Protein Engineering, Vol. 13, No. 8, 527-533,
August 2000
© 2000 Oxford University Press
The initial step of the thermal unfolding of 3-isopropylmalate dehydrogenase detected by the temperature-jump Laue method
1 Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8501, 2 RIKEN Harima Institute, Kouto 1–1–1, Mikazuki-cho, Sayo-gun, Hyogo 679-5148, 3 Experimental Facilities Division, Japan Synchrotron Radiation Research Institute, SPring-8, Kouto 1–1–1, Mikazuki-cho, Sayo-gun, Hyogo 679-5198 and 6 Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan
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Abstract |
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A temperature-jump (T-jump) time-resolved X-ray crystallographic technique using the Laue method was developed to detect small, localized structural changes of proteins in crystals exposed to a temperature increase induced by laser irradiation. In a chimeric protein between thermophilic and mesophilic 3-isopropylmalate dehydrogenases (2T2M6T), the initial structural change upon T-jump to a denaturing temperature (~90°C) was found to be localized at a region which includes a ß-turn and a loop located between the two domains of the enzyme. A mutant, 2T2M6T-E110P/S111G/S113E, having amino acid replacements in this ß-turn region with the corresponding residues of the thermophilic enzyme, showed greater stability than the original chimera (increase of Tm by ~10°C) and no T-jump-induced structural change in this region was detected by our method. These results indicate that thermal unfolding of the original chimeric enzyme, 2T2M6T, is triggered in this ß-turn region.
Keywords: crystal structure/3-isopropylmalate dehydrogenase/temperature-jump Laue method/thermal unfolding/thermostability
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The mechanisms of protein folding and stability are of particular importance as the sequences of large numbers of proteins are being established in various genome projects. Characterization of the intermediates and transition states involved in the folding process is a key step in understanding the mechanisms. The molten globule state is a well-known intermediate in the folding process (Kuwajima, 1989
) and can exist stably under certain conditions. Recently, the kinetics of protein folding/unfolding have been evaluated by means of time-resolved analyses at the nanosecond–microsecond–second time-scale (reviewed in Eaton et al., 1991; Plaxco and Dobson, 1996) utilizing hydrogen–deuterium exchange (Jacobs and Fox, 1994; Gladwin and Evans, 1996), small-angle X-ray scattering (Pollock et al., 1999), fluorescence spectroscopy (Royer, 1995) and IR spectroscopy (Phillips et al., 1995; Reinstädler et al., 1996). Many attempts have also been made to determine in detail the structures of intermediate species, for instance, by NMR (Balbach et al., 1995; Eliezer et al., 1998; Udgaonkar and Baldwin, 1988), X-ray diffraction (Gillbert et al., 1982) and molecular dynamics simulations (reviewed in Daggett and Levitt, 1994). However, it is still difficult to elucidate the structural dynamics of protein folding/unfolding processes at the atomic level, especially for relatively large proteins, to which NMR analysis is not applicable.
We have developed an X-ray crystallographic structural analysis technique coupled with a temperature jump (T-jump) induced by laser irradiation to determine the structural change of proteins induced by the T-jump, at the atomic level. Gillbert et al. (1982) previously attempted to identify the conformational changes of crystalline ribonuclease A induced by a stepwise temperature increase, by following the changes in X-ray diffraction and resonance Raman signals. Their results suggested the existence of an initial denaturation site in the protein from which all further denaturation propagates and they observed several structural changes as crystals were warmed close to the denaturation temperature. However, loss of diffraction and radiation damage to the crystals prevented detailed structural analysis at higher temperatures. We bypassed these problems by utilizing polychromatic Laue crystallographic methods, which permit an increased rate of data collection, so that the structural details of even an unstable intermediate can be visualized. This technique has been applied to reveal the crystal structures of several transient catalytic intermediates (Schlichting et al., 1990; Bolduc et al., 1995; Stoddard, 1998; Stoddard et al., 1998). In the present T-jump Laue method, a protein crystal was heated extremely quickly by YAG laser radiation and Laue images were recorded within 40 ms. The T-jump generated by the laser was estimated to be ~70°C, which is sufficient to initiate thermal unfolding of some proteins. The structural changes can be detected by a comparison of the structures solved by the Laue method with and without the T-jump. If any localized and specific structural change(s), which should be small enough to allow the crystal lattice to remain intact, occurs at the very early stage of thermal unfolding upon T-jump, it would be observable at the atomic level with the present method.
