The progressive development of structure and stability during the equilibrium folding of the alpha subunit of tryptophan synthase from Escherichia coli.


Abstract

The urea-induced equilibrium unfolding of the alpha subunit of tryptophan synthase (alphaTS), a single domain alpha/beta barrel protein, displays a stable intermediate at approximately 3.2 M urea when monitored by absorbance and circular dichroism (CD) spectroscopy (Matthews CR, Crisanti MM, 1981, Biochemistry 20:784-792). The same experiment, monitored by one-dimensional proton NMR, shows another cooperative process between 5 and 9 M urea that involves His92 (Saab-Rincón G et al., 1993, Biochemistry 32:13,981-13,990). To further test and quantify the implied four-state model, N <--> I1 <--> I2 <--> U, the urea-induced equilibrium unfolding process was followed by tyrosine fluorescence total intensity, tyrosine fluorescence anisotropy and far-UV CD. All three techniques resolve the four stable states, and the transitions between them when the FL total intensity and CD spectroscopy data were analyzed by the singular value decomposition method. Relative to U, the stabilities of the N, I1, and I2 states are 15.4, 9.4, and 4.9 kcal mol(-1), respectively. I2 partially buries one or more of the seven tyrosines with a noticeable restriction of their motion; it also recovers approximately 6% of the native CD signal. This intermediate, which is known to be stabilized by the hydrophobic effect, appears to reflect the early coalescence of nonpolar side chains without significant organization of the backbone. I1 recovers an additional 43% of the CD signal, further sequesters tyrosine residues in nonpolar environments, and restricts their motion to an extent similar to N. The progressive development of a higher order structure as the denaturant concentration decreases implies a monotonic contraction in the ensemble of conformations that represent the U, I2, I1, and N states of alphaTS. Study holds ProTherm entries: 6724, 6725, 6726, 6727, 6728, 6729 Extra Details: additive : K2EDTA(0.2 mM),alphaTS: the alpha subunit of tryptophan synthase from Escherichia coli,transition 1 anisotropy; circular dichroism; fluorescence;,singular value decomposition; stable folding intermediates

Submission Details

ID: fnoR8WoE4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:32 p.m.

Version: 1

Publication Details
Gualfetti PJ;Bilsel O;Matthews CR,Protein Sci. (1999) The progressive development of structure and stability during the equilibrium folding of the alpha subunit of tryptophan synthase from Escherichia coli. PMID:10452606
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