Folding of a three-stranded coiled coil.

Area of Science:

• Biochemistry
• Structural Biology
• Protein Folding

Background:

• Coiled coils are protein structures formed by alpha-helices.
• Dimeric coiled coils often form via a single-step association and folding process.
• The folding mechanism of three-stranded coiled coils remains less understood.

Purpose of the Study:

• To investigate the folding mechanism of a designed three-stranded coiled coil peptide (LZ16A).
• To determine if a simple, coupled folding and association mechanism applies to trimeric coiled coil formation.
• To quantify the kinetic and thermodynamic parameters governing LZ16A assembly.

Main Methods:

• Time-resolved fluorescence spectroscopy to monitor folding kinetics.
• Pulsed hydrogen-exchange experiments to assess structural intermediates.
• Far-ultraviolet circular dichroism to determine equilibrium binding constants.

Main Results:

• LZ16A folding to dimer (D) and trimer (T) follows a two-step mechanism: 2M (k1)<==>(k(-1)) D and M + D (k2)<==>(k(-2)) T.
• Rate constants for association and dissociation were determined: k1 = 7.8 x 10(4) M(-1) s(-1), k(-1) = 0.015 s(-1), k2 = 6.5 x 10(5) M(-1) s(-1), k(-2) = 1.1 s(-1).
• Equilibrium binding constants derived from kinetics matched those from circular dichroism; only unfolded monomers, dimers, and trimers were significantly populated.

Conclusions:

• The formation of three-stranded coiled coils, like LZ16A, proceeds via a sequential, two-step mechanism.
• Association-competent monomers, potentially partially helical, drive the formation of dimeric and trimeric structures.
• This contrasts with the single-step mechanism often observed for dimeric coiled coils, highlighting differences in assembly pathways.