Tracking Disordered Extracellular Domains of Membrane Proteins in the Cell with Cu(II)-Based Spin Labels.

In-cell electron paramagnetic resonance (EPR) spectroscopy experiments provide high-resolution data about conformational changes of proteins within the cell. However, one of the limitations of EPR is the requisite of stable paramagnetic centers in a reducing environment. We recently showed that histidine-rich sites in proteins hold a high affinity to Cu(II) ions complexed with a chelator. Using a chelator prevents the reduction of Cu(II) ions. Moreover, this spin-labeling methodology can be performed within the native cellular environment on any overexpressed protein without protein purification and delivery to the cell. Herein, we use this novel methodology to gain spatial information on the extracellular domain of the human copper transporter, hCtr1. Limited structural information on the transmembrane domain of the human Ctr1 (hCtr1) was obtained using X-ray crystallography and cryo-EM. However, these structures are missing information on the disordered extracellular domains of hCtr1. Extracellular domains are sensing or interacting with the environment outside of the cell and therefore play an essential role in any transmembrane protein. Especially in hCtr1, the extracellular domain functions as a gating mechanism for copper ions. Here, we performed EPR experiments revealing structural information about the extracellular N-terminal domain of the full-length hCtr1 in vitro and in situ in insect cells and cell membrane fragments. The comparison revealed that the extracellular domains of the in situ and native membrane hCtr1 are further apart than the structure of the purified protein. These method-related differences highlight the significance of studying membrane proteins in their native environment.

Treating late-onset Tay Sachs disease: Brain delivery with a dual trojan horse protein.

Tay-Sachs (TS) disease is a neurodegenerative disease resulting from mutations in the gene encoding the α-subunit (HEXA) of lysosomal ß-hexosaminidase A (HexA). We report that (1) recombinant HEXA alone increased HexA activity and decreased GM2 content in human TS glial cells and peripheral mononuclear blood cells; 2) a recombinant chimeric protein composed of HEXA linked to two blood-brain barrier (BBB) entry elements, a transferrin receptor binding sequence and granulocyte-colony stimulating factor, associates with HEXB in vitro; reaches human cultured TS cells lysosomes and mouse brain cells, especially neurons, in vivo; lowers GM2 in cultured human TS cells; lowers whole brain GM2 concentration by approximately 40% within 6 weeks, when injected intravenously (IV) to adult TS-mutant mice mimicking the slow course of late-onset TS; and increases forelimbs grip strength. Hence, a chimeric protein equipped with dual BBB entry elements can transport a large protein such as HEXA to the brain, decrease the accumulation of GM2, and improve muscle strength, thereby providing potential treatment for late-onset TS.

An efficient EPR spin-labeling method enables insights into conformational changes in DNA.

Electron paramagnetic resonance (EPR) is a powerful tool for elucidating both static and dynamic conformational alterations in macromolecules. However, to effectively utilize EPR for such investigations, the presence of paramagnetic centers, known as spin labels, is required. The process of spin labeling, particularly for nucleotides, typically demands intricate organic synthesis techniques. In this study, we introduce a unique addition-elimination reaction method with a simple spin-labeling process, facilitating the monitoring of structural changes within nucleotide sequences. Our investigation focuses on three distinct labeling positions with a DNA sequence, allowing the measurement of distance between two spin labels. The experimental mean distances obtained agreed with the calculated distances, underscoring the efficacy of this straightforward spin-labeling approach in studying complex biological processes such as transcription mechanism using EPR measurements.

The Dynamic Plasticity of P. aeruginosa CueR Copper Transcription Factor upon Cofactor and DNA Binding.

Bacteria use specialized proteins, like transcription factors, to rapidly control metal ion balance. CueR is a Gram-negative bacterial copper regulator. The structure of E. coli CueR complexed with Cu(I) and DNA was published, since then many studies have shed light on its function. However, P. aeruginosa CueR, which shows high sequence similarity to E. coli CueR, has been less studied. Here, we applied room-temperature electron paramagnetic resonance (EPR) measurements to explore changes in dynamics of P. aeruginosa CueR in dependency of copper concentrations and interaction with two different DNA promoter regions. We showed that P. aeruginosa CueR is less dynamic than the E. coli CueR protein and exhibits much higher sensitivity to DNA binding as compared to its E. coli CueR homolog. Moreover, a difference in dynamical behavior was observed when P. aeruginosa CueR binds to the copZ2 DNA promoter sequence compared to the mexPQ-opmE promoter sequence. Such dynamical differences may affect the expression levels of CopZ2 and MexPQ-OpmE proteins in P. aeruginosa. Overall, such comparative measurements of protein-DNA complexes derived from different bacterial systems reveal insights about how structural and dynamical differences between two highly homologous proteins lead to quite different DNA sequence-recognition and mechanistic properties.

Revealing the DNA Binding Modes of CsoR by EPR Spectroscopy.

In pathogens, a unique class of metalloregulator proteins, called gene regulatory proteins, sense specific metal ions that initiate gene transcription of proteins that export metal ions from the cell, thereby preventing toxicity and cell death. CsoR is a metalloregulator protein found in various bacterial systems that "sense" Cu(I) ions with high affinity. Upon copper binding, CsoR dissociates from the DNA promoter region, resulting in initiation of gene transcription. Crystal structures of CsoR in the presence and absence of Cu(I) from various bacterial systems have been reported, suggesting either a dimeric or tetrameric structure of these helical proteins. However, structural information about the CsoR-DNA complex is missing. Here, we applied electron paramagnetic resonance (EPR) spectroscopy to follow the conformational and dynamical changes that Mycobacterium tuberculosis CsoR undergoes upon DNA binding in solution. We showed that the quaternary structure is predominantly dimeric in solution, and only minor conformational and dynamical changes occur in the DNA bound state. Also, labeling of the unresolved C- terminus revealed no significant change in dynamics upon DNA binding. These observations are unique, since for other bacterial copper metalloregulators, such as the MerR and CopY families, major conformational changes were observed upon DNA binding, indicating a different mode of action for this protein family.

