Rational redesign of the folding pathway of a modular protein.
The modular structures of repeat proteins afford them distinct properties compared with globular proteins, enabling them to function in a large and diverse range of cellular processes. Here, we show that they can also have different folding mechanisms. Myotrophin comprises four ankyrin repeats stacked linearly to form an elongated structure. Using site-directed mutagenesis, we find that folding of wild-type myotrophin is initiated at the C-terminal repeats. However, close examination of the mutant chevron plots reveals that simple models are insufficient to describe all of the data, and double mutant analysis subsequently confirms that there are parallel folding pathways. Destabilizing mutations in the C-terminal repeats reduce flux through the wild-type pathway, making a new route accessible in which folding is initiated at the N-terminal repeats. Thus, the folding mechanism of the repeat protein is poised on a fulcrum: When one end of the molecule is perturbed, the balance shifts between the different nucleation sites. The vast majority of studies on small globular proteins indicate a single, well defined route between the denatured and native states. By contrast, the potential to initiate folding at more than one site may be a general feature of repeat proteins arising from the symmetry inherent in their structures. We show that this simple architecture makes it straightforward to direct the folding pathway of a repeat protein by design.
Lowe
Alan R
AR
MRC Cancer Cell Unit, Hutchison/Medical Research Council Research Centre, Hills Road, Cambridge CB2 2XZ, United Kingdom.
Itzhaki
Laura S
LS
D017089:Ankyrin Repeat; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding
D036341:Intercellular Signaling Peptides and Proteins; D050505:Mutant Proteins; C065700:myotrophin