We applied this new method to 3-isopropylmalate dehydrogenase (EC 1.1.1.85) (IPMDH), one of the enzymes in the leucine biosynthetic pathway. IPMDH has very high sequence homology over various organisms, but differences in stability exist (Kagawa et al., 1984; Yamada et al., 1990; Kirino and Oshima, 1991; Kirino et al., 1994; Wallon et al., 1997a; Závodszky et al., 1998). The mechanism of the thermostability of IPMDH has been investigated extensively by several methods, including X-ray crystallography (Omada et al., 1991; Onodera et al., 1994; Moriyama et al., 1995; Wallon et al., 1997b), site-directed mutagenesis (Kirino et al., 1994; Numata et al., 1995) and several biophysical analyses (Hayashi-Iwasaki et al., 1996; Motono et al., 1999). The enzyme is a homodimer and each subunit has two domains (Figure 1b; Imada et al., 1991), so it has sufficient complexity to be of interest as a model enzyme. In the present study, we used a chimeric enzyme between thermophile, Thermus thermophilus, and mesophile, Bacillus subtilis, IPMDHs owing to its moderate stability (Numata et al., 1995). This chimeric IPMDH, 2T2M6T (Figure 1a), has residues 1–73 and 133–345 from the thermophile (T) and the remaining residues 74–132 from the mesophile (M) sequences. The unfolding process of the chimera has been investigated by circular dichroism (CD) spectroscopy and differential scanning calorimetry (Hayashi-Iwasaki et al., 1996). The chimera shows a biphasic unfolding process with a dimeric intermediate and the first transition temperature is ~67°C at neutral pH (Figure 2). We have previously found that one particular region located in the mesophilic portion of the chimera is important for the stability of the enzyme and the replacement of amino acid residues in this portion with those of the thermophilic sequence (E110P/S111G/S113E) increases the unfolding temperature of the first phase by ~10°C (Figure 2; J.Kawaguchi, Y.Hayashi-Iwasaki and T.Oshima, in preparation) (Tm of T.thermophilus IPMDH is 86°C).
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In this study, the T-jump Laue method was applied to the original chimera, 2T2M6T, to detect the specific structural changes that occurred upon T-jump. The stabilized mutant of 2T2M6T (2T2M6T-E110P/S111G/S113E) was then subjected to T-jump Laue experiments as a reference protein. The T-jump induced by the laser in this method was estimated to be from room temperature (~20°C) to ~90°C. A small, localized structural change was detected by the present method and this may trigger the unfolding reaction of the whole protein.
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Materials and methods |
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Enzyme purification and crystallization
2T2M6T and the mutant, 2T2M6T-E110P/S111G/S113E, were overexpressed in Escherichia coli and purified as described previously (Yamada et al., 1990; Numata et al., 1995). The IPMDH was stored as a precipitate in fully saturated ammonium sulfate in 20 mM potassium phosphate buffer (pH 7.6). For crystallization, protein solution was centrifuged at 10 000 g for 3 min to collect the precipitates. Then the supernatant was completely removed and the protein was dissolved in the same buffer to obtain a final concentration of 30 mg/ml. To remove insoluble materials and to unify the molecular volume distribution, the protein solution was passed through a 0.1 µm filter. All crystals were prepared by the vapor diffusion hanging drop method using ammonium sulfate as a precipitant (Onodera et al., 1991).
The T-jump Laue method
Experiments involving laser irradiation followed by diffraction analysis with the Laue method using white synchrotron X-ray radiation were performed at station BL18B in the Photon Factory (Tsukuba, Japan) (Watanabe et al., 1995). The diode-pumped solid-state laser system mediated by YAG (Spectra-Physics, San Jose, CA, USA) gave an output power of 1 W at a wavelength of 1064 nm, with a beam diameter of 0.51 mm. The absorption by the crystal, measured with a semiconductor counter (Model D3MM, THORLABS), was 0.32. Note that the maximum energy of a laser shot at 1 W for 80 ms corresponds to 19.1 mcal. The maximum temperature jump generated by the laser was calculated to be about 150°C based on a crystal size of about 40x10–3 mm3. Because of energy loss due to scattering and reflection, the actual increase was estimated to be about 70°C. The Laue experiments were performed at room temperature and therefore the temperature change was estimated to be from ~20 to ~90°C. The laser was pumped prior to irradiation and regulated by a bistable electronic shutter (Newport, Irvine, CA, USA). The laser shutter system was directly connected to the X-ray gallery swing shutter system of the beam line. Once the laser irradiation was complete, the X-ray shutter was opened for 40 ms to take a Laue image of the crystal (the dead time was estimated to be ~1 ms). The diffraction images were recorded on a 400x 800 mm imaging plate using a Weissenberg camera (Campbell et al., 1995; Sakabe et al., 1997). The resulting images were indexed using the software lauegen (Campbell, 1995) and processed using the Photon Factory in-house program genlaue (Dr T.Higashi, Rigaku, Japan).
