P61A mutation in the factor for inversion stimulation results in a thermostable dimeric intermediate

Area of Science:

• Protein biochemistry
• Molecular biology
• Structural biology

Background:

• The factor for inversion stimulation (FIS) is a conserved homodimeric DNA-binding protein in enteric bacteria.
• FIS has a flexible N-terminus and four alpha-helices, with a conserved Proline 61 at the center of helix B.
• Previous studies indicated that P61A mutation leads to nonuniform protein stabilization and a dimeric intermediate.

Purpose of the Study:

• To investigate the impact of the P61A mutation on the thermal and chemical denaturation of FIS.
• To characterize the stability and structural properties of the dimeric intermediate formed by P61A FIS.
• To explore the functional implications of Proline 61 conservation in FIS.

Main Methods:

• Thermal denaturation experiments at varying protein and urea concentrations.
• Urea denaturation equilibrium studies.
• Glutaraldehyde cross-linking experiments.
• Low pH denaturation studies.

Main Results:

• P61A FIS exhibited incomplete thermal denaturation, forming a concentration-independent dimeric intermediate at 90°C.
• Urea addition rendered P61A FIS thermal denaturation concentration-dependent, confirming intermediate denaturation.
• Glutaraldehyde cross-linking supported the existence of a thermostable dimeric intermediate.
• P61A FIS showed resistance to low pH denaturation, with biphasic thermal denaturation at pH 3.5.

Conclusions:

• The P61A mutation stabilizes a dimeric intermediate with a protected hydrophobic core.
• Proline 61's conservation likely regulates FIS stability and proteolytic resistance.
• The study provides insights into the structural basis of FIS stability and regulation.