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


Abstract

The urea-induced equilibrium unfolding of the alpha-subunit of tryptophan synthase (alphaTS) from Escherichia coli can be described by a four-state model, N right harpoon over left harpoon I1 right harpoon over left harpoon I2 right harpoon over left harpoon 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 approximately 24 A for the native state of alphaTS. The small-angle X-ray scattering (SAXS) experiment yields a radius of gyration, R(g), of 19.1 A, consistent with the value predicted from the X-ray structure and the Stokes radius. As the equilibrium is shifted to favor I1 at approximately 3.2 M and I2 at 5.0 M urea, SEC and UC show that R(s) increases from approximately 38 to approximately 52 A. 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 approximately 54 A 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 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 right harpoon over left harpoon I1 and I1 right harpoon over left harpoon 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 right harpoon over left harpoon 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) 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 respective conformational ensembles. Study holds ProTherm entries: 5864, 5865, 5866, 5867, 5868, 5869, 5870, 5871 Extra Details: additive : K2EDTA(0.2 mM),transition is from native to intermediate 1 four-state model; apparent radius; Stokes radius;,radius of gyration; thermodynamic parameter

Submission Details

ID: vu2kFvLW4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:31 p.m.

Version: 1

Publication Details
Gualfetti PJ;Iwakura M;Lee JC;Kihara H;Bilsel O;Zitzewitz JA;Matthews CR,Biochemistry (1999) Apparent radii of the native, stable intermediates and unfolded conformers of the alpha-subunit of tryptophan synthase from E. coli, a TIM barrel protein. PMID:10529212
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1XCF 2004-11-02 1.8 Crystal structure of P28L/Y173F tryptophan synthase alpha-subunits from Escherichia coli
1WQ5 2005-02-15 2.3 Crystal structure of tryptophan synthase alpha-subunit from Escherichia coli
1V7Y 2005-02-15 2.5 Crystal structure of tryptophan synthase alpha-subunit from Escherichia coli at room temperature
1XC4 2004-11-02 2.8 Crystal structure of wild-type tryptophan synthase alpha-subunits from Escherichia coli

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
97.0 Tryptophan synthase alpha chain B7LS20 TRPA_ESCF3
97.8 Tryptophan synthase alpha chain B7NVN0 TRPA_ECO7I
98.1 Tryptophan synthase alpha chain B1LH32 TRPA_ECOSM
98.5 Tryptophan synthase alpha chain B7N473 TRPA_ECOLU
98.5 Tryptophan synthase alpha chain Q0TIB0 TRPA_ECOL5
98.1 Tryptophan synthase alpha chain B7MU99 TRPA_ECO81
98.9 Tryptophan synthase alpha chain B7UR66 TRPA_ECO27
98.5 Tryptophan synthase alpha chain Q1RCA7 TRPA_ECOUT
98.5 Tryptophan synthase alpha chain A1AAN0 TRPA_ECOK1
98.5 Tryptophan synthase alpha chain B7ML76 TRPA_ECO45
98.9 Tryptophan synthase alpha chain B5YZP0 TRPA_ECO5E
98.9 Tryptophan synthase alpha chain Q8X7B5 TRPA_ECO57
99.3 Tryptophan synthase alpha chain Q32GT0 TRPA_SHIDS
98.9 Tryptophan synthase alpha chain Q3Z108 TRPA_SHISS
99.6 Tryptophan synthase alpha chain Q0T5D6 TRPA_SHIF8
99.6 Tryptophan synthase alpha chain Q31ZV3 TRPA_SHIBS
99.3 Tryptophan synthase alpha chain Q8FHW0 TRPA_ECOL6
99.6 Tryptophan synthase alpha chain B2U0F1 TRPA_SHIB3
99.3 Tryptophan synthase alpha chain A7ZL78 TRPA_ECO24
99.6 Tryptophan synthase alpha chain B7L492 TRPA_ECO55
99.6 Tryptophan synthase alpha chain B6I9X4 TRPA_ECOSE
99.6 Tryptophan synthase alpha chain B7LY16 TRPA_ECO8A
100.0 Tryptophan synthase alpha chain P0A878 TRPA_SHIFL
100.0 Tryptophan synthase alpha chain P0A877 TRPA_ECOLI
100.0 Tryptophan synthase alpha chain B1ITJ5 TRPA_ECOLC
100.0 Tryptophan synthase alpha chain A7ZZJ6 TRPA_ECOHS
100.0 Tryptophan synthase alpha chain B1XBK9 TRPA_ECODH
100.0 Tryptophan synthase alpha chain C4ZTV3 TRPA_ECOBW