Phosphorylation as Conformational Switch from the Native to Amyloid State: Trp-Cage as a Protein Aggregation Model

Abstract

The 20 residue long Trp-cage miniprotein is an excellent model both for computational and experimental studies of protein folding and stability. Recently, a great attention emerged to study disease-related protein misfolding, aggregation and amyloid formation, with the aim of revealing their structural and thermodynamic background. Trp-cage is sensitive to both environmental and structure-modifying effects, and aggregates with ease upon structure destabilization and thus, it is an ideal model of aggregation and amyloid formation. Here, we characterize the amyloid formation of several sequence modified and side-chain phosphorylated Trp-cage variants. We applied NMR, CD, FTIR spectroscopy, MD simulations, transmission electron microscopy in conjunction with thioflavin-T fluorescence measurements to reveal the structural consequences of side-chain phosphorylation. We demonstrate that the native fold is destabilized upon serine phosphorylation and the resultant highly dynamic structures form amyloid-like ordered aggregates with high intermolecular β-structure content. The only exception is the D9S(P) variant which follows an alternative aggregation process by forming thin fibrils, presenting a CD spectrum of PPII helix, and showing low ThT binding capability. We propose a complex aggregation model for these Trp-cage miniproteins. This model assumes an additional aggregated state, a collagen triple helical form which can precede amyloid formation. The phosphorylation of a single serine residue serves as a conformational switch, triggering aggregation, otherwise mediated by kinases in cell. We show that Trp-cage miniprotein is indeed a realistic model of larger globular systems of composite folding and aggregation landscapes and helps us to understand the fundamentals of deleterious protein aggregation and amyloid formation.3

Phosphorylation at Ser8 as an Intrinsic Regulatory Switch to Regulate the Morphologies and Structures of Alzheimer's 40-residue β-Amyloid (Aβ40) Fibrils

“…Phosphorylation can alter the structures of a protein and modulate its activities (33). Using Trp-cage as a model protein, Kardos et al (34) reported that phosphorylation could serve as a conformational switch to trigger the transition from native to amyloid state. Significantly, we and other groups have demonstrated that phosphorylation is involved in the formation of low barrier hydrogen bond (35) and turn conformations (36) as well as the destabilization of ␤-hairpin structure (37).…”

Hydration shell differentiates folded and disordered states of a Trp-cage miniprotein, allowing characterization of structural heterogeneity by wide-line NMR measurements

“…Polypeptide and protein drugs are becoming ever more common in the pharmaceutical industry (over 130 different products now approved for clinical use by FDA) but the purification, formulation and storage of such drugs is a continuous challenge. Both cooling to subzero temperatures and heating above physiological temperatures results in the unfolding of globular proteins, in a great variety of solvents 1,2 . While the loss of global fold at high temperatures has been studied in detail, the significance and mechanism of cold denaturing are still under debate 3–8 .…”

Progressive Phosphorylation Modulates the Self-Association of a Variably Modified Histone H3 Peptide

“…This question has been experimentally addressed in several systems (Wright and Dyson, 2015). For example, it has been suggested that phosphorylation can induce aggregation from the native to the amyloid state (Kardos et al, 2015). In contrast, progressive phosphorylation of the FRQ protein has been shown to support the time-delayed regulative mechanism of the circadian clock in Neurospora.…”