Position-dependent interactions between cysteine residues and the helix dipole

Area of Science:

• Biochemistry
• Structural Biology
• Protein Science

Background:

• The N-terminus of alpha-helices possesses a partial positive charge due to the helix dipole effect.
• Understanding this charge's influence on nearby residues is crucial for protein stability and function.
• Cysteine residues, with their reactive thiol groups, serve as valuable probes for studying electrostatic interactions.

Purpose of the Study:

• To quantify the interaction energy between cysteine thiols and the helix dipole.
• To investigate the position-dependence of the helix dipole effect on cysteine reactivity.
• To elucidate the distinct components contributing to the experimentally observed helix dipole effect.

Main Methods:

• Development of a protein model using recombinant sperm whale myoglobin.
• Site-directed mutagenesis to introduce cysteine residues at specific N-terminal positions of helix H.
• Determination of thiol pKa values in folded proteins and unfolded peptides to calculate interaction energies.

Main Results:

• Stabilization energies for N1, N2, and Ncap thiolate positions were determined as 0.3, 0.7, and 2.8 kcal/mole, respectively.
• The Ncap position showed significant stabilization, suggesting a potential hydrogen bond involvement.
• The helix dipole effect was resolved into charge-dipole and hydrogen bond components, varying with residue position.

Conclusions:

• The helix dipole effect is position-dependent, with distinct charge-dipole and hydrogen bond contributions.
• Cysteine reactivity is modulated by the helix dipole, with stronger effects at the N-terminus and capping positions.
• These findings provide insights into cysteine behavior in proteins like hemoglobins and thioredoxins.