CooA, a heme-containing transcriptional activator, binds CO to the heme moiety and then undergoes... more CooA, a heme-containing transcriptional activator, binds CO to the heme moiety and then undergoes a structural change that promotes the specific binding to the target DNA. To elucidate the activation mechanism coupled to CO binding, we investigated the CO-dependent structural transition of CooA with small-angle X-ray scattering (SAXS). In the absence of CO, the radius of gyration Rg and the second virial coefficient (A2) were 25.3(+/-0.5)A and -0.39(+/-0.25) x 10(-4)ml mol g(-2), respectively. CO binding caused a slight increase in Rg (by 0.5A) and a marked decrease in A2 (by 5.09 x 10(-4)ml mol g(-2)). The observed decrease in A2 points to higher attractive interactions between CO-bound CooA molecules in solution compared with CO-free CooA. Although the minor alternation of Rg rules out changes in the overall structure, the marked change in the surface properties points to a CO-induced conformational transition. The experimental Rg and SAXS curves of the two states did not agree with the crystal structure of CO-free CooA. We thus simulated the solution structures of CooA based on the experimental data using rigid-body refinements as well as low-resolution model reconstructions. Both results demonstrate that the hinge region connecting the N-terminal heme domain and C-terminal DNA-binding domain is kinked in CO-free CooA, so that the two domains are positioned close to each other. The CO-dependent structural change observed by SAXS corresponds to a slight swing of the DNA-binding domains away from the heme domains coupled with their rotation by about 8 degrees around the axis of 2-fold symmetry.
To examine the effects of aggregation-inducing motifs related to neurodegenerative diseases on am... more To examine the effects of aggregation-inducing motifs related to neurodegenerative diseases on amyloid formation of host protein, we prepared several chimera myoglobins, in which various aggregation-inducing motifs were inserted. The focused aggregation-inducing motifs included five (R5) or two (R2) oligopeptide repeats in yeast Sup35p, five octapeptide repeats (OPR) in the human prion protein, a nonamyloid beta component (NAC) in alpha-synuclein, and tandem repeats of 50 glutamines (Q50). Circular dichroism and infrared spectroscopies suggested that the OPR, R5, and Q50 motifs formed an antiparallel beta sheet as well as a random coil, whereas the R2 and NAC motifs mainly formed random coils. The OPR, R5, and Q50 mutants, but not the R2 and NAC mutants, readily formed the SDS-resistant aggregates under physiological condition, and electron microscopy revealed that the aggregates contained amyloid fibrils. The destabilization and increase in gyration radius of the OPR, R5, and Q50 mutants correlated with the tendency to form amyloid fibrils. A control mutant bearing a nonamyloidgenic sequence was also moderately destabilized but did not form amyloid fibrils. Therefore, we concluded that the OPR, R5, and Q50 motifs, even in a quite stable protein such as myoglobin, led the host protein to formation of amyloid fibrils under physiological condition.
Proceedings of The National Academy of Sciences, 2005
Characterization of the conformational landscapes for proteins with different secondary structure... more Characterization of the conformational landscapes for proteins with different secondary structures is important in elucidating the mechanism of protein folding. The folding trajectory of single-chain monellin composed of a five-stranded beta-sheet and a helix was investigated by using a pH-jump from the alkaline unfolded to native state. The kinetic changes in the secondary structures and in the overall size and shape were measured by circular dichroism spectroscopy and small-angle x-ray scattering, respectively. The formation of the tertiary structure was monitored by intrinsic and extrinsic fluorescence. A significant collapse was observed within 300 micros after the pH-jump, leading to the intermediate with a small amount of secondary and tertiary structures but with an overall oblate shape. Subsequently, the stepwise formation of secondary and tertiary structures was detected. The current observation was consistent with the theoretical prediction that a more significant collapse precedes the formation of secondary structures in the folding of beta-sheet proteins than that of helical proteins [Shea, J. E., Onuchic, J. N. & Brooks, C. L., III (2002) Proc. Natl. Acad. Sci. USA 99, 16064-16068]. Furthermore, it was implied that the initial collapse was promoted by the formation of some specific structural elements, such as tight turns, to form the oblate shape.