Noninvasive Treatment of Alzheimer's Disease with Scintillating Nanotubes.

Effective and accessible treatments for Alzheimer's disease (AD) are urgently needed. Soluble Aß oligomers are identified as neurotoxic species in AD and targeted in antibody-based drug development to mitigate cognitive decline. However, controversy exists concerning their efficacy and safety. In this study, an alternative strategy is proposed to inhibit the formation of Aß oligomers by selectively oxidizing specific amino acids in the Aß sequence, thereby preventing its aggregation. Targeted oxidation is achieved using biocompatible and blood-brain barrier-permeable multicomponent nanoscintillators that generate singlet oxygen upon X-ray interaction. Surface-modified scintillators interact selectively with Aß and, upon X-ray irradiation, inhibit the formation of neurotoxic aggregates both in vitro and in vivo. Feeding transgenic Caenorhabditis elegans expressing human Aß with the nanoscintillators and subsequent irradiation with soft X-ray reduces Aß oligomer levels, extends lifespan, and restores memory and behavioral deficits. These findings support the potential of X-ray-based therapy for AD and warrant further development.

An in-cell spin-labelling methodology provides structural information on cytoplasmic proteins in bacteria.

EPR in-cell spin-labeling was applied to CueR in E. coli. The methodology employed a Cu(II)-NTA complexed with dHis. High resolved in-cell distance distributions were obtained revealing minor differences between in vitro and in-cell data. This methodology allows study of structural changes of any protein in-cell, independent of size or cellular system.

Conformations and Local Dynamics of the CopY Metal Sensor Revealed by EPR Spectroscopy.

Metal transcription factors regulate metal concentrations in eukaryotic and prokaryotic cells. Copper is a metal ion that is being tightly regulated, owing to its dual nature. Whereas copper is an essential nutrient for bacteria, it is also toxic at high concentrations. CopY is a metal-sensitive transcription factor belonging to the copper-responsive repressor family found in Gram-positive bacteria. CopY represses transcription in the presence of Zn(II) ions and initiates transcription in the presence of Cu(I) ions. The complete crystal structure of CopY has not been reported yet, therefore most of the structural information on this protein is based on its similarity to the well-studied MecI protein. In this study, electron paramagnetic resonance (EPR) spectroscopy was used to characterize structural and local dynamical changes in Streptococcus pneumoniae CopY as a function of Zn(II), Cu(I), and DNA binding. We detected different conformations and changes in local dynamics when CopY bound Zn(II), as opposed to Cu(I) ions. Furthermore, we explored the effects of metal ions and DNA on CopY conformation. Our results revealed the sensitivity and selectivity of CopY towards metal ions and provide new insight into the structural mechanism of the CopY transcription factor.

Disrupting Cu trafficking as a potential therapy for cancer.

Copper ions play a crucial role in various cellular biological processes. However, these copper ions can also lead to toxicity when their concentration is not controlled by a sophisticated copper-trafficking system. Copper dys-homeostasis has been linked to a variety of diseases, including neurodegeneration and cancer. Therefore, manipulating Cu-trafficking to trigger selective cancer cell death may be a viable strategy with therapeutic benefit. By exploiting combined in silico and experimental strategies, we identified small peptides able to bind Atox1 and metal-binding domains 3-4 of ATP7B proteins. We found that these peptides reduced the proliferation of cancer cells owing to increased cellular copper ions concentration. These outcomes support the idea of harming copper trafficking as an opportunity for devising novel anti-cancer therapies.

Allostery-driven changes in dynamics regulate the activation of bacterial copper transcription factor.

Metalloregulators bind and respond to metal ions by regulating the transcription of metal homeostasis genes. Copper efflux regulator (CueR) is a copper-responsive metalloregulator that is found in numerous Gram-negative bacteria. Upon Cu(I) coordination, CueR initiates transcription by bending the bound DNA promoter regions facilitating interaction with RNA polymerase. The structure of Escherichia coli CueR in presence of DNA and metal ion has been reported using X-ray crystallography and cryo-EM, providing information about the mechanism of action. However, the specific role of copper in controlling this transcription mechanism remains elusive. Herein, we use room temperature electron paramagnetic resonance (EPR) experiments to follow allosterically driven dynamical changes in E. coli CueR induced by Cu(I) binding. We suggest that more than one Cu(I) ion binds per CueR monomer, leading to changes in site-specific dynamics at the Cu(I) binding domain and at the distant DNA binding site. Interestingly, Cu(I) binding leads to an increase in dynamics about 27 Å away at the DNA binding domain. These changes in the dynamics of the DNA binding domain are important for exact coordination with the DNA. Thus, Cu(I) binding is critical to initiate a series of conformational changes that regulate and initiate gene transcription. BROAD AUDIENCE STATEMENT: The dynamics of metal transcription factors as a function of metal and DNA binding are complex. In this study, we use EPR spectroscopy to measure dynamical changes of Escherichia coli CueR as a function of copper and DNA binding. We show that copper controls the activation of the transcription processes by initiation a series of dynamical changes over the protein.

Dynamical interplay between the human high-affinity copper transporter hCtr1 and its cognate metal ion.

Abnormal cellular copper levels have been clearly implicated in genetic diseases, cancer, and neurodegeