Inverse electrostatic effect: electrostatic repulsion in the unfolded state stabilizes a leucine zipper

Area of Science:

• Protein Biochemistry
• Structural Biology
• Biophysics

Background:

• Protein stability is critically dependent on electrostatic interactions involving ionizable residues.
• The pH-dependent behavior of proteins is a key factor in their function and stability.
• Leucine zippers are model systems for studying protein folding and stability.

Purpose of the Study:

• To characterize the contributions of charged Glu and His residues to the stability of a designed leucine zipper.
• To determine the NMR structure of a heterodimeric leucine zipper (AB) and compare it to a disulfide-linked variant (AB(SS)).
• To investigate the differing pH-dependent stabilities and electrostatic contributions in the two leucine zipper constructs.

Main Methods:

• Nuclear Magnetic Resonance (NMR) spectroscopy to determine protein structure.
• Thermodynamic analysis to quantify stability parameters.
• pKa shift analysis to evaluate the energetic contributions of charged residues.

Main Results:

• The NMR structures of AB and AB(SS) were determined and found to be similar, with both forming six interchain salt bridges.
• Leucine zipper AB exhibits peak stability around pH 4.5 and is more stable at pH 8 than pH 2, unlike AB(SS).
• Charged Glu residues stabilize AB but destabilize AB(SS), with non-paired charges destabilizing AB(SS) more significantly.

Conclusions:

• The higher stability of AB at neutral pH is primarily due to unfavorable electrostatic interactions in the unfolded state (inverse electrostatic effect).
• This study underscores the crucial role of residual interactions and energetics in the unfolded state for overall protein stability.
• Understanding unfolded state properties is essential for predicting and engineering protein stability.