Apparent radii of the native, stable intermediates and unfolded conformers of the alpha-subunit of tryptophan synthase from E. coli, a TIM barrel protein.
Apparent radii of the native, stable intermediates and unfolded conformers of the alpha-subunit of tryptophan synthase from E. coli, a TIM barrel protein.
复制标题
大肠杆菌色氨酸合酶（TIM 桶状蛋白）α 亚基的天然稳定中间体和未折叠构象异构体的表观半径。
DOI：
10.1021/bi991296s
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发表时间：
1999
期刊：
影响因子：
2.9
通讯作者：
Matthews,CR
中科院分区：
文献类型：
--
作者：
Gualfetti,PJ;Iwakura,M;Lee,JC;Kihara,H;Bilsel,O;Zitzewitz,JA;Matthews,CR
The urea-induced equilibrium unfolding of the α-subunit of tryptophan synthase (αTS) fromEscherichia colican 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,Rs, of ∼24 Å for the native state of αTS. The small-angle X-ray scattering (SAXS) experiment yields a radius of gyration,Rg, 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 thatRsincreases 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 thatRsincreases linearly with increasing urea concentration to ∼54 Å at 8 M urea. The measurements ofRsby DLS andRgby SAXS are sufficiently imprecise that both values appear to be identical for 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-Rincón, G., Gualfetti, P. J., and Matthews, C. R. (1996)Biochemistry35, 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 respective conformational ensembles.
