Folding Pathway of a Two-Domain Protein Studied with Single Molecule Three-Color FRET

Abstract

Every protein needs to undergo a folding process after it's synthesis in the cell. Most in vitro protein folding sudies are carried out on small proteins. To understand complex folding processes in large proteins, we studied maltose binding protein (MBP) as a model system. MBP is a two domain, 42 kDa protein natively folds within a minute. With the two mutations V8G and Y283D, the spontaneous folding process is slowed down to half an hour. Here, we apply single molecule three-color FRET which allows us to monitor three distances at a time. With the method in hand, we asked the question whether the two domains fold together or independently. For three color FRET, MBP is labeled with three fluorophores at specific positions to have three coordinates that monitor the folding of the N-terminal (NTD) and C-terminal (CTD) domains simultaneously. Three color FRET measurements as well as control measurements on double labeled MBP revealed that both domains folds simultaneously in a cooperative fashion with a intermediate state present in both the domains.