A truncated peptide model of the mutant P61A FIS forms a stable dimer.
Factor for inversion stimulation (FIS) is a 98-residue homodimeric DNA-binding protein involved in several different cellular processes including DNA inversion and the regulation of multiple genes. FIS contains a flexible and functionally important N-terminus followed by four helices (A-D), the last two of which consist of the DNA-binding region. Helix B, which comprises the main dimerization interface has a 20 degrees kink at its center that was originally thought to be caused by the presence of a proline at position 61. However, it was later shown that the kink remained largely intact and that FIS retained its native-like function when the proline was mutated to an alanine. We previously showed that the P61A mutation increased the stability of FIS, but decreased its equilibrium denaturation cooperativity apparently due to preferential stabilization of the B-helix. Here we studied a peptide of P61A FIS, corresponding to residues 26-71 (26-71(A3) FIS), which encompasses the dimer interface (helices A and B). Circular dichroism (CD) and size-exclusion chromatography/multi-angle light scattering showed that the peptide was alpha-helical and dimeric, respectively, as expected based on the 3D structure of FIS. Urea-induced equilibrium denaturation experiments monitored by far-UV CD revealed a concentration-dependent transition, and data analysis based on a N2<-->2U model yielded a DeltaG of approximately -10 kcal/mol. Our results suggest that 26-71(A3) FIS can form a stable dimeric structure despite lacking the N- and C-terminus of native FIS.
Moriarty
Daniel F
DF
Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY 12180, USA. dmoriarty@siena.edu
Fiorillo
Christine
C
Miller
Charmi
C
Colón
Wilfredo
W
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D019281:Dimerization; D035021:Factor For Inversion Stimulation Protein / Q000737:chemistry* / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary
D035021:Factor For Inversion Stimulation Protein; D010446:Peptide Fragments