Inter-peptidic metal exchange is becoming increasingly apparent in neurological disorders like Alzheimer’s disease as amyloid-beta and other intrinsically disordered peptides sequester metals in the brain even from other metalloproteins. The short peptides GHK and DAHK and their fluorescent counterparts GHW and DAHW are known Cu(II) amyloid-beta binding models. These peptides are all strong Cu(II) chelators that have conditional (pH 7.4, 0.1 M HEPES) binding constants, Ka, of GHK (3N+1O binding), DAHK (4N binding) and amyloid-beta at 1.4 x 1013, 3.9 x 1013 M-1 and at 2.4 x 109 M-1, respectively. Using sensitive fluorescence spectroscopy and taking advantage of proximity fluorescence quenching of tryptophan by Cu(II), we have monitored the exchange from Cu(II)-GHW to GHK and DAHK rather efficiently. The conditional (pH 7.4, 0.1M HEPES) second order rate constants of two exchanges from Cu(II)-GHW to GHK and DAHK were determined to be 1.4 ± 0.2 x 102 and 4.7 ± 1.9 x 101 M-1s-1, respectively. We propose an intermediate ternary GHW-Cu(II)-GHK/DAHK complex likely facilitates the exchange as the hydrated coordination spot in the Cu(II)-GHW complex can undergo a ligand-exchange with the incoming peptide’s N terminus or imidazole N’s. Changes in the superhyperfine coupling lines in the EPR spectra in experimental conditions (1:2 vs 1:1 Cu:GHK) do support the formation of the ternary complex where in 1:2 Cu:GHK conditions a 4th N-donor ligand binds when the second peptide is added to Cu(II)-GHK instead of an O-donor as seen in 1:1 Cu:GHK conditions. These studies show that metal coordination plays an important role in metal exchange kinetics and we plan to extend this method development to study inter-peptidic metal exchange of amyloid-beta with itself and other biologically relevant peptides.

Cu(II) coordination differences in the amyloid-beta protein binding models GHK and DAHK result in varied conditional inter-peptidic metal exchange rate constants

253rd National Meeting (2017)

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