P61A mutation in the factor for inversion stimulation results in a thermostable dimeric intermediate.
The factor for inversion stimulation (FIS) is a homodimeric DNA-binding protein found in enteric bacteria. FIS consists of 98 residues and self-assembles into an entwined dimer containing a flexible and mostly disordered N-terminus followed by four alpha-helices. Proline 61, which is 100% conserved in FIS homologues, is located at the center of helix B, and its substitution for alanine (P61A) was previously shown to result in nonuniform stabilization of the protein, leading to the appearance of a marginally populated dimeric intermediate in urea denaturation equilibrium studies. Here we show that, in contrast to WT FIS, the thermal denaturation of P61A FIS was incomplete and yielded a transition curve that was independent of FIS concentration, suggesting the presence of a dimeric intermediate at 90 degrees C. In the presence of urea, the thermal denaturation of P61A FIS became concentration dependent, consistent with the denaturation of the dimeric intermediate. The existence of a thermostable dimeric intermediate of P61A FIS was further confirmed by glutaraldehyde cross-linking experiments at 95 degrees C. Urea denaturation experiments at 90 degrees C revealed a cooperative transition, indicating that the dimeric intermediate of P61A FIS has a solvent-protected hydrophobic core. P61A FIS, unlike the WT protein, was found to be resistant to denaturation by low pH, but its thermal denaturation at pH 3.5 revealed a biphasic transition, providing clues about the structure of the dimeric intermediate. From a functional perspective, it is plausible that the full conservation of proline 61 in FIS may serve to limit the stability and proteolytic resistance of this highly regulated transcription factor.
Meinhold
Derrick
D
Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, 110 Eighth Street, Troy, New York 12180, USA.
Boswell
Sarah
S
Colón
Wilfredo
W
D000409:Alanine / Q000235:genetics; D019943:Amino Acid Substitution; D003432:Cross-Linking Reagents; D019281:Dimerization; D035021:Factor For Inversion Stimulation Protein / Q000737:chemistry* / Q000235:genetics; D005976:Glutaral / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D009154:Mutation; D011392:Proline / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D013696:Temperature; D014508:Urea / Q000737:chemistry
D003432:Cross-Linking Reagents; D035021:Factor For Inversion Stimulation Protein; D014508:Urea; D011392:Proline; D000409:Alanine; D005976:Glutaral