On the mechanism of protein fold-switching by a molecular sensor.
Alternate frame folding (AFF) is a mechanism by which conformational change can be engineered into a protein. The protein structure switches from the wild-type fold (N) to a circularly-permuted fold (N'), or vice versa, in response to a signaling event such as ligand binding. Despite the fact that the two native states have similar structures, their interconversion involves folding and unfolding of large parts of the molecule. This rearrangement is reported by fluorescent groups whose relative proximities change as a result of the order-disorder transition. The nature of the conformational change is expected to be similar from protein to protein; thus, it may be possible to employ AFF as a general method to create optical biosensors. Toward that goal, we test basic aspects of the AFF mechanism using the AFF variant of calbindin D(9k). A simple three-state model for fold switching holds that N and N' interconvert through the unfolded state. This model predicts that the fundamental properties of the switch--calcium binding affinity, signal response (i.e., fluorescence change upon binding), and switching rate--can be controlled by altering the relative stabilities of N and N'. We find that selectively destabilizing N or N' changes the equilibrium properties of the switch (binding affinity and signal response) in accordance with the model. However, kinetic data indicate that the switching pathway does not require whole-molecule unfolding. The rate is instead limited by unfolding of a portion of the protein, possibly in concert with folding of a corresponding region.
Stratton
Margaret M
MM
Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.
Loh
Stewart N
SN
D000595:Amino Acid Sequence; D015374:Biosensing Techniques / Q000379:methods*; D064026:Calbindins; D002118:Calcium; D005453:Fluorescence; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011162:Porphobilinogen / Q000031:analogs & derivatives / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D064030:S100 Calcium Binding Protein G / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics
C538930:3-anilino difluoroboron dipyrromethene; D064026:Calbindins; D050505:Mutant Proteins; D064030:S100 Calcium Binding Protein G; D011162:Porphobilinogen; D002118:Calcium
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