Apparent radii of the native, stable intermediates and unfolded conformers of the α-subunit of tryptophan synthase from E. coli, a TIM barrel protein

Peter J. Gualfetti, Masahiro Iwakura, James Lee, Hiroshi Kihara, Osman Bilsel, Jill A. Zitzewitz, C. Robert Matthews

Research output: Contribution to journalArticle

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Abstract

The urea-induced equilibrium unfolding of the α-subunit of tryptophan synthase (αTS) from Escherichia coli can be described by a four-state model, N ⇆ I1 ⇆ I2 ⇆ U, involving two highly populated intermediates, I1 and I2 [Gualfetti, P.J., Bilsel, O., and Matthews, C.R. (1999) Protein Sci. 8, 1623- 1635]. To extend the physical characterization of these stable forms, the apparent radius was measured by several techniques. size-exclusion chromatography (SEC), analytical ultracentrifugation (UC), and dynamic light scattering (DLS) experiments yield an apparent Stokes radius, R(s), of ~24 Å for the native state of αTS. The small-angle X-ray scattering (SAXS) experiment yields a radius of gyration, R(g), of 19.1 Å, consistent with the value predicted from the X-ray structure and the Stokes radius. As the equilibrium is shifted to favor I1 at ~3.2 M and I2 at 5.0 M urea, SEC and UC show that R(s) increases from ~38 to ~52 Å. Measurements of the radius by DLS and SAXS between 2 and 4.5 M urea were complicated by the self- association of the I1 species at the relatively high concentrations required by those techniques. Above 6 M urea, SEC and UC reveal that R(s) increases linearly with increasing urea concentration to ~54 Å at 8 M urea. The measurements of R(s) by DLS and R(g) by SAXS are sufficiently imprecise that both values appear to be identical to the I2 and U states and, considering the errors, are in good agreement with the results from SEC and UC. Thermodynamic parameters extracted from the SEC data for the N ⇆ I1 and I1 ⇆ I2 transitions agree with those from the optical data, showing that this technique accurately monitors a part of the equilibrium model. The lack of sensitivity to the I2 ⇆ U transition, beyond a simple swelling of both species with increasing urea concentration, implies that the Stokes radii for the I2 and U states are not distinguishable. Surprisingly, the hydrophobic core known to stabilize I2 at 5.0 M urea [Saab-Rincon, G., Gualfetti, P.J., and Matthews, C.R. (1996) Biochemistry 35, 1988-1994] develops without a significant contraction of the polypeptide, i.e., beyond that experienced by the unfolded form at decreasing urea concentrations. Kratky plots of the SAXS data, however, reveal that I2, similar to N and I1, has a globular structure while U has a more random coil-like form. By contrast, the formation of substantial secondary structure and the burial of aromatic side chains in I1 and, eventually, N are accompanied by substantial decreases in their Stokes radii and, presumably, the size of their respectively conformational ensembles.

Original languageEnglish (US)
Pages (from-to)13367-13378
Number of pages12
JournalBiochemistry
Volume38
Issue number40
DOIs
StatePublished - Oct 5 1999

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Tryptophan Synthase
Escherichia coli
Urea
Size exclusion chromatography
Ultracentrifugation
Gel Chromatography
X ray scattering
Proteins
X-Rays
Dynamic light scattering
Burial
Biochemistry
Thermodynamics
Swelling
Experiments
Association reactions

ASJC Scopus subject areas

  • Biochemistry

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Gualfetti, P. J., Iwakura, M., Lee, J., Kihara, H., Bilsel, O., Zitzewitz, J. A., & Matthews, C. R. (1999). Apparent radii of the native, stable intermediates and unfolded conformers of the α-subunit of tryptophan synthase from E. coli, a TIM barrel protein. Biochemistry, 38(40), 13367-13378. https://doi.org/10.1021/bi991296s

Apparent radii of the native, stable intermediates and unfolded conformers of the α-subunit of tryptophan synthase from E. coli, a TIM barrel protein. / Gualfetti, Peter J.; Iwakura, Masahiro; Lee, James; Kihara, Hiroshi; Bilsel, Osman; Zitzewitz, Jill A.; Matthews, C. Robert.

In: Biochemistry, Vol. 38, No. 40, 05.10.1999, p. 13367-13378.

