Many proteins have the potential to aggregate into amyloid fibrils, protein polymers associated with a wide range of human disorders such as Alzheimer’s and Parkinson’s disease. The thermodynamic stability of amyloid fibrils, in contrast to that of folded proteins, is not well understood: the balance between entropic and enthalpic terms, including the chain entropy and the hydrophobic effect, are poorly characterised. Using a combination of theory, in vitro experiments, simulations of a coarse-grained protein model and meta-data analysis, we delineate the enthalpic and entropic contributions that dominate amyloid fibril elongation. Our prediction of a characteristic temperature-dependent enthalpic signature is confirmed by the performed calorimetric experiments and a meta-analysis over published data. From these results we are able to define the necessary conditions to observe cold denaturation of amyloid fibrils. Overall, we show that amyloid fibril elongation is associated with a negative heat capacity, the magnitude of which correlates closely with the hydrophobic surface area that is buried upon fibril formation, highlighting the importance of hydrophobicity for fibril stability. Most proteins fold in the cell into stable, compact structures. Nevertheless, many proteins also have the ability to stick together, forming long fibrillar structures that are associated with a wide range of human disorders including Alzheimer’s and Parkinson’s disease. The exact nature of the amyloid-causing stickiness is not well understood, nevertheless amyloid fibrils show some very specific thermodynamic characteristics. Some fibrils even destabilise at low temperatures. In this work we translate hydrophobic theory previously used to model protein folding to fibril formation. We combine this theory with experimental measurements, simulations and meta-data analysis of different types of fibrils. This allowed us to unravel the nature of the stickiness in amyloid fibrils by observing the effect of temperature changes, specifically at low temperatures, on hydrophobicity.

Plants employ a number of phosphorylation cascades in response to a wide range of environmental stimuli. Previous studies in Arabidopsis and yeast indicate that histidine kinase AHK1 is a positive regulator of drought and osmotic stress responses. Based on these studies AHK1 was proposed a plant osmosensor, although the molecular basis of plant osmosensing still remains unknown. To understand the molecular role and signaling mechanism of AHK1 in osmotic stress, we have expressed and purified full-length AHK1 from Arabidopsis in a bacterial host to allow for studies on the isolated transmembrane receptor. Purification of the recombinant protein solubilized from the host membranes was achieved in a single step by metal-affinity chromatography. Analysis of the purified AHK1 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting show a single band indicating that the preparation is highly pure and devoid of contaminants or degradation products. In addition, gel filtration experiments indicate that the preparation is homogenous and monodisperse. Finally, CD-spectroscopy, phosphorylation activity, dimerization studies, and protein–protein interaction with plant phosphorylation targeting AHP2 demonstrate that the purified protein is functionally folded and acts as phospho-His or phospho-Asp phosphatase. Hence, the expression and purification of recombinant AHK1 reported here provide a basis for further detailed functional and structural studies of the receptor, which might help to understand plant osmosensing and osmosignaling on the molecular level.