Long‐range side‐chain–main‐chain interactions play crucial roles in stabilizing the (βα)₈ barrel motif of the alpha subunit of tryptophan synthase

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 (ba) 8 , TIM barrel protein, the alpha subunit of tryptophan synthase (aTS), was probed by mutational analysis. The F19-D46 and I97-D124 hydrogen bonds link the N terminus of a b-strand with the C terminus of the succeeding antiparallel ahelix, and the A103-D130 hydrogen bond links the N terminus of an a-helix with the C terminus of the succeeding antiparallel b-strand, forming clamps for the respective ba or ab 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 aTS, 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: circular dichroism; protein folding; site-directed mutagenesis; thermodynamic and kinetic mechanisms; ba and ab hairpins Side chains stabilize the native, functional conformations of proteins by van der Waals interactions between nonpolar partners, hydrogen bonds (H-bonds) between polar donors and acceptors, and electrostatic interactions between opposing charged groups. Mutational analysis of buried nonpolar side chains has shown that replacements with alanine can lead to destabilizations of up to 7 kcal mol À1 (Shortle et al. 1990;Eriksson et al. 1992;Serrano et al. 1992). The magnitudes of the effects correlate with the local packing density (Shortle et al. 1990) and the burial of nonpolar surface area (Jackson et al. 1993

βα-Hairpin Clamps Brace βαβ Modules and Can Make Substantive Contributions to the Stability of TIM Barrel Proteins

“…The absence of the βα-hairpin clamps in the I states of all three TIM barrel proteins demonstrates that the potent effects of these clamps only appear as the N state appears [6]. Kinetic folding studies on αTS revealed further that each clamp is crucial for accessing the transition state ensemble required to reach the properly-folded structure [6].…”

“…Kinetic folding studies on αTS revealed further that each clamp is crucial for accessing the transition state ensemble required to reach the properly-folded structure [6]. Although the local connectivity of the βαβ modules might have been expected to enable the clamp to have a role in the early stages of the folding reaction, the primary role of the potent set of clamps is to drive the final stage of the reaction to completion and fully develop global cooperativity.…”

The Structure of a Truncated Phosphoribosylanthranilate Isomerase Suggests a Unified Model for Evolution of the (βα)8 Barrel Fold

“…37 A survey of 71 (βα) 8 barrels revealed a 4-fold symmetry in the preferred locations of these βα-hairpin clamps, and mutagenesis demonstrated their importance for stabilizing both αTS and IGPS. 37,38 The structure of PRAI 26 reveals a canonical βα-hairpin clamp, between the main chain amide hydrogen of Ile355 and a side chain oxygen of Asp374. This interaction secures the N-terminus of strand β 5 to the loop that immediately precedes strand β 6 .…”

Structural Analysis of Kinetic Folding Intermediates for a TIM Barrel Protein, Indole-3-glycerol Phosphate Synthase, by Hydrogen Exchange Mass Spectrometry and Gō Model Simulation

“…The possibility of misfolding at the periphery of an N-terminal core is supported by the results of another mutational analysis of a pair of long-range H-bonds in the N-terminal region between aspartic acid side chains at the C-termini of two helices and main chain amide hydrogens near the N-termini of their preceding β-strands. 56 These βα-hairpin clamps are disrupted in the I1 intermediate. However, the very strong protection of these amide hydrogens against exchange, > weeks-months at pH 7.8 and 25 °C, implies that both have found alternative partners in the I1 state and only exchange through other very high energy intermediates.…”