Probing ribosome-nascent chain complexes by NMR spectroscopy and molecular dynamics. (360G-Wellcome-086712_Z_08_Z)

Protein synthesis occurs on ribosomes within all living organisms. The newly synthesized 'nascent chain' emerges from the ribosome one amino acid at a time and explores conformations that eventually lead to its folded structure. This project aims to use structural methods, in particular NMR spectroscopy combined with molecular dynamics simulations to determine structures of emerging nascent chains on their parent ribosomes. Biochemical/biophysical studies have shown that nascent chains can obtain native-like structure on their ribosomes and display activity. We have shown [e.g. 1 ,2] that NMR can uniquely provide structure and dynamics of ribosome nascent chain complexes (RNCs), allowing monitoring the emergence of folded structure. Among the RNCs to be considered here will be the biosynthesis of a disease-associated intrinsically disordered proteins (lOPs). We will answer questions such as how they avoid aggregation/degradation, what conformations are sampled on the pathway, how these differ from those in isolated solution, whether molecular chaperones interact with them, and whether protein targets of these proteins can interact with them before their ribosomal release. Protein conformational diseases include a range of degenerative disorders in which specific peptides or proteins - often lOPs - misfold and aberrantly self-assemble, often in the form of amyloid fibrils. These include Alzheimer's, Parkinson's, and Huntington's diseases. a-synuclein is a well-studied and increasingly prominent protein, the aggregation of which is linked to the pathogenesis of Parkinson's disease. Indeed, a-synuclein is the major component of Lewy bodies, the protein-rich aggregates found post-mortem in the brains of patients suffering from Parkinson's disease or a number of related diseases. By preparing a-synuclein-RNCs, high-resolution snapshots of the conformational properties sampled by asyn as it is synthesized on the ribosome will be obtained as well as it's interactions with the molecular chaperone, Trigger Factor (TF). Such structural data will be combined with novel MD simulations to provide a detailed structural/dynamical information the ensemble of structures sampled during the progressive emergence of the nascent chain and it's interactions with the ribosome. Additionally, the pathological conversion of misfolded proteins into cytotoxic species is modulated by interactions with several proteins, among them molecular chaperones such as the Trigger Factor (TF}, which will also be studied. This work will provide detailed insights into protein folding at the level of synthesis and will have applications to understanding protein misfolding and its links to biology and the onset of disease.