Fluorescence Determination of Inter-peptidic Cu(II) Exchange Rate Constants of Amyloid-beta Binding Models
Research Seminar abstract
It is relatively agreed upon that most to all biological Cu(II) ions are chelated in some fashion. Inter-peptidic metal exchange is becoming increasingly apparent in neurodegenerative disorders like Alzheimer’s disease as amyloid-beta and other intrinsically disordered proteins sequester metals in the brain from other proteins. In the past, Cu(II) exchange rate constants of small peptide complexes to other non-peptide ligands have been almost exclusively studied through stopped-flow absorbance; however, distinguishing between peptide complexes of the same binding is difficult to spectrally resolve by this technique. The short peptides GHK and DAHK are known Cu(II) amyloid-beta binding models as ATCuN binding (Amino Terminus Copper and Nickel) is reserved as well as imidazole N chelation and stabilizing ring formation. These peptides are 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 and 2.4 x 109 M-1, respectively. As the lysine containing peptides are fluorescence silent, we used the same peptide sequences with the amino acid tryptophan (λexcite 290 nm, λemission 360 nm) in place of lysine to be able to distinguish the two peptides using fluorescence spectroscopy. The tryptophan containing peptides were shown to have the same Cu(II) binding as the lysine counterparts as shown by EPR and UV-vis spectroscopies and are suitable to determine the rate constant between two peptides of identical binding. Using sensitive fluorescence spectroscopy and taking advantage of proximity fluorescence quenching of tryptophan by Cu(II), we have monitored the exchange rate constant of Cu(II) from Cu(H-1GHW) to GHK and DAHK without the use of stopped-flow absorbance. The conditional (pH 7.4, 0.1 M HEPES) second order rate constants of two exchanges from Cu(H-1GHW) 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 observe an intermediate ternary DAHK/GHK-Cu(H-1GHW) complex which facilitates the exchange as the hydrated coordination spot in Cu(H-1GHW) can easily undergo a ligand-exchange with the incoming peptide. Unfortunately, fluorescence spectroscopy is unable to differentiate between the initial Cu(H-1GHW) and the ternary species due to the quenching of tryptophan fluorescence by Cu(II). However, changes in the superhyperfine coupling in EPR spectra of experimental conditions of 1:2 Cu(II):GHK vs. 1:1 Cu(II):GHK do support the formation of the ternary complex. We observed 9 superhyperfine lines in the 1:2 ratio and 7 lines in the 1:1 ratio which indicates 4N binding (9 lines) versus 3N+1O binding (7 lines). We plan to employ this method to determine the inter-peptidic exchange rate constants in amyloid-beta with itself and other proteins associated with neurodegeneration.
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Speaker: Cheryle Beuning
Speaker Institution: Colorado State University
Event Date: 04-19-2017
Event Time: 4:00 PM
Event Location: Chemistry A101
Mixer Time: 3:45 PM
Mixer Location: Chemistry B101E
Host: D. Crans