To improve completeness, four Laue images at different spindle angles from a single crystal, obtained at intervals of 1 min, were merged. Further laser irradiation caused damage to the crystal. Consequently, the completeness of diffraction data in all data sets was ~70% (Table I).
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Static and monochromatic diffractometry
The diffraction data were collected on an R-AXIS IIc diffractometer with Cu K
radiation generated by an RU300 unit (Rigaku, Japan). The oscillation images were processed using the built-in software PROCESS (Higashi, 1990).
Structure refinement
The structures of all proteins were located by the rigid body refinement and refined using the program X-PLOR (Brünger, 1990; Brünger et al., 1990) with iterative modification using FRODO (Jones, 1985). The statistics of refinements are summarized in Table I
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CD measurements
Protein solution (0.2 mg/ml) in 20 mM potassium phosphate buffer, pH 7.6, was warmed at a constant rate of 1°C/min as described previously (Hayashi-Iwasaki et al., 1996). The change of CD at 222 nm was recorded.
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Results |
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Structure of 2T2M6T solved by the Laue method
To evaluate the reliability of the structures solved by the present method, the Laue experiment without T-jump was first applied to 2T2M6T (Table I). The overall structure of 2T2M6T solved by the Laue method was found to be identical with the `static' structure (Onodera et al., 1994) previously solved by monochromatic diffractometry. The average r.m.s. deviation of the main chains between the two was 0.07 Å and the maximum r.m.s. deviation of the main chain was 0.39 Å, which is insignificant for structures solved at 2.5 Å resolution. The reliability of the structure of 2T2M6T solved by the Laue method was thus confirmed.
Structural change in 2T2M6T detected by the T-jump Laue technique
Suitable exposure times for the laser shot and white X-ray irradiation were determined as follows. Long laser exposure caused destruction of the crystal lattice and the Laue spots became elliptical, resulting in very high R-values. An exposure time of 80 ms gave the best result for the crystal size of about 40x10–3 mm3 and caused a T-jump of ~70°C. The optimum exposure time to white X-rays was determined as 40 ms to obtain data with a good signal-to-noise ratio. Thus, the initial structural change, occurring within 40 ms, of 2T2M6T caused by the laser-induced T-jump (from ~20 to ~90°C) could be detected by the Laue technique.
The 2T2M6T structure after a T-jump thus obtained (Table I) was compared with the structure at room temperature. The r.m.s. deviation of the main chain between the two structures (Figure 3a) indicated that significant structural changes occurred specifically in two regions (from 109 to 114 and from 252 to 255). The electron density maps around residues 110–113 with and without T-jump are shown in Figure 4a. Because the average r.m.s. deviation of the main chain was only 0.08 Å, the changes in this region (>0.6 Å) should not be attributable to native fluctuations such as domain movement. The two regions are very close to each other in the enzyme structure and are located between the two domains (Figure 1c). This result implies that the thermal unfolding of 2T2M6T is triggered at this particular region between the two domains of the enzyme.
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. Left and right are the maps obtained without and with T-jump, respectively.The region from 109 to 114 includes the ß-turn structure (109–112) between the ß-strand F and a loop followed by the ß-strand G between the two domains (Imada et al., 1991
) and is exposed to solvent (Figure 1c) in the original 2T2M6T structure. This ß-turn structure is categorized as type I, because the dihedral angles (
, 
) of residue i + 1 (110) and residue i + 2 (111) are (–40, –23°) and (–88, –61°), respectively (Hutchinson and Thornton, 1994). In the T-jump structure of 2T2M6T, the corresponding turn was distorted and had dihedral angles (, ) of (–49, 87°) and (152, –30°) at residues 110 and 111, respectively, indicating that it can no longer be classified as a ß-turn (Wilmot and Thornton, 1990). In addition, while the orientation of the Ser113 side chain was maintained, the O of Glu110 was shifted in 2T2M6T after the T-jump (Figures 4a, 5a and b). This change caused a loss of the hydrogen bond between O
of Ser113 and O of Glu110. Hence the results obtained here indicate that the T-jump to a denaturing temperature caused deformation of the turn structure with loss of a hydrogen bond in 2T2M6T IPMDH. The residues from 252 to 255 include loop regions connecting the two domains of IPMDH and are located close to the ß-turn from 109 to 114 described above. This part has relatively high B-factor values (Figure 3c) and seems to be a very flexible region even in the static structure of 2T2M6T.
