A luminescent ATCUN peptide variant with enhanced properties for copper(II) sensing in biological media

Enrico Falcone,Bertrand Vileno,Mai Hoang,Laurent Raibaut,Peter Faller

doi:10.1016/j.jinorgbio.2021.111478

Abstract

The measurement of labile CuII in biological samples is fundamental for understanding Cu metabolism and has been emerging as a promising diagnostic marker for Cu-related pathologies such as Wilson's and Alzheimer's diseases. The use of fluorescent chelators may be useful to circumvent separation steps employed by current methods. For this purpose, we recently designed a selective and suited-affinity turn-off luminescent probe based on a peptide bearing the CuII-binding Xxx-Zzz-His (Amino-Terminal CuII- and NiII-binding, ATCUN) motif and a TbIII-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) complex. Here, we present an analogue probe bearing the ATCUN motif variant Xxx-His-His. This probe showed much faster response in biologically-relevant media and higher stability than the previous motif at low pH. These features could be beneficial to the measurement of dynamic CuII fluctuations and the application in slightly acidic media, such as urine.

Highlights

Most living organisms need copper to survive, owing to its involvement as a redox cofactor in essential biochemical processes, such as cellular respiration
The thermodynamic and kinetic stability of Amino-Terminal CuIIand NiII-binding (ATCUN) complexes depends on the identity of Xxx, Zzz and the sequence downstream His3.[16]. For instance, CuII binds to Human Serum Albumin (HSA) motif (Asp-Ala-His, DAH-) with log cK7.4 = 13, while it binds to Neuromedin C, whose Gly-Asn-His-Trp (GNHW) motif was adopted in the probe GNHW/Tb, with log cK7.4 = 13.6. [17,18] CuII-XZH complexes are stable over a large pH range, classically from above pH 10 down to about 5.5, where decoordination starts to occur.[19,20,21]
In order to explore the competitiveness of the probes in the presence of biological CuII-ligands and to compare their kinetic response in a simpler system, we investigated the ability of the probes to compete with Human Serum Albumin (HSA), the most abundant component of the exchangeable copper (CuEXC) pool in the blood

Summary

Introduction

Most living organisms need copper to survive, owing to its involvement as a redox cofactor in essential biochemical processes, such as cellular respiration. The main requisites are (i) high selectivity over other physiological metal ions (especially ZnII, as it is more available than CuII, and the most similar to CuII in term of coordination properties), (ii) a suited affinity, e.g. a conditional stability constant at pH 7.4 (log cK7.4) higher than HSA (≈13) for application in the blood, and (iii) a signal-to-noise ratio apt to measure relatively low sensor concentrations (e.g. lower than ≈10 μM to ensure a significant Cu-loading in the blood, where CuEXC is at most ≈5 μM) despite high signal background, such as autofluorescence.[8] For this purpose, we recently developed a luminescent peptide probe, hereafter denoted GNHW/Tb, bearing the Amino-Terminal CuIIand NiII-binding (ATCUN) motif and a TbIII-DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10tetraacetic acid) complex.

Objectives

Results

Conclusion