome conformation to the closed state upon the ATP hydrolysis. Only at 50◦C, the

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Fig. 10.5. Experimental data for the open and closed thermosome structure

approach can also be used in SANS, simply by measuring the overall scattering power, which is proportional to the molecular mass of the scattering particle (see Sect. 10.2).

However, much more information can be obtained by taking advantage of the di erent contrast of protonated and deuterated proteins. Let us look at the formation of the symmetric chaperonin complex GroEL–GroEL–GroES, a reaction intermediate that is found in the presence of high concentrations of ATP [37]. Although the physiological relevance of the symmetric complex for substrate folding is not proven, it is interesting for the study of the cooperative interactions of the GroE/nucleotide system

For the steady-state titration analysis, ATP was substituted by its nonhydrolysable analogue AMP-PNP. In order to override the anticooperativity of the GroEL the concentration of ANP-PNP was in a 100-fold excess with respect to GroEL. Under these conditions the second GroEL ring is switched into the GroES acceptor state. Protonated GroEL was matched by 40% D2O, and the concentration of perdeuterated GroES was increased stepwise. Figure 10.7 shows the p(r) functions derived from the scattering data.

As described above, only the deuterated GroES protein contributes to the scattering signal. Thus, the first maximum in the p(r) function is due to the increasing amount of GroES; it stems from all distances within single, bound or unbound, GroES molecules. Besides this dominant maximum a smaller

Fig. 10.7. Formation of the symmetrical GroES–GroEL–GroES complex. With increasing GroES concentration the amount of symmetric complex increases

10.4.3 Chasing Experiments (Slow Kinetics)

An elegant way of employing the contrast matching technique is materialized in so-called “chasing” experiments. GP31 a protein found in the bacteriophage T4 [38], is able to substitute itself for GroES in bacteria and to bind in its place to one end of the GroEL cylinder. Despite a low sequence homology the two high-resolution crystal structures available for GP31 and GroES are rather similar. Like the co-chaperonin GroES, GP31 consists of seven identical subunits arranged in a ring. GP31 has a larger molecular weight (12 kDa; GroES 9 kDa), and the elongated flexible loops of GP31 could help to enlarge the refolding cavity after binding to the GroEL cylinder. The replacement of GroES by GP31 is maybe necessary for the folding of GP23, an important protein for the assembly of the T4 phage head that folds only in the presence of GP31 into its active conformation. In Fig. 10.8 a comparison of GroES and GP31 exhibits the di erent structures derived by high-resolution protein crystallography.

Chasing can be observed when an excess of partially deuterated GroES is added to GroEL that is prebound with GroES or GP31 in a bu er solution containing an excess of ADP and that matches the scattering-length density of the partially deuterated GroES, rendering the latter “invisible” (see Fig. 10.9). Varying the relative concentration of GroES and GP31 allows one to determine their binding constants, since the scattering intensity of the sample decreases with time, as the apparent scattering mass of the H-GroEL–D-GroES complex is lower than that of the initial H-GroEL–H-GroES or H-GroEL–H-GP31 [41].

The data can be fitted with a double-exponential revealing two di erent dissociation constants (see Fig. 10.9). For the fast reaction a value of 1.3 × 10−3 s−1 for the chasing of protonated GroES by deuterated GroES (control) and 4.9 × 10−4 s−1 for the corresponding GP31 experiment. The second constants were 2.3 × 10−5 s−1 for the chasing of GP31 by GroES and 3.9 × 10−5 s−1 for the control. The double exponential behavior indicates two di erent reaction mechanisms. First, in the fast reaction the real chasing of

Fig. 10.8. Crystal structures of GP31 (outer left) [39] from phage T4 in comparison with GroES (inner left) [40]. The “chasing” principle is shown at the right picture (see text)

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Fig. 10.9. Pair-distance distribution function of protonated GroEL and protonated GP31 with partially deuterated GroES in 80% D2O (left), and data fitted with double-exponential (right)

bound GroES or GP31 by invisible GroES takes place. The already chased GroES or GP31 starts to compete with the invisible GroES. This competition slows down the apparent dissociation constant and can be described by a second exponential decay function. The di erent dissociation constants in the fast process indicate a higher stability of the GroEL –GP31 complex than the native GroEL –GroES assembly. GP31 is necessary for the folding of GP23, a special protein in the phage cycle one can argue that the higher stability of the GroEL –GP31 complex indicates an enhanced folding rate of the GP23 in the phage-infected bacteria. These higher stability and binding a nity are facilitated by the di erent binding sites of GP31 and GroES to the interface of GroEL.

10.4.4 Time Resolved Small-Angle Neutron Scattering

Since the advent of third-generation synchrotron sources (e.g., ESRF, APS, Spring 8) time resolved small-angle scattering (TR-SAS) was used successfully for solution scattering of biological macromolecules. In general, the time resolved small-angle scattering technique is not restricted to X-rays, but the limiting quantity is the flux of scattered particles. While third-generation synchrotron sources produce a flux of 5 × 1012 ph s−1 (at λ = 1 ˚A), the SANS instrument D22 at the Institut Laue-Langevin in Grenoble, France, provides a neutron flux of 3 ×107 s−1 cm−2 (λ = 8 ˚A, collimation length 4 m). The flux can be significantly higher if smaller proteins are to be examined, because then shorter collimation lengths and/or wavelengths can be used. Another di erence between TR-SAXS and TR-SANS is the size of the neutron and X-ray beams. The beam size at the SAXS beamline ID02 of the ESRF is 0.6 × 0.2 mm2 while the beam size of D22 is 55 × 40 mm2. For the optimal use of these neutron beam parameters a flat cell has to be employed, and the experiment has to be repeated several times.

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Fig. 10.11. Neutron scattering intensities of a single experiment and the averaging over 10 independent experiments

The decrease of the Rg value with time can be used to follow the binding reaction of the two proteins. This stratagem is safer than following an increase of intensity that can also be due to aggregation. In Fig. 10.12 the time dependence of radius of gyration for the binding of H-GroES to D-GroEL in the presence of a high concentration (30 mM) of the nonhydrolysable ATP analogue AMP-PNP is shown. Under these conditions the symmetric GroES– GroEL–GroES complex is formed. The time progression can be fitted to a double exponential function, indicating the successive binding of the second GroES molecule to GroEL.

Fig. 10.12. Time dependence of the radius of gyration for the formation of the symmetric GroEL–(GroES)2 complex [30]