Artificial sequences of the 153 amino acids have been designed to fit the main-chain framework of... more Artificial sequences of the 153 amino acids have been designed to fit the main-chain framework of the sperm whale myoglobin (Mb) structure based on a knowledge-based 3D-1D compatibility method. The previously designed artificial globin (DG1) folded into a monomeric, compact, highly helical and globular form with overall dimensions similar to those of the target structure, but it lacked structural uniqueness at the side-chain level [Isogai, Y., Ota, M., Fujisawa, T. , Izuno, H., Mukai, M., Nakamura, H., Iizuka, T., and Nishikawa, K. (1999) Biochemistry 38, 7431-7443]. In this study, we redesigned hydrophobic sites of DG1 to improve the structural specificity. Several Leu and Met residues in DG1 were replaced with beta-branched amino acids, Ile and Val, referring to the 3D profile of DG1 to produce three redesigned globins, DG2-4. These residue replacements resulted in no significant changes of their compactness and alpha-helical contents in the absence of denaturant, whereas they significantly affected the dependence of the secondary structure on the concentration of guanidine hydrochloride. The analyses of the denaturation curves revealed higher global stabilities of the designed globins than that of natural apoMb. Among DG1-4, DG3, in which 11 Leu residues of DG1 are replaced with seven Ile and four Val residues, and one Met residue is replaced with Val, displayed the lowest stability but the most cooperative folding-unfolding transition and the most dispersed NMR spectrum with the smallest line width. The present results indicate that the replacements of Leu (Met) with the beta-branched amino acids at appropriate sites reduce the freedom of side-chain conformation and improve the structural specificity at the expense of stability.
Static and time-resolved two-dimensional x-ray diffraction patterns, recorded from the living mou... more Static and time-resolved two-dimensional x-ray diffraction patterns, recorded from the living mouse diaphragm muscle, were compared with those from living frog sartorius muscle. The resting pattern of mouse muscle was similar to that of frog muscle, and consisted of actin- and myosin-based reflections with spacings basically identical to those of frog. As a notable exception, the sampling pattern of the myosin layer lines (MLL's) indicated that the mouse myofilaments were not organized into a superlattice as in frog. The intensity changes of reflections upon activation were also similar. The MLL's of both muscles were markedly weakened. Stereospecific (rigorlike) actomyosin species were not significantly populated in either muscle, as was evidenced by the 6th actin layer line (ALL), which was substantially enhanced but without a shift in its peak position or a concomitant rise of lower order ALL's. On close examination of the mouse pattern, however, a few lower order ALL's were found to rise, slightly but definitely, at the position expected for stereospecific binding. Their quick rise after the onset of stimulation indicates that this stereospecific complex is generated in the process of normal contraction. However, their rise is still too small to account for the marked enhancement of the 6th ALL, which is better explained by a myosin-induced structural change of actin. Since the forces of the two muscles are comparable regardless of the amount of stereospecific complex, it would be natural to consider that most of the force of skeletal muscle is supported by nonstereospecific actomyosin species.
The effect of high pressure on lactate dehydrogenase (LDH) was studied using small-angle X-ray sc... more The effect of high pressure on lactate dehydrogenase (LDH) was studied using small-angle X-ray scattering (SAXS). The SAXS results are interpreted in terms of the dissociation and association of LDH within a compression and decompression cycle and its temperature dependence. LDH consists of four identical subunits. At 120 MPa and 25 degrees C, 50% of the LDH dissociates into subunits, while at 10 degrees C, this occurs at 78 MPa. The hysteresis in the dissociation and association under pressure was confirmed in terms of the radius of gyration and was seen to be more conspicuous at low temperature. Forward scattering, I(0)/C, which is proportional to molecular weight, showed that LDH dissociated into dimer (not monomer) subunits under pressure. The application of high pressure to dissociated dimers induced irreversible aggregation. This result is in sharp contrast with the result of fluorescence spectroscopy suggesting a dissociated monomer [King, L., and Weber, G. (1986) Biochemistry 25, 3637-3640]. As for structural change after reassociation, there was little structural difference between native and drifted LDH. The difference was smaller than the structure change by ligand binding. At 200 MPa, the presence of five scattering peaks in the medium-angle region indicates that the dissociated dimer does not have a molten globule-like structure but a core structure. We propose a model of the dissociated dimer, based on the SAXS profile, in which the volume is reduced without disrupting the core structure.
