Folding mechanism of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus: a test of the conservation of folding mechanisms hypothesis in (beta(alpha))(8) barrels.


Abstract

As a test of the hypothesis that folding mechanisms are better conserved than sequences in TIM barrels, the equilibrium and kinetic folding mechanisms of indole-3-glycerol phosphate synthase (sIGPS) from the thermoacidophilic archaebacterium Sulfolobus solfataricus were compared to the well-characterized models of the alpha subunit of tryptophan synthase (alphaTS) from Escherichia coli. A multifaceted approach combining urea denaturation and far-UV circular dichroism, tyrosine fluorescence total intensity, and tyrosine fluorescence anisotropy was employed. Despite a sequence identity of only 13%, a stable intermediate (I) in sIGPS was found to be similar to a stable intermediate in alphaTS in terms of its thermodynamic properties and secondary structure. Kinetic experiments revealed that the fastest detectable folding event for sIGPS involves a burst-phase (<5ms) reaction that leads directly to the stable intermediate. The slower of two subsequent phases reflects the formation/disruption of an off-pathway dimeric form of I. The faster phase reflects the conversion of I to the native state and is limited by folding under marginally stable conditions and by isomerization or rearrangement under strongly folding conditions. By contrast, alphaTS is thought to fold via an off-pathway burst-phase intermediate whose unfolding controls access to a set of four on-pathway intermediates that comprise the stable equilibrium intermediate. At least three proline isomerization reactions are known to limit their interconversions and lead to a parallel channel mechanism. The simple sequential mechanism deduced for sIGPS reflects the dominance of the on-pathway burst-phase intermediate and the absence of prolyl residues that partition the stable intermediate into kinetically distinguishable species. Comparison of the results for sIGPS and alphaTS demonstrates that the thermodynamic properties and the final steps of the folding reaction are better conserved than the early events. The initial events in folding appear to be more sensitive to the sequence differences between the two TIM barrel proteins. Study holds ProTherm entries: 15457, 15458 Extra Details: N->I; 0.2mM K2EDTA was added in the experiment conserved folding mechanisms; TIM barrel

Submission Details

ID: LJJUdFAS4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:46 p.m.

Version: 1

Publication Details
Forsyth WR;Matthews CR,J. Mol. Biol. (2002) Folding mechanism of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus: a test of the conservation of folding mechanisms hypothesis in (beta(alpha))(8) barrels. PMID:12126630
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
1A53 1998-02-19T00:00:00+0000 2.0 COMPLEX OF INDOLE-3-GLYCEROLPHOSPHATE SYNTHASE FROM SULFOLOBUS SOLFATARICUS WITH INDOLE-3-GLYCEROLPHOSPHATE AT 2.0 A RESOLUTION
1IGS 1995-08-11T00:00:00+0000 2.0 INDOLE-3-GLYCEROLPHOSPHATE SYNTHASE FROM SULFOLOBUS SOLFATARICUS AT 2.0 A RESOLUTION
1JUK 1996-05-03T00:00:00+0000 2.5 INDOLE-3-GLYCEROLPHOSPHATE SYNTHASE FROM SULFOLOBUS SOLFATARICUS IN A TRIGONAL CRYSTAL FORM
1JUL 1996-05-03T00:00:00+0000 2.0 INDOLE-3-GLYCEROLPHOSPHATE SYNTHASE FROM SULFOLOBUS SOLFATARICUS IN A SECOND ORTHORHOMBIC CRYSTAL FORM
1LBF 2002-04-03T00:00:00+0000 2.05 CRYSTAL STRUCTURE OF INDOLE-3-GLYCEROL PHOSPHATE SYNTASE (IGPS)WITH REDUCED 1-(O-CABOXYPHENYLAMINO)-1-DEOXYRIBULOSE 5-PHOSPHATE (RCDRP)
1LBL 2002-04-04T00:00:00+0000 2.4 Crystal structure of indole-3-glycerol phosphate synthase (IGPS) in complex with 1-(o-carboxyphenylamino)-1-deoxyribulose 5'-phosphate (CdRP)
2C3Z 2005-10-13T00:00:00+0000 2.8 Crystal structure of a truncated variant of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus
3NYZ 2010-07-15T00:00:00+0000 1.51 Crystal Structure of Kemp Elimination Catalyst 1A53-2
3NZ1 2010-07-15T00:00:00+0000 1.56 Crystal Structure of Kemp Elimination Catalyst 1A53-2 Complexed with Transition State Analog 5-Nitro Benzotriazole
3TC6 2011-08-08T00:00:00+0000 1.6 Crystal Structure of Engineered Protein. Northeast Structural Genomics Consortium Target OR63.

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Indole-3-glycerol phosphate synthase Q06121 TRPC_SACS2
100.0 Multifunctional tryptophan biosynthesis protein P25170 TRPG_PHACH