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Structural analysis of the mutant enzyme 2T2M6T-E110P/S111G/S113E
2T2M6T has mesophilic sequences around the ß-turn region of 109–112 (Figure 1a). We have recently found that the replacement of amino acid residues at this site with the thermophilic sequence (E110P/S111G/S113E) significantly increases the stability of the enzyme (J.Kawaguchi, Y.Hayashi-Iwasaki and T.Oshima, in preparation). The thermostability of 2T2M6T-E110P/S111G/S113E was found to be improved by 10°C by means of CD measurement (Figure 2) or by 7.5°C on the basis of a remaining activity experiment (data not shown).
The monochromatic structure of 2T2M6T-E110P/S111G/S113E was determined prior to the Laue experiment (Table I). The average r.m.s. deviation of the main chain between 2T2M6T and 2T2M6T-E110P/S111G/S113E was 0.04 Å, indicating that the overall folding topology of the mutant enzyme was almost identical with that of 2T2M6T (data not shown). As expected, 2T2M6T-E110P/S111G/S113E had a type II ß-turn (109–112) with a hydrogen bond between O
of Glu113 and N of Gly111 in the mutated region, which is very similar to the corresponding region of T.thermophilus wild-type IPMDH (Figure 5c) (Imada et al., 1991). The values of the dihedral angles (, ) of residues i + 1 (110) and i + 2 (111) in 2T2M6T-E110P/S111G/S113E were (–55, 134°) and (91, –10°), respectively. These values correspond to a type II ß-turn structure.
The averaged B-factor of 2T2M6T-E110P/S111G/S113E is 27.6 Å2 and this value is lower than that of 2T2M6T (32.4 Å2). The B-factor distribution of the mutant enzyme was similar to that of 2T2M6T (Figure 3c), except for the differences in the B-factor values at the mutation sites; the B-factor around the type II ß-turn (109–112) in the mutant enzyme was lower by 25 Å2 than that in 2T2M6T. This implies that the ß-turn in 2T2M6T-E110P/S111G/S113E is more rigid than that in 2T2M6T.
The T-jump Laue experiment with the mutant enzyme 2T2M6T-E110P/S111G/S113E
The overall structure of 2T2M6T-E110P/S111G/S113E solved by the Laue method without T-jump (Table I) was identical with the `static' monochromatic structure as described above. The average main chain r.m.s. deviation between them was 0.08 Å (data not shown).
The T-jump Laue method was applied to the mutant enzyme under the same conditions as employed with 2T2M6T (Table I). The average r.m.s. deviation of the main chain between the Laue structure at room temperature and that after T-jump was 0.09 Å in 2T2M6T-E110P/S111G/S113E. In contrast to the result for 2T2M6T, the T-jump caused no observable structural change at the mutation site (109–114) or at the neighboring region (252–255) in the mutant enzyme (Figures 3b and 4b). The changes in dihedral angles in residue i + 1 (110) and residue i + 2 (111) are small and the hydrogen bond between O of Glu113 and N of Gly111 remains intact (Figure 4b). This indicates that the ß-turn structure was retained after the T-jump in the mutant enzyme.
The r.m.s. deviation plot showed two local structural changes at positions different from those observed in 2T2M6T, i.e. at residues 272–273 and 328–329 (Figure 3b).
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Discussion |
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We have developed a new method to observe the dynamic structural changes in proteins upon T-jump by X-ray crystallography and employed it to monitor the initial step in the thermal unfolding of the chimeric IPMDH 2T2M6T, and also its stabilized mutant, 2T2M6T-E110P/S111G/S113E. In this experiment, significant structural change was localized at a particular region between the two domains of 2T2M6T (Figure 3a
), implying that the thermal unfolding is triggered at this particular region. Similar results have been obtained in the previous X-ray crystallographic analysis of ribonuclease A at a temperature close to the denaturation temperature by Gillbert et al. (1982), who observed a significant displacement at residues 31–32; they pointed out that this site is one of the regions which become susceptible to cleavage by trypsin at high temperature and suggested that it is the initial denaturation site in the protein from which all further denaturation propagates.
