Vectorial Folding of Proteins and Nascent Polypeptide Chains by AFM and Computer Simulations

Duke University, Durham NC

Investigators

Abstract

Significant progress has been made toward understanding how proteins acquire their structure through in vitro experiments and computer simulations, but much less is known about protein folding in vivo. During co-translational folding in vivo, the nascent polypeptide chain (NPC) is extruded sequentially in a vectorial manner from the ribosome exit tunnel and starts folding under severe conformational constraints. It is presently unknown how such one dimensional (1D) constraints affect the folding pathway. The long-term objective of this research is to advance understanding of protein folding by: a) studying the vectorial folding of single proteins under 1D constraints by Atomic Force Microscopy-based single-molecule force spectroscopy (AFM-SMFS) and steered molecular dynamics computer simulations (SMD); b) directly examining the folding behavior of the NPC itself, using AFM. This project will examine folding behavior of proteins composed primarily of alpha-helical repeats that stack and form extended, solenoid-like "vectorial" structures making them ideal model systems for vectorial folding studies. The objectives are to a) engineer repeat proteins for vectorial folding studies; b) examine by AFM vectorial folding of repeat proteins under 1D constraints; c) use SMD simulations to examine vectorial folding pathways of repeat proteins. SMD-derived force-extension relationships will be compared with the AFM data. Unfolding and refolding trajectories will be reconstructed and analyzed by building a native contact map, and monitoring its time evolution during stretching and relaxing; d) examine the folding behavior of the nascent polypeptide chain by AFM. Stalled ribosome-NPC complexes will be produced using an in vitro protein expression system. These stalled NPCs will be picked up by the AFM tip and stretched to examine their folding status. The project promises to narrow the gap between an understanding of protein folding in vitro and in vivo. This project will provide interdisciplinary education and research opportunities for graduate and undergraduate students. The graduate students working on this project will participate in a unique international research and educational exchange experience. Outreach activities by involving K12 students, their parents and teachers will raise the scientific literacy of the public. This project is jointly supported by Molecular Biophysics in the Division of Molecular and Cellular Biosciences in the Directorate for Biological Sciences and the Physics of Living Systems Program in the Division of Physics in the Mathematical and Physical Sciences Directorate.