Amyloid-like Fibrils from an α-Helical Transmembrane Protein.

Karen Stroobants,Nicola J Harris,Paula J Booth,Janet R Kumita,Dimitri Y Chirgadze,Michele Vendruscolo,Christopher M Dobson

doi:10.1021/acs.biochem.7b00157

Karen Stroobants, Nicola J Harris + Show 5 more

Open Access

https://doi.org/10.1021/acs.biochem.7b00157

Copy DOI

Journal: Biochemistry	Publication Date: Jun 12, 2017
Citations: 19	License type: cc-by

Affiliation: King's College London, University of Cambridge

Abstract

The propensity to misfold and self-assemble into stable aggregates is increasingly being recognized as a common feature of protein molecules. Our understanding of this phenomenon and of its links with human disease has improved substantially over the past two decades. Studies thus far, however, have been almost exclusively focused on cytosolic proteins, resulting in a lack of detailed information about the misfolding and aggregation of membrane proteins. As a consequence, although such proteins make up approximately 30% of the human proteome and have high propensities to aggregate, relatively little is known about the biophysical nature of their assemblies. To shed light on this issue, we have studied as a model system an archetypical representative of the ubiquitous major facilitator superfamily, the Escherichia coli lactose permease (LacY). By using a combination of established indicators of cross-β structure and morphology, including the amyloid diagnostic dye thioflavin-T, circular dichroism spectroscopy, Fourier transform infrared spectroscopy, X-ray fiber diffraction, and transmission electron microscopy, we show that LacY can form amyloid-like fibrils under destabilizing conditions. These results indicate that transmembrane α-helical proteins, similarly to cytosolic proteins, have the ability to adopt this generic state.

Highlights

A wide range of medical conditions, including neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases, are associated with the misfolding and aggregation of specific proteins into amyloid fibrils.[1−4] In vivo and in vitro studies of these disease-related aggregates have led to the recognition of their common structural features, despite the diversity of the amino acid sequences and native state structures of the proteins by which they are formed.[4]
Most studies report a fast degradation of dysfunctional CFTR rather than formation of deposits,[22] it has been indicated that misfolded aggregates with highly organized arrays of β-strands can form for the P205 variant.[23]
The detergent was used at a concentration of 1 mM in all experiments, which is well above its critical micelle concentration (CMC) of 0.16 mM.[51]

Summary

Introduction

A wide range of medical conditions, including neurodegenerative disorders such as Alzheimer’s and Parkinson’s diseases, are associated with the misfolding and aggregation of specific proteins into amyloid fibrils.[1−4] In vivo and in vitro studies of these disease-related aggregates have led to the recognition of their common structural features, despite the diversity of the amino acid sequences and native state structures of the proteins by which they are formed.[4]. LacY in 50 mM sodium phosphate and 0.05% DDM (pH 7.4) was used directly as obtained from the purification (2 μM, 4 μM, and occasionally 8 μM samples) or concentrated (to 8, 16, or 32 μM), and aggregation was initiated by increasing the temperature of the sample to 37 °C.

Results

Conclusion