SPring-8 RIKEN beamline I has been designed and developed for structural biology research by the ... more SPring-8 RIKEN beamline I has been designed and developed for structural biology research by the Institute of Physical and Chemical Research (RIKEN). The beamline consists of two experimental stations for protein crystallography and small-angle X-ray scattering. Both types of experiments can be carried out simultaneously, with dichromatic synchrotron radiation emitted from two coaxial undulators with vertical polarization. The branched beams are generated by a transparent diamond crystal. With synchrotron radiation, the multiple-wavelength anomalous-dispersion (MAD) method, which gives phases from a single anomalous scatterer, has been developed. Anomalous scattering contributes a small proportion of the diffraction intensity so that the accuracy of intensity data is important. The protein crystallography branch of RIKEN beamline I has been designed based on a 'trichromatic concept' to optimize MAD data collection. This concept requires the quasi-simultaneous collection, by use of a 'trichromator', of three intensity data sets at three different wavelengths from a single protein crystal without changing any settings. The main feature of the concept is the minimization of systematic errors in the measurement of anomalous diffraction for the MAD method. Initial commissioning of the beamline has provided three different monochromated undulator beams, which were successfully observed on the phosphor screen located at the near end of the trichromator.
Tropomodulin, the P-end (slow-growing end)-capping protein of the actin-tropomyosin filament, and... more Tropomodulin, the P-end (slow-growing end)-capping protein of the actin-tropomyosin filament, and its fragment (C20) of the C-terminal half were studied by synchrotron small-angle X-ray scattering, restoring low-resolution shapes using an ab initio shape-determining procedure. Tropomodulin is elongated (115 Å long) and consists of two domains, one of 65 Å in length and the other being similar to C20 in shape and size if the long axes of the two are tilted by about 40° relative to each other. We propose a model for tropomodulin in association with tropomyosin and actin: the N-terminal half of tropomodulin, a rod, binds to the N-terminus of tropomyosin and the C-terminal triangle domain protrudes from the P-end being slightly bent towards the actin subunit at the end, thereby blocking the P-end.
CooA, a heme-containing transcriptional activator, binds CO to the heme moiety and then undergoes... more CooA, a heme-containing transcriptional activator, binds CO to the heme moiety and then undergoes a structural change that promotes the specific binding to the target DNA. To elucidate the activation mechanism coupled to CO binding, we investigated the CO-dependent structural transition of CooA with small-angle X-ray scattering (SAXS). In the absence of CO, the radius of gyration Rg and the second virial coefficient (A2) were 25.3(+/-0.5)A and -0.39(+/-0.25) x 10(-4)ml mol g(-2), respectively. CO binding caused a slight increase in Rg (by 0.5A) and a marked decrease in A2 (by 5.09 x 10(-4)ml mol g(-2)). The observed decrease in A2 points to higher attractive interactions between CO-bound CooA molecules in solution compared with CO-free CooA. Although the minor alternation of Rg rules out changes in the overall structure, the marked change in the surface properties points to a CO-induced conformational transition. The experimental Rg and SAXS curves of the two states did not agree with the crystal structure of CO-free CooA. We thus simulated the solution structures of CooA based on the experimental data using rigid-body refinements as well as low-resolution model reconstructions. Both results demonstrate that the hinge region connecting the N-terminal heme domain and C-terminal DNA-binding domain is kinked in CO-free CooA, so that the two domains are positioned close to each other. The CO-dependent structural change observed by SAXS corresponds to a slight swing of the DNA-binding domains away from the heme domains coupled with their rotation by about 8 degrees around the axis of 2-fold symmetry.
