Molecular dissection of the folding mechanism of the alpha subunit of tryptophan synthase: an amino-terminal autonomous folding unit controls several rate-limiting steps in the folding of a single domain protein.


Abstract

The alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli is a 268-residue 8-stranded beta/alpha barrel protein. Two autonomous folding units, comprising the first six strands (residues 1-188) and the last two strands (residues 189-268), have been previously identified in this single structural domain protein by tryptic digestion [Higgins, W., Fairwell, T., and Miles, E. W. (1979) Biochemistry 18, 4827-4835]. The larger, amino-terminal fragment, alphaTS(1-188), was overexpressed and independently purified, and its equilibrium and kinetic folding properties were studied by absorbance, fluorescence, and near- and far-UV circular dichroism spectroscopies. The native state of the fragment unfolds cooperatively in an apparent two-state transition with a stability of 3.98 +/- 0.19 kcal mol(-1) in the absence of denaturant and a corresponding m value of 1.07 +/- 0.05 kcal mol(-1) M(-1). Similar to the full-length protein, the unfolding of the fragment shows two kinetic phases which arise from the presence of two discrete native state populations. Additionally, the fragment exhibits a significant burst phase in unfolding, indicating that a fraction of the folded state ensemble under native conditions has properties similar to those of the equilibrium intermediate populated at 3 M urea in full-length alphaTS. Refolding of alphaTS(1-188) is also complex, exhibiting two detectable kinetic phases and a burst phase that is complete within 5 ms. The two slowest isomerization phases observed in the refolding of the full-length protein are absent in the fragment, suggesting that these phases reflect contributions from the carboxy-terminal segment. The folding mechanism of alphaTS(1-188) appears to be a simplified version of the mechanism for the full-length protein [Bilsel, O., Zitzewitz, J. A., Bowers, K.E, and Matthews, C. R.(1999) Biochemistry 38, 1018-1029]. Four parallel channels in the full-length protein are reduced to a pair of channels that most likely reflect a cis/trans proline isomerization reaction in the amino-terminal fragment. The off- and on-pathway intermediates that exist for both full-length alphaTS and alphaTS(1-188) may reflect the preponderance of local interactions in the beta/alpha barrel motif. Study holds ProTherm entries: 23835, 23836, 23837, 23838, 23839, 23840 Extra Details: Tryptophan synthase alpha-subunit (1-188), Absorbance at 287 nm, Added additives: 1) 0.2 mM K2EDTA, 2) 1mM beta-mercaptoethanol, State:Folding to unfolding folding mechanism, amino-terminal fragment, tryptophan synthase alpha-subunit

Submission Details

ID: AQ8pKzWm3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:54 p.m.

Version: 1

Publication Details
Zitzewitz JA;Matthews CR,Biochemistry (1999) Molecular dissection of the folding mechanism of the alpha subunit of tryptophan synthase: an amino-terminal autonomous folding unit controls several rate-limiting steps in the folding of a single domain protein. PMID:10433729
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 UniProtKB Entries

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