KINETIC-STUDY ON THE FORMATION OF A DE-NOVO DESIGNED HETERODIMERIC COILED-COIL - USE OF SURFACE-PLASMON RESONANCE TO MONITOR THE ASSOCIATION AND DISSOCIATION OF POLYPEPTIDE-CHAINS

The surface plasmon resonance (SPR) technique was used to study the fo rmation 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 hep tad (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 linke r C-nL-G-G-G (nL = norleucine) is present at the C-terminus of the E p eptide and at the N-terminus of the K peptide for the SPR studies, Het erodimer formation involves both electrostatic and hydrophobic interac tions at the dimer interface, Under conditions that favor the heterodi mer formation, the CD signal ([Theta](222)) varied as a function of pe ptide concentration, The estimated dissociation constant (K-d) was 2.4 5 +/- 0.71 nM, Denaturation studies with guanidine-HCl (GdnHCl(1/2) = 3.9 M) suggested a value of 3.53 +/- 0.48 nM, For the SPR investigatio n, 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-co il formation based on CD measurements, The biosensor study revealed a slow dissociation rate constant for the heterodimer (k(d) approximate to 2 x 10(-4) s(-1)) and a moderately fast association rate constant [ k(d) approximate to (4.27-4.53) x 10(5) M(-1) s(-1)), This gives a cal culated K-d of 0.47-0.50 nM, which agrees reasonably well with the equ ilibrium CD studies, Therefore, based on the SPR data, the preference for heterodimer formation is due to a combination of moderately fast a ssociation and slow dissociation rates.