title: Protein unfolding with a steric trap.
abstract: The study of protein folding requires a method to drive unfolding, which is typically accomplished by altering solution conditions to favor the denatured state. This has the undesirable consequence that the molecular forces responsible for configuring the polypeptide chain are also changed. It would therefore be useful to develop methods that can drive unfolding without the need for destabilizing solvent conditions. Here we introduce a new method to accomplish this goal, which we call steric trapping. In the steric trap method, the target protein is labeled with two biotin tags placed close in space so that both biotin tags can only be bound by streptavidin when the protein unfolds. Thus, binding of the second streptavidin is energetically coupled to unfolding of the target protein. Testing the method on a model protein, dihydrofolate reductase (DHFR), we find that streptavidin binding can drive unfolding and that the apparent binding affinity reports on changes in DHFR stability. Finally, by employing the slow off-rate of wild-type streptavidin, we find that DHFR can be locked in the unfolded state. The steric trap method provides a simple method for studying aspects of protein folding and stability in native solvent conditions, could be used to specifically unfold selected domains, and could be applicable to membrane proteins.
authors.affiliation: Department of Chemistry and Biochemistry, UCLA-DOE Institute for Genomics and Proteomics, University of California, Los Angeles, California 90095, USA.
authors.forename: Tracy M
authors.initials: TM
authors.lastname: Blois
authors.forename: Heedeok
authors.initials: H
authors.lastname: Hong
authors.forename: Tae H
authors.initials: TH
authors.lastname: Kim
authors.forename: James U
authors.initials: JU
authors.lastname: Bowie
mesh_terms: D000818:Animals; D019921:Biotinylation; D051379:Mice; D008958:Models, Molecular; D011487:Protein Conformation; D011489:Protein Denaturation / Q000502:physiology; D017510:Protein Folding; D019809:Streptavidin / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000378:metabolism
chemical_list: D019809:Streptavidin; D013762:Tetrahydrofolate Dehydrogenase