Abstract
Electron paramagnetic resonance (EPR) spectroscopy has emerged as an ideal biophysical tool to study complex biological processes. EPR spectroscopy can follow minor conformational changes in various proteins as a function of ligand or protein binding or interactions with high resolution and sensitivity. Resolving cellular mechanisms, involving small ligand binding or metal ion transfer, is not trivial and cannot be studied using conventional biophysical tools. In recent years, our group has been using EPR spectroscopy to study the mechanism underlying copper ion transfer in eukaryotic and prokaryotic systems. This mini-review focuses on our achievements following copper metal coordination in the diamagnetic oxidation state, Cu(I), between biomolecules. We discuss the conformational changes induced in proteins upon Cu(I) binding, as well as the conformational changes induced in two proteins involved in Cu(I) transfer. We also consider how EPR spectroscopy, together with other biophysical and computational tools, can identify the Cu(I)-binding sites. This work describes the advantages of EPR spectroscopy for studying biological processes that involve small ligand binding and transfer between intracellular proteins.
Highlights
Transition metals are key nutrients for almost all organisms, given their roles in critical biochemical pathways, such as respiration, photosynthesis, and nitrogen fixation [1,2,3]
While electron paramagnetic resonance (EPR) spectroscopy cannot provide information on residues directly involved in the coordination of diamagnetic metal ions, it can, probe the dynamics and conformational changes in a biomolecule in solution upon metal ion binding, thereby providing important structural and functional information on the biomolecule as a function of metal coordination
In the presence of these peptides, Atox1 accommodates only one conformation, the open conformation. These results indicate that DEER experiments can follow interactions between two biomolecules that are sensitive to metal ion transfer between them
Summary
Copper is an essential trace metal for living organisms. Many proteins that participate in cellular respiration, anti-oxidant defense, neurotransmitter biosynthesis, connective tissue biosynthesis, and pigment formation use copper as their prosthetic active group [2,4]. Free copper can participate in Fenton-like chemical reactions to generate highly toxic hydroxyl radicals from hydrogen peroxide and superoxide [2,7] Both prokaryotic and eukaryotic systems have developed highly regulated mechanisms for copper transport and intracellular distribution to allow only negligible changes (subfemtomolar concentrations) in the intracellular copper levels [10,11,18]. The first mechanism involves proteins that export copper from the cytoplasm Such proteins are P1B -type ATPase membrane transporters responsible for pumping Cu(I) ions from the plasma membrane to the periplasmic space. These correspond to gene regulatory proteins, coined metalloregulatory proteins, or metal sensor proteins that tolerate the metal transfer rate [32] These specialized “metal receptor” proteins have evolved metal coordination sites that “sense” specific metal ion(s) by forming specific coordination complexes that can present zeptomolar (~10−21 M) level affinities. In E. coli, the Cu(I) metal sensor CueR activates the transcription of CopA and CueO metalloproteins [36,37,38,39]
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