Date Published: June 28, 2010
Publisher: Public Library of Science
Author(s): Amit Bhardwaj, Sadhu Leelavathi, Sudeshna Mazumdar-Leighton, Amit Ghosh, Suryanarayanarao Ramakumar, Vanga S. Reddy, Andreas Hofmann. http://doi.org/10.1371/journal.pone.0011347
Abstract: Stabilization strategies adopted by proteins under extreme conditions are very complex and involve various kinds of interactions. Recent studies have shown that a large proportion of proteins have their N- and C-terminal elements in close contact and suggested they play a role in protein folding and stability. However, the biological significance of this contact remains elusive.
In the present study, we investigate the role of N- and C-terminal residue interaction using a family 10 xylanase (BSX) with a TIM-barrel structure that shows stability under high temperature, alkali pH, and protease and SDS treatment. Based on crystal structure, an aromatic cluster was identified that involves Phe4, Trp6 and Tyr343 holding the N- and C-terminus together; this is a unique and important feature of this protein that might be crucial for folding and stability under poly-extreme conditions.
A series of mutants was created to disrupt this aromatic cluster formation and study the loss of stability and function under given conditions. While the deletions of Phe4 resulted in loss of stability, removal of Trp6 and Tyr343 affected in vivo folding and activity. Alanine substitution with Phe4, Trp6 and Tyr343 drastically decreased stability under all parameters studied. Importantly, substitution of Phe4 with Trp increased stability in SDS treatment. Mass spectrometry results of limited proteolysis further demonstrated that the Arg344 residue is highly susceptible to trypsin digestion in sensitive mutants such as ΔF4, W6A and Y343A, suggesting again that disruption of the Phe4-Trp6-Tyr343 (F-W-Y) cluster destabilizes the N- and C-terminal interaction. Our results underscore the importance of N- and C-terminal contact through aromatic interactions in protein folding and stability under extreme conditions, and these results may be useful to improve the stability of other proteins under suboptimal conditions.
Partial Text: Thermal stability of proteins is one of the most extensively documented properties in protein biochemistry, and yet we still do not have a complete understanding of the stabilization strategies adopted by proteins. Stabilization becomes even more difficult under poly-extreme conditions, such as extreme pH, protease treatment and SDS treatment. Each class of proteins appears to have evolved its own mechanism for establishing protein stability under extreme conditions rather than converging on a single universal mechanism. It is therefore necessary to identify the determinants of protein stability for each class of proteins. The TIM-barrel structure (β/α)8 provides an excellent model to address the function and stability of an enzyme. It is the most common protein fold and is present in approximately 10% of all known enzyme structures. Several investigations have been carried out to better understand the principles underlying the folding and stability of the TIM-barrel fold , [ 2], [ 3]. The major interactions reported to be responsible for these properties include ionic bonding, hydrogen bonding and hydrophobic interactions. Aromatic interactions have also been reported to enhance the stability of proteins, mainly via dimer or higher order polymer formation . Nevertheless, a recent in silico analysis based on protein structures available in the protein structure database has pointed out the correlation between the presence of large numbers of aromatic clusters and the thermal stability of proteins . However, until recently, no experimental evidence was available concerning the significant role, if any, played by any specific aromatic interaction(s) between the N- and C-terminus in protein folding and stability under poly-extreme conditions.
Many factors affect protein stability, including hydrophobic clusters , oligomerization , [ 26], salt bridges , [ 27], [ 28], an increased number of side-chain–side-chain hydrogen bonds, a smaller number of glutamine and asparagines, improved packing of the hydrophobic core , aromatic clusters , [ 30], [ 31], [ 32], and cation-pi interactions , [ 33], [ 34]. A comprehensive in silico analysis by Gromiha et al. showed that most of the bona fide stabilizing residues are oriented toward the interior of the TIM-barrel fold  but did not predict much about the interactions between the very N- and C-terminal residues in the stability of these proteins. In this study, for the first time we have experimentally established the crucial role played by the N- and C-terminal contacts involving Phe4-Trp6-Tyr343 (F-W-Y) aromatic cluster in folding, as well as the stability of BSX under extreme conditions.