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.
The question "Can one predict how proteins will fold?", that is, how do proteins assume their functional structures, was in 2005 listed as one of the most important unsolved scientific problems by the Science magazine. The significance of understanding how proteins fold lies in the fundamental role of proteins in all aspects of cellular processes, and in the fact that the misfolding of proteins, or assuming of an incorrect or non-functional protein structure, underlies the onset of several human illnesses such as Parkinson's and Alzheimer diseases. Even though significant progress has been made toward understanding the protein folding mechanisms through traditional biochemical experiments and computer simulations, much less is known about folding behavior in vivo. As a protein is being produced in a living cell, a nascent polypeptide chain (NPC) is extruded from a ribosome sequentially, one amino acid after another, in a linear, vectorial fashion. The protein likely starts to assume its final structure when it emerges from a long and narrow ribosomal exit tunnel, even before its synthesis is complete— the so-called "co-translational folding". It is presently unknown how these geometrical and temporal conditions of protein synthesis affect the folding process, because up to now protein folding has been examined primarily on free proteins in test tubes away from the ribosomal interfaces present when the proteins are actually produced in a cell. The long-term goal of this project has been to advance an understanding of protein folding, including co-translational folding by: a) Studying the vectorial (linear) folding of single protein particles under geometrical constraints imposed by Atomic Force Microscopy-based single-molecule force spectroscopy (AFM-SMFS), simulating the conditions experienced by the NPC in the ribosome exit tunnel b) Performing computer simulations of the mechanical unfolding and refolding process c) Directly examining the folding behavior of the Nascent Polypeptide Chain (NPC) itself, using AFM force spectroscopy and imaging techniques. The activities within this Project resulted in several very significant results and observations such as: a) Capturing by AFM imaging, for the first time, the folded structure of a nascent polypeptide chain still attached to the ribosome b) Providing direct evidence using several representative proteins that large proteins do indeed fold sequentially under geometrical constraints and not in the "all-or-none" fashion which had been previously assumed. c) Identifying evidence that the sequential folding of large proteins may have evolved as a means to avoid misfolding into incorrect structures. d) Developing new, powerful peptide-based probes for studying the folding status of individual domains within complex multi-domain proteins These results and observations strongly suggest that the proposed understanding of protein folding as "all-or-none" need to be revised for majority of proteins, which possess multiple structural domains, to include their vectorial and sequential folding behaviors. These observations may also help to better understand the mechanisms leading to protein misfolding into incorrect structures. The results of this study were presented at 10 scientific conferences and published in one book chapter and 12 peer-reviewed journal publications. The computer algorithms and methodologies to improve experimental throughput and data acquisition of single-molecule force spectroscopy experiments have been published on-line and made available to other researchers. Altogether, the project trained two post-doctoral associates, five PhD students and five undergraduate students contributing to the development of a new cadre of researchers familiar with the cutting edge nanotechnology and biophysics problems and armed with state-of-the-art research tools to address them.
