Aggregation Gatekeeper and Controlled Assembly of Trpzip β-Hairpins

Abstract

Protein and peptide aggregation is an important issue both in vivo and in vitro. Herein, we examine the aggregation behaviors of two well studied β-hairpins, Trpzip1 and Trpzip2. Previous studies suggest that Trpzip2 remains monomeric up to a concentration of ~15 mM whereas Trpzip1 readily aggregates at micromolar concentrations at acidic or neutral pH. This disparity is puzzling considering that these two peptides only differ in their turn sequences (i.e., GN versus NG). We hypothesize that these peptides can aggregate from their folded states via native edge-to-edge interactions and that the Lys8 residue in Trpzip2 is a more effective aggregation gatekeeper, due to a more favorable orientation. In support of this hypothesis, we find that raising the pH to 13 or replacing Lys8 with a hydrophobic and photolabile Lys analog, Lys(nvoc), leads to a significant increase in the aggregation propensity of Trpzip2, and that the aggregation of this Trpzip2 mutant can be reversed upon restoring the native Lys sidechain via photocleavage of the nvoc moiety. In addition, we find that while both Trpzip1 and Trpzip2 form parallel β-sheet aggregates, the Lys(nvoc) Trpzip2 mutant forms antiparallel β-sheets and more stable fibrils. Taken together, these findings provide another example showing how sensitive peptide and protein aggregation is to minor sequence variation and that it is possible to use a photolabile non-natural amino acid, such as Lys(nvoc), to tune the rate of peptide aggregation and to control fibrillar structure.

Autonomous aggregation suppression by acidic residues explains why chaperones favour basic residues

“…We chose the denomination "aggregation gatekeepers" rather than "structural gatekeepers" to emphasize the specific position of these residues at the flanks of APRs. Several other studies confirmed the prevalence and importance of the GK pattern (Monsellier et al, 2008;Tartaglia & Vendruscolo, 2008;Buell et al, 2009;Wang et al, 2010b;Markiewicz et al, 2014;Sant'Anna et al, 2014;Estácio et al, 2015). We later confirmed that GKs constitute a bona fide and ubiquitous functional class specifically devoted to protein homeostasis.…”

Vibrational Approach to the Dynamics and Structure of Protein Amyloids

“…In addition to the aforementioned amyloidogenic proteins, the backbone amide I spectra have been widely used in aggregation studies of other disease-associated amyloidogenic proteins, e.g., crystallin [83], prion [84,85], polyglutamine (polyQ) [86], and model peptides [87,88]. For instance, deposits of aggregated crystallin on the lens of the eye cause blurred vision or blindness in cataracts.…”

Structural Characterization of Self‐Assembled Tetra‐Tryptophan Based Nanostructures: Variations on a Common Theme

“…In this PEGylated tetra‐peptide the Phe residue was replaced with the tryptophan (Trp, W). W is one of the essential amino acids and the aromatic ring in its side chain can represent fruitful chemical tool to address the aggregation in short peptides sequences . In addition, it is well‐known that Trp in long peptides or proteins can create structural motifs, called tryptophan zippers (Trpzip), which are stabilizing elements for the β‐hairpin conformation .…”

“…W is one of the essential amino acids and the aromatic ring in its side chain can represent fruitful chemical tool to address the aggregation in short peptides sequences. [30] In addition, it is well-known that Trp in long peptides or proteins can create structural motifs, called tryptophan zippers (Trpzip), which are stabilizing elements for the b-hairpin conformation. [31] Indeed, analogously to the previously studied PEG 6 -F4, also PEG 6 -W4 possesses a high tendency to aggregate.…”