The surface plasmon resonance (SPR) technique was used to study the formation kinetics of a de novo designed coiled-coil (E/K coil). The E/K coil is made up of two distinct peptides (E and K) each with five heptad (g-a-b-c-d-e-f) repeats. The E peptide's heptad sequence is E-V-S-A-L-E-K, and the K peptide's heptad sequence is K-V-S-A-L-K-E. A linker C-nL-G-G-G (nL = norleucine) is present at the C-terminus of the E peptide and at the N-terminus of the K peptide for the SPR studies. Heterodimer formation involves both electrostatic and hydrophobic interactions at the dimer interface. Under conditions that favor the heterodimer formation, the CD signal ([theta]222) varied as a function of peptide concentration. The estimated dissociation constant (Kd) was 2.45 +/- 0.71 nM. Denaturation studies with guanidine-HCI (GdnHC11/2 = 3.9 M) suggested a value of 3.53 +/- 0.48 nM. For the SPR investigation, the peptides were biotinylated and linked to streptavidin in order to increase their effective molecular weight and consequently enhance the signal intensity. Biotinylation in itself did not impede coiled-coil formation based on CD measurements. The biosensor study revealed a slow dissociation rate constant for the heterodimer (kd approximately 2 x 10(-4) s-1) and a moderately fast association rate constant [ka approximately (4.27-4.53) x 10(5) M-1 s-1). This gives a calculated Kd of 0.47-0.50 nM, which agrees reasonably well with the equilibrium CD studies. Therefore, based on the SPR data, the preference for heterodimer formation is due to a combination of moderately fast association and slow dissociation rates.