Protein Nanopore-Based, Single-Molecule Exploration of Copper Binding to an Antimicrobial-Derived, Histidine-Containing Chimera Peptide

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• 作者：Loredana Mereuta ; Irina Schiopu ; Alina Asandei ; Yoonkyung Park ; Kyung-Soo Hahm ; Tudor Luchian
• 刊名：Langmuir
• 出版年：2012
• 出版时间：December 11, 2012
• 年：2012
• 卷：28
• 期：49
• 页码：17079-17091
• 全文大小：560K
• 年卷期：v.28,no.49(December 11, 2012)
• ISSN：1520-5827

文摘

Metal ions binding exert a crucial influence upon the aggregation properties and stability of peptides, and the propensity of folding in various substates. Herein, we demonstrate the use of the 伪-HL protein as a powerful nanoscopic tool to probe Cu²⁺-triggered physicochemical changes of a 20 aminoacids long, antimicrobial-derived chimera peptide with a His residue as metal-binding site, and simultaneously dissect the kinetics of the free- and Cu²⁺-bound peptide interaction to the 伪-HL pore. Combining single-molecule electrophysiology on reconstituted lipid membranes and fluorescence spectroscopy, we show that the association rate constant between the 伪-HL pore and a Cu²⁺-free peptide is higher than that of a Cu²⁺-complexed peptide. We posit that mainly due to conformational changes induced by the bound Cu²⁺ on the peptide, the resulting complex encounters a higher energy barrier toward its association with the protein pore, stemming most likely from an extra entropy cost needed to fit the Cu²⁺-complexed peptide within the 伪-HL lumen region. The lower dissociation rate constant of the Cu²⁺-complexed peptide from 伪-HL pore, as compared to that of Cu²⁺-free peptide, supports the existence of a deeper free energy well for the protein interaction with a Cu²⁺-complexed peptide, which may be indicative of specific Cu²⁺-mediated contributions to the binding of the Cu²⁺-complexed peptide within the pore lumen.