Long‐range side‐chain–main‐chain interactions play crucial roles in stabilizing the (βα)8 barrel motif of the alpha subunit of tryptophan synthase
- 1 July 2007
- journal article
- Published by Wiley in Protein Science
- Vol. 16 (7) , 1398-1409
- https://doi.org/10.1110/ps.062704507
Abstract
The role of hither-to-fore unrecognized long-range hydrogen bonds between main-chain amide hydrogens and polar side chains on the stability of a well-studied (betaalpha)8, TIM barrel protein, the alpha subunit of tryptophan synthase (alphaTS), was probed by mutational analysis. The F19-D46 and I97-D124 hydrogen bonds link the N terminus of a beta-strand with the C terminus of the succeeding antiparallel alpha-helix, and the A103-D130 hydrogen bond links the N terminus of an alpha-helix with the C terminus of the succeeding antiparallel beta-strand, forming clamps for the respective betaalpha or alphabeta hairpins. The individual replacement of these aspartic acid side chains with alanine leads to what appear to be closely related partially folded structures with significantly reduced far-UV CD ellipticity and thermodynamic stability. Comparisons with the effects of eliminating another main-chain-side-chain hydrogen bond, G26-S33, and two electrostatic side-chain-side-chain hydrogen bonds, D38-H92 and D112-H146, all in the same N-terminal folding unit of alphaTS, demonstrated a unique role for the clamp interactions in stabilizing the native barrel conformation. Because neither the asparagine nor glutamic acid variant at position 46 can completely reproduce the spectroscopic, thermodynamic, or kinetic folding properties of aspartic acid, both size and charge are crucial to its unique role in the clamp hydrogen bond. Kinetic studies suggest that the three clamp hydrogen bonds act in concert to stabilize the transition state leading to the fully folded TIM barrel motif.Keywords
This publication has 25 references indexed in Scilit:
- A Tightly Packed Hydrophobic Cluster Directs the Formation of an Off-pathway Sub-millisecond Folding Intermediate in the α Subunit of Tryptophan Synthase, a TIM Barrel ProteinJournal of Molecular Biology, 2006
- Strength and co-operativity of contributions of surface salt bridges to protein stabilityPublished by Elsevier ,2006
- Multi-state Unfolding of the Alpha Subunit of Tryptophan Synthase, a TIM Barrel Protein: Insights into the Secondary Structure of the Stable Equilibrium Intermediates by Hydrogen Exchange Mass SpectrometryJournal of Molecular Biology, 2004
- Salt-bridges can Stabilize but do not Accelerate the Folding of the Homodimeric Coiled-coil Peptide GCN4-p1Journal of Molecular Biology, 2004
- Partial NMR assignments and secondary structure mapping of the isolated α subunit of Escherichia coli tryptophan synthase, a 29‐kD TIM barrel proteinProtein Science, 2003
- Molecular Dissection of the Folding Mechanism of the α Subunit of Tryptophan Synthase: An Amino-Terminal Autonomous Folding Unit Controls Several Rate-Limiting Steps in the Folding of a Single Domain ProteinBiochemistry, 1999
- The progressive development of structure and stability during the equilibrium folding of the α subunit of tryptophan synthase from Escherichia coliProtein Science, 1999
- Satisfying Hydrogen Bonding Potential in ProteinsJournal of Molecular Biology, 1994
- Effect of cavity-creating mutations in the hydrophobic core of chymotrypsin inhibitor 2Biochemistry, 1993
- The folding of an enzyme: II. Substructure of barnase and the contribution of different interactions to protein stabilityJournal of Molecular Biology, 1992