To examine the effects of aggregation-inducing motifs related to neurodegenerative diseases on am... more To examine the effects of aggregation-inducing motifs related to neurodegenerative diseases on amyloid formation of host protein, we prepared several chimera myoglobins, in which various aggregation-inducing motifs were inserted. The focused aggregation-inducing motifs included five (R5) or two (R2) oligopeptide repeats in yeast Sup35p, five octapeptide repeats (OPR) in the human prion protein, a nonamyloid beta component (NAC) in alpha-synuclein, and tandem repeats of 50 glutamines (Q50). Circular dichroism and infrared spectroscopies suggested that the OPR, R5, and Q50 motifs formed an antiparallel beta sheet as well as a random coil, whereas the R2 and NAC motifs mainly formed random coils. The OPR, R5, and Q50 mutants, but not the R2 and NAC mutants, readily formed the SDS-resistant aggregates under physiological condition, and electron microscopy revealed that the aggregates contained amyloid fibrils. The destabilization and increase in gyration radius of the OPR, R5, and Q50 mutants correlated with the tendency to form amyloid fibrils. A control mutant bearing a nonamyloidgenic sequence was also moderately destabilized but did not form amyloid fibrils. Therefore, we concluded that the OPR, R5, and Q50 motifs, even in a quite stable protein such as myoglobin, led the host protein to formation of amyloid fibrils under physiological condition.
Proceedings of The National Academy of Sciences, 2005
Characterization of the conformational landscapes for proteins with different secondary structure... more Characterization of the conformational landscapes for proteins with different secondary structures is important in elucidating the mechanism of protein folding. The folding trajectory of single-chain monellin composed of a five-stranded beta-sheet and a helix was investigated by using a pH-jump from the alkaline unfolded to native state. The kinetic changes in the secondary structures and in the overall size and shape were measured by circular dichroism spectroscopy and small-angle x-ray scattering, respectively. The formation of the tertiary structure was monitored by intrinsic and extrinsic fluorescence. A significant collapse was observed within 300 micros after the pH-jump, leading to the intermediate with a small amount of secondary and tertiary structures but with an overall oblate shape. Subsequently, the stepwise formation of secondary and tertiary structures was detected. The current observation was consistent with the theoretical prediction that a more significant collapse precedes the formation of secondary structures in the folding of beta-sheet proteins than that of helical proteins [Shea, J. E., Onuchic, J. N. & Brooks, C. L., III (2002) Proc. Natl. Acad. Sci. USA 99, 16064-16068]. Furthermore, it was implied that the initial collapse was promoted by the formation of some specific structural elements, such as tight turns, to form the oblate shape.
Artificial sequences of the 153 amino acids have been designed to fit the main-chain framework of... more Artificial sequences of the 153 amino acids have been designed to fit the main-chain framework of the sperm whale myoglobin (Mb) structure based on a knowledge-based 3D-1D compatibility method. The previously designed artificial globin (DG1) folded into a monomeric, compact, highly helical and globular form with overall dimensions similar to those of the target structure, but it lacked structural uniqueness at the side-chain level [Isogai, Y., Ota, M., Fujisawa, T. , Izuno, H., Mukai, M., Nakamura, H., Iizuka, T., and Nishikawa, K. (1999) Biochemistry 38, 7431-7443]. In this study, we redesigned hydrophobic sites of DG1 to improve the structural specificity. Several Leu and Met residues in DG1 were replaced with beta-branched amino acids, Ile and Val, referring to the 3D profile of DG1 to produce three redesigned globins, DG2-4. These residue replacements resulted in no significant changes of their compactness and alpha-helical contents in the absence of denaturant, whereas they significantly affected the dependence of the secondary structure on the concentration of guanidine hydrochloride. The analyses of the denaturation curves revealed higher global stabilities of the designed globins than that of natural apoMb. Among DG1-4, DG3, in which 11 Leu residues of DG1 are replaced with seven Ile and four Val residues, and one Met residue is replaced with Val, displayed the lowest stability but the most cooperative folding-unfolding transition and the most dispersed NMR spectrum with the smallest line width. The present results indicate that the replacements of Leu (Met) with the beta-branched amino acids at appropriate sites reduce the freedom of side-chain conformation and improve the structural specificity at the expense of stability.
