Proteins are biological molecules composed of amino acid building blocks connected into polymer-like chains. They are critically important in life processes by providing structural elements to cells and by performing many active cellular functions, for example as oxygen carriers in red blood cells, by generating contractile forces in muscles and by supporting the immune system because they are able to recognize and bind foreign molecules. Their ability to perform their cellular functions is dependent upon proteins folding into the appropriate structure. When proteins misfold, not only do they loose function but they can also cause diseases such as Alzheimer's and Parkinson's disease. To date, significant progress has been made toward understanding of how small (~100 amino acids), model proteins acquire their three-dimensional structures, however, very little is known about folding mechanisms of large, complex, multi-domain proteins that dominate all forms of life. The long-term objective of this project is to advance understanding of the folding mechanisms of large, complex, multi-domain proteins by using a combination of experimental and computational techniques. This project will provide an exciting interdisciplinary education and research opportunity for 3 graduate and 6 undergraduate students. This project also combines several experimental disciplines and it will provide a rich learning experience for all people involved, junior and senior researchers alike. Outreach activities will involve K12 students and their teachers.
In this project the mechanism of folding of large, complex, multi-domain proteins will be examines using a combination of experimental and computational techniques. The hypothesis that large multi-domain proteins fold sequentially under vectorial constraint conditions to avoid misfolding, and not in a two-state-fashion typical of small proteins, will be examined. Single-molecule protein folding data will be acquired by combining DNA and protein engineering with Atomic Force Microscopy-based and optical trap-based single-molecule force spectroscopy (AFM-SMFS, OT-SMFS) measurements. Computational methods such as Steered Molecular Dynamics (SMD) will be performed in parallel to experimental measurements and aid in interpreting data. Multi-domain proteins present a unique problem for interpreting AFM-SMFS and OT-SMFS data: the domain that gives rise to a signal must be identified out of the multiple domains in the protein. To circumvent this issue, novel polypeptide-based 'folding probes' such as antiparallel coiled-coils will be developed that can be inserted into large proteins at critical positions to report on the folding status of the surrounding structure when interrogated by SMFS. These studies will examine mechanical folding of a number of large proteins with increasing size and structural complexity that supports their important biological functions such as ATP synthesis or DNA replication. The research will advance the knowledge about one of the most fundamental unsolved problems in biology; mechanisms allowing long amino acid chains to fold into complex, multidomain structures.