Research output: Contribution to journalArticle

Gualfetti, PJ, Iwakura, M, Lee, J, Kihara, H, Bilsel, O, Zitzewitz, JA & Matthews, CR 1999, 'Apparent radii of the native, stable intermediates and unfolded conformers of the α-subunit of tryptophan synthase from E. coli, a TIM barrel protein', Biochemistry, vol. 38, no. 40, pp. 13367-13378. https://doi.org/10.1021/bi991296s
Gualfetti, Peter J. ; Iwakura, Masahiro ; Lee, James ; Kihara, Hiroshi ; Bilsel, Osman ; Zitzewitz, Jill A. ; Matthews, C. Robert. / Apparent radii of the native, stable intermediates and unfolded conformers of the α-subunit of tryptophan synthase from E. coli, a TIM barrel protein. In: Biochemistry. 1999 ; Vol. 38, No. 40. pp. 13367-13378.
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abstract = "The urea-induced equilibrium unfolding of the α-subunit of tryptophan synthase (αTS) from Escherichia coli can be described by a four-state model, N ⇆ I1 ⇆ I2 ⇆ U, involving two highly populated intermediates, I1 and I2 [Gualfetti, P.J., Bilsel, O., and Matthews, C.R. (1999) Protein Sci. 8, 1623- 1635]. To extend the physical characterization of these stable forms, the apparent radius was measured by several techniques. size-exclusion chromatography (SEC), analytical ultracentrifugation (UC), and dynamic light scattering (DLS) experiments yield an apparent Stokes radius, R(s), of ~24 {\AA} for the native state of αTS. The small-angle X-ray scattering (SAXS) experiment yields a radius of gyration, R(g), of 19.1 {\AA}, consistent with the value predicted from the X-ray structure and the Stokes radius. As the equilibrium is shifted to favor I1 at ~3.2 M and I2 at 5.0 M urea, SEC and UC show that R(s) increases from ~38 to ~52 {\AA}. Measurements of the radius by DLS and SAXS between 2 and 4.5 M urea were complicated by the self- association of the I1 species at the relatively high concentrations required by those techniques. Above 6 M urea, SEC and UC reveal that R(s) increases linearly with increasing urea concentration to ~54 {\AA} at 8 M urea. The measurements of R(s) by DLS and R(g) by SAXS are sufficiently imprecise that both values appear to be identical to the I2 and U states and, considering the errors, are in good agreement with the results from SEC and UC. Thermodynamic parameters extracted from the SEC data for the N ⇆ I1 and I1 ⇆ I2 transitions agree with those from the optical data, showing that this technique accurately monitors a part of the equilibrium model. The lack of sensitivity to the I2 ⇆ U transition, beyond a simple swelling of both species with increasing urea concentration, implies that the Stokes radii for the I2 and U states are not distinguishable. Surprisingly, the hydrophobic core known to stabilize I2 at 5.0 M urea [Saab-Rincon, G., Gualfetti, P.J., and Matthews, C.R. (1996) Biochemistry 35, 1988-1994] develops without a significant contraction of the polypeptide, i.e., beyond that experienced by the unfolded form at decreasing urea concentrations. Kratky plots of the SAXS data, however, reveal that I2, similar to N and I1, has a globular structure while U has a more random coil-like form. By contrast, the formation of substantial secondary structure and the burial of aromatic side chains in I1 and, eventually, N are accompanied by substantial decreases in their Stokes radii and, presumably, the size of their respectively conformational ensembles.",
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T1 - Apparent radii of the native, stable intermediates and unfolded conformers of the α-subunit of tryptophan synthase from E. coli, a TIM barrel protein

AU - Gualfetti, Peter J.

AU - Iwakura, Masahiro

AU - Lee, James

AU - Kihara, Hiroshi

AU - Bilsel, Osman

AU - Zitzewitz, Jill A.