Static and time-resolved two-dimensional x-ray diffraction patterns, recorded from the living mou... more Static and time-resolved two-dimensional x-ray diffraction patterns, recorded from the living mouse diaphragm muscle, were compared with those from living frog sartorius muscle. The resting pattern of mouse muscle was similar to that of frog muscle, and consisted of actin- and myosin-based reflections with spacings basically identical to those of frog. As a notable exception, the sampling pattern of the myosin layer lines (MLL's) indicated that the mouse myofilaments were not organized into a superlattice as in frog. The intensity changes of reflections upon activation were also similar. The MLL's of both muscles were markedly weakened. Stereospecific (rigorlike) actomyosin species were not significantly populated in either muscle, as was evidenced by the 6th actin layer line (ALL), which was substantially enhanced but without a shift in its peak position or a concomitant rise of lower order ALL's. On close examination of the mouse pattern, however, a few lower order ALL's were found to rise, slightly but definitely, at the position expected for stereospecific binding. Their quick rise after the onset of stimulation indicates that this stereospecific complex is generated in the process of normal contraction. However, their rise is still too small to account for the marked enhancement of the 6th ALL, which is better explained by a myosin-induced structural change of actin. Since the forces of the two muscles are comparable regardless of the amount of stereospecific complex, it would be natural to consider that most of the force of skeletal muscle is supported by nonstereospecific actomyosin species.
The effect of high pressure on lactate dehydrogenase (LDH) was studied using small-angle X-ray sc... more The effect of high pressure on lactate dehydrogenase (LDH) was studied using small-angle X-ray scattering (SAXS). The SAXS results are interpreted in terms of the dissociation and association of LDH within a compression and decompression cycle and its temperature dependence. LDH consists of four identical subunits. At 120 MPa and 25 degrees C, 50% of the LDH dissociates into subunits, while at 10 degrees C, this occurs at 78 MPa. The hysteresis in the dissociation and association under pressure was confirmed in terms of the radius of gyration and was seen to be more conspicuous at low temperature. Forward scattering, I(0)/C, which is proportional to molecular weight, showed that LDH dissociated into dimer (not monomer) subunits under pressure. The application of high pressure to dissociated dimers induced irreversible aggregation. This result is in sharp contrast with the result of fluorescence spectroscopy suggesting a dissociated monomer [King, L., and Weber, G. (1986) Biochemistry 25, 3637-3640]. As for structural change after reassociation, there was little structural difference between native and drifted LDH. The difference was smaller than the structure change by ligand binding. At 200 MPa, the presence of five scattering peaks in the medium-angle region indicates that the dissociated dimer does not have a molten globule-like structure but a core structure. We propose a model of the dissociated dimer, based on the SAXS profile, in which the volume is reduced without disrupting the core structure.
SPring-8 RIKEN beamline I has been designed and developed for structural biology research by the ... more SPring-8 RIKEN beamline I has been designed and developed for structural biology research by the Institute of Physical and Chemical Research (RIKEN). The beamline consists of two experimental stations for protein crystallography and small-angle X-ray scattering. Both types of experiments can be carried out simultaneously, with dichromatic synchrotron radiation emitted from two coaxial undulators with vertical polarization. The branched beams are generated by a transparent diamond crystal. With synchrotron radiation, the multiple-wavelength anomalous-dispersion (MAD) method, which gives phases from a single anomalous scatterer, has been developed. Anomalous scattering contributes a small proportion of the diffraction intensity so that the accuracy of intensity data is important. The protein crystallography branch of RIKEN beamline I has been designed based on a 'trichromatic concept' to optimize MAD data collection. This concept requires the quasi-simultaneous collection, by use of a 'trichromator', of three intensity data sets at three different wavelengths from a single protein crystal without changing any settings. The main feature of the concept is the minimization of systematic errors in the measurement of anomalous diffraction for the MAD method. Initial commissioning of the beamline has provided three different monochromated undulator beams, which were successfully observed on the phosphor screen located at the near end of the trichromator.
Tropomodulin, the P-end (slow-growing end)-capping protein of the actin-tropomyosin filament, and... more Tropomodulin, the P-end (slow-growing end)-capping protein of the actin-tropomyosin filament, and its fragment (C20) of the C-terminal half were studied by synchrotron small-angle X-ray scattering, restoring low-resolution shapes using an ab initio shape-determining procedure. Tropomodulin is elongated (115 Å long) and consists of two domains, one of 65 Å in length and the other being similar to C20 in shape and size if the long axes of the two are tilted by about 40° relative to each other. We propose a model for tropomodulin in association with tropomyosin and actin: the N-terminal half of tropomodulin, a rod, binds to the N-terminus of tropomyosin and the C-terminal triangle domain protrudes from the P-end being slightly bent towards the actin subunit at the end, thereby blocking the P-end.
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Papers by Tetsuro Fujisawa