Cavity-based free energy analysis of osmolyte effects on protein denaturation.

Experimental measurements of the thermal effects of the same osmolytes on two different globular proteins, C-reactive protein (CRP) and tumor necrosis factor alpha (TNFα), have shown that quantifying the change in the denaturing temperature leads to some results that are unique to each protein. In order to find osmolyte-dependent parameters that can be applied more consistently from protein to protein, this work considers, instead, the overall free energy change associated with that denaturation using coarse-grained models. This is enabled by using theoretical fluid equations that take into account the exclusion of water and osmolyte from the volume occupied by the protein in both its native and denatured forms. Assuming ideal geometric models of the two protein states whose sizes are based on the protein's surface area in each form, and taking into account the density of the aqueous osmolyte solution, the free energy change due to the change in geometry can be calculated. The overall change in free energy of the system is found from that quantity and other protein- and osmolyte-specific parameters, which are determined using the experimental concentration and temperature results. We find that these fitted parameters accurately reproduce experimental results and also show consistent patterns from protein to protein. We also consider two different model geometries of the denatured protein and find little impact on the use of one or the other. Defining the effects of the osmolyte in terms of free energy also allows for prediction of overall phase change behavior, including cold denaturation.

Cryo-EM confirms a common fibril fold in the heart of four patients with ATTRwt amyloidosis.

ATTR amyloidosis results from the conversion of transthyretin into amyloid fibrils that deposit in tissues causing organ failure and death. This conversion is facilitated by mutations in ATTRv amyloidosis, or aging in ATTRwt amyloidosis. ATTRv amyloidosis exhibits extreme phenotypic variability, whereas ATTRwt amyloidosis presentation is consistent and predictable. Previously, we found an unprecedented structural variability in cardiac amyloid fibrils from polyneuropathic ATTRv-I84S patients. In contrast, cardiac fibrils from five genotypically-different patients with cardiomyopathy or mixed phenotypes are structurally homogeneous. To understand fibril structure's impact on phenotype, it is necessary to study the fibrils from multiple patients sharing genotype and phenotype. Here we show the cryo-electron microscopy structures of fibrils extracted from four cardiomyopathic ATTRwt amyloidosis patients. Our study confirms that they share identical conformations with minimal structural variability, consistent with their homogenous clinical presentation. Our study contributes to the understanding of ATTR amyloidosis biopathology and calls for further studies.

Multi-organ structural homogeneity of amyloid fibrils in ATTRv-T60A amyloidosis patients, revealed by Cryo-EM.

ATTR amyloidosis is a degenerative disorder characterized by the systemic deposition of the protein transthyretin. These amyloid aggregates of transthyretin (ATTR) can deposit in different parts of the body causing diverse clinical manifestations. Our laboratory aims to investigate a potential relationship between the different genotypes, organ of deposition, clinical phenotypes, and the structure of ATTR fibrils. Using cryo-electron microscopy, we have recently described how the neuropathic related mutations ATTRv-I84S and ATTRv-V122∆ can drive structural polymorphism in ex vivo fibrils. Here we question whether the mutation ATTRv-T60A, that commonly triggers cardiac and neuropathic symptoms, has a similar effect. To address this question, we extracted and determined the structure of ATTR-T60A fibrils from multiple organs (heart, thyroid, kidney, and liver) from the same patient and from the heart of two additional patients. We have found a consistent conformation among all the fibril structures, acquiring the "closed-gate morphology" previously found in ATTRwt and others ATTRv related to cardiac or mixed manifestations. The closed-gate morphology is composed by two segments of the protein that interact together forming a polar channel, where the residues glycine 57 to isoleucine 68 act as a gate of the polar cavity. Our study indicates that ATTR-T60A fibrils present in peripheral organs adopt the same structural conformation in all patients, regardless of the organ of deposition.