The present structural analysis revealed a deformation of a ß-turn structure (109–112) and the loss of a hydrogen bond upon T-jump to the unfolding temperature in 2T2M6T (Figures 4a, 5a and b). These changes were not observed in the mutant, 2T2M6T-E110P/S111G/S113E, which has the thermophilic sequence in this ß-turn and exhibits greater thermostability (Figures 3b and 4b). These results indicate that this ß-turn region is the least stable structure in 2T2M6T. This further suggests that the deformation of this region occurs at a very early stage of thermal unfolding, prior to loss of the overall structure of the enzyme. In other words, the unfolding of 2T2M6T is initiated at this particular region under the conditions of our experiment. The introduction of the thermophilic sequence at this region increases the stability of the ß-turn, leading to the stabilization of the mutant enzyme.
In the `static' structures solved by the monochromatic analyses, the ß-turn (109–112) of the original chimera, 2T2M6T, at room temperature was categorized as type I, whereas 2T2M6T-E110P/S111G/S113E has a type II ß-turn, which is structurally very similar to that of the T.thermophilus IPMDH structure. The stability of ß-turns has been investigated as a function of the amino acid sequence and the turn type (Scully and Hermans, 1994; Yang et al., 1996). These studies indicate that the type II ß-turn with the sequence –Xaa–Pro–Gly–Xaa– is the most stable among various ß-turns (such as types I, II, I' and II'). The ß-turn at residues 109–112 in 2T2M6T-E110P/S111G/S113E has the sequence –Phe–Pro–Gly–Leu– derived from the thermophilic wild-type enzyme and is categorized as type II, whereas the turn in 2T2M6T is type I with the sequence –Phe–Glu–Ser–Leu–. Our finding that the type I ß-turn structure in 2T2M6T is less stable than the type II ß-turn in 2T2M6T-E110P/S111G/S113E and the wild-type enzyme is thus consistent with the previous studies (Scully and Hermans, 1994; Yang et al., 1996).
In the T-jump structure of 2T2M6T-E110P/S111G/S113E, the structural changes at residues 272–273 and 328–329 are located at loop regions in the outside domain (domain 1, Figure 1c) of IPMDH, implying that the unfolding is triggered in these portions in the mutant enzyme. A significant contribution of short, compact, surface loop structures to protein stability has been inferred from experimental results (Nagi and Regan, 1997) and from a comparative analysis of complete genomes of thermophilic and mesophilic organisms (Thompson and Eisenberg, 1999), suggesting the importance of loop structures for stability. However, at present, it is difficult to decide whether the deviations observed in these regions of the mutant enzyme represent allowable fluctuations in the native conformation or the destruction of specific interactions, because these loop regions seem to be very flexible. Further analysis with the T-jump Laue method combined with mutational study and other biophysical methods is required.
The importance of the investigation of unfolding kinetics for the purpose of protein stabilization is great, because for many proteins, the folded and unfolded proteins are not in rapid equilibrium and the stability is likely to be controlled by kinetics (Shaw and Bott, 1996). It has also been suggested that the identification of structural regions connected with early unfolding events could be the basis for rational strategies of protein stabilization (Mansfeld et al., 1999). The results obtained in this study suggest that the present technique is applicable to the analysis of T-jump-induced structural changes and can reveal the very early events in the protein thermal unfolding process. Furthermore, our findings with the chimeric IPMDH indicate that the destruction of a specific ß-turn between the two domains occurs in the initial stage of its thermal unfolding and that stabilization in this region by the introduction of a thermophile sequence results in a marked increase in the stability of the whole protein.
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Notes |
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5 Present address: Centre for Genome Research, University of Edinburgh, Kings Buildings, West Mains Road, Edinburgh EH9 3JQ, UK
7 Present address: Department of Bioengineering, Nagaoka University of Technology, Kamitomioka-cho 1603-1, Nagaoka, Niigata 940-2188, Japan
4 To whom correspondence should be addressed. E-mail: aki5{at}sp8sun.spring8.or.jp
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Acknowledgments |
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The authors thank Drs Nobuhisa Watanabe, Mamoru Suzuki, Noriyuki Igarashi, Kiwako Sakabe and Noriyoshi Sakabe of the Photon Factory for Laue data collection. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan and Japan Space Forum.
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Received April 13, 2000; revised June 9, 2000; accepted June 12, 2000.
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