AU - Matthews, C. Robert

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N2 - The urea-induced equilibrium unfolding of the α-subunit of tryptophan synthase (αTS) from Escherichia coli can be described by a four-state model, N ⇆ I1 ⇆ I2 ⇆ U, involving two highly populated intermediates, I1 and I2 [Gualfetti, P.J., Bilsel, O., and Matthews, C.R. (1999) Protein Sci. 8, 1623- 1635]. To extend the physical characterization of these stable forms, the apparent radius was measured by several techniques. size-exclusion chromatography (SEC), analytical ultracentrifugation (UC), and dynamic light scattering (DLS) experiments yield an apparent Stokes radius, R(s), of ~24 Å for the native state of αTS. The small-angle X-ray scattering (SAXS) experiment yields a radius of gyration, R(g), of 19.1 Å, consistent with the value predicted from the X-ray structure and the Stokes radius. As the equilibrium is shifted to favor I1 at ~3.2 M and I2 at 5.0 M urea, SEC and UC show that R(s) increases from ~38 to ~52 Å. Measurements of the radius by DLS and SAXS between 2 and 4.5 M urea were complicated by the self- association of the I1 species at the relatively high concentrations required by those techniques. Above 6 M urea, SEC and UC reveal that R(s) increases linearly with increasing urea concentration to ~54 Å at 8 M urea. The measurements of R(s) by DLS and R(g) by SAXS are sufficiently imprecise that both values appear to be identical to the I2 and U states and, considering the errors, are in good agreement with the results from SEC and UC. Thermodynamic parameters extracted from the SEC data for the N ⇆ I1 and I1 ⇆ I2 transitions agree with those from the optical data, showing that this technique accurately monitors a part of the equilibrium model. The lack of sensitivity to the I2 ⇆ U transition, beyond a simple swelling of both species with increasing urea concentration, implies that the Stokes radii for the I2 and U states are not distinguishable. Surprisingly, the hydrophobic core known to stabilize I2 at 5.0 M urea [Saab-Rincon, G., Gualfetti, P.J., and Matthews, C.R. (1996) Biochemistry 35, 1988-1994] develops without a significant contraction of the polypeptide, i.e., beyond that experienced by the unfolded form at decreasing urea concentrations. Kratky plots of the SAXS data, however, reveal that I2, similar to N and I1, has a globular structure while U has a more random coil-like form. By contrast, the formation of substantial secondary structure and the burial of aromatic side chains in I1 and, eventually, N are accompanied by substantial decreases in their Stokes radii and, presumably, the size of their respectively conformational ensembles.

AB - The urea-induced equilibrium unfolding of the α-subunit of tryptophan synthase (αTS) from Escherichia coli can be described by a four-state model, N ⇆ I1 ⇆ I2 ⇆ U, involving two highly populated intermediates, I1 and I2 [Gualfetti, P.J., Bilsel, O., and Matthews, C.R. (1999) Protein Sci. 8, 1623- 1635]. To extend the physical characterization of these stable forms, the apparent radius was measured by several techniques. size-exclusion chromatography (SEC), analytical ultracentrifugation (UC), and dynamic light scattering (DLS) experiments yield an apparent Stokes radius, R(s), of ~24 Å for the native state of αTS. The small-angle X-ray scattering (SAXS) experiment yields a radius of gyration, R(g), of 19.1 Å, consistent with the value predicted from the X-ray structure and the Stokes radius. As the equilibrium is shifted to favor I1 at ~3.2 M and I2 at 5.0 M urea, SEC and UC show that R(s) increases from ~38 to ~52 Å. Measurements of the radius by DLS and SAXS between 2 and 4.5 M urea were complicated by the self- association of the I1 species at the relatively high concentrations required by those techniques. Above 6 M urea, SEC and UC reveal that R(s) increases linearly with increasing urea concentration to ~54 Å at 8 M urea. The measurements of R(s) by DLS and R(g) by SAXS are sufficiently imprecise that both values appear to be identical to the I2 and U states and, considering the errors, are in good agreement with the results from SEC and UC. Thermodynamic parameters extracted from the SEC data for the N ⇆ I1 and I1 ⇆ I2 transitions agree with those from the optical data, showing that this technique accurately monitors a part of the equilibrium model. The lack of sensitivity to the I2 ⇆ U transition, beyond a simple swelling of both species with increasing urea concentration, implies that the Stokes radii for the I2 and U states are not distinguishable. Surprisingly, the hydrophobic core known to stabilize I2 at 5.0 M urea [Saab-Rincon, G., Gualfetti, P.J., and Matthews, C.R. (1996) Biochemistry 35, 1988-1994] develops without a significant contraction of the polypeptide, i.e., beyond that experienced by the unfolded form at decreasing urea concentrations. Kratky plots of the SAXS data, however, reveal that I2, similar to N and I1, has a globular structure while U has a more random coil-like form. By contrast, the formation of substantial secondary structure and the burial of aromatic side chains in I1 and, eventually, N are accompanied by substantial decreases in their Stokes radii and, presumably, the size of their respectively conformational ensembles.

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