title: On the mechanism of protein fold-switching by a molecular sensor.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, State University of New York Upstate Medical University, Syracuse, New York 13210, USA.
authors.forename: Margaret M
authors.initials: MM
authors.lastname: Stratton
authors.forename: Stewart N
authors.initials: SN
authors.lastname: Loh
mesh_terms: 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
chemical_list: C538930:3-anilino difluoroboron dipyrromethene; D064026:Calbindins; D050505:Mutant Proteins; D064030:S100 Calcium Binding Protein G; D011162:Porphobilinogen; D002118:Calcium
references.citation: Proteins. 2002 May 15;47(3):323-33
references.pmid: 11948786
references.citation: Proc Natl Acad Sci U S A. 2007 May 15;104(20):8311-5
references.pmid: 17494761
references.citation: J Mol Biol. 2006 Apr 7;357(4):1058-62
references.pmid: 16483603
references.citation: J Mol Biol. 2009 Feb 27;386(3):854-68
references.pmid: 19038264
references.citation: Proc Natl Acad Sci U S A. 2010 Feb 16;107(7):2824-9
references.pmid: 20133757
references.citation: Proc Natl Acad Sci U S A. 2004 May 25;101(21):7976-81
references.pmid: 15150406
references.citation: Curr Opin Struct Biol. 2002 Feb;12(1):54-60
references.pmid: 11839490
references.citation: J Proteome Res. 2006 Oct;5(10):2839-48
references.pmid: 17022655
references.citation: Biochemistry. 1966 Jan;5(1):365-85
references.pmid: 5938952
references.citation: Curr Opin Struct Biol. 2009 Feb;19(1):31-8
references.pmid: 19157855
references.citation: PMC Biophys. 2008 Dec 18;1(1):6
references.pmid: 19351428
references.citation: Biochemistry. 1998 Jun 23;37(25):8915-25
references.pmid: 9636033
references.citation: Proc Natl Acad Sci U S A. 2005 Aug 2;102(31):10841-5
references.pmid: 16046542
references.citation: Biochemistry. 1998 Jun 23;37(25):8926-37
references.pmid: 9636034
references.citation: Eur J Biochem. 1988 Oct 15;177(1):47-52
references.pmid: 3181158
references.citation: J Mol Biol. 1993 May 20;231(2):415-30
references.pmid: 8389885
references.citation: Nat Rev Mol Cell Biol. 2005 Mar;6(3):197-208
references.pmid: 15738986
references.citation: J Am Chem Soc. 2009 Sep 23;131(37):13347-54
references.pmid: 19719116
references.citation: J Mol Biol. 2007 Aug 10;371(2):308-16
references.pmid: 17572441
references.citation: ACS Chem Biol. 2008 Nov 21;3(11):723-32
references.pmid: 18947182
references.citation: J Mol Biol. 2003 Sep 19;332(3):529-36
references.pmid: 12963365
references.citation: J Mol Biol. 1965 May;12:88-118
references.pmid: 14343300
references.citation: Trends Biochem Sci. 1995 Dec;20(12):495-7
references.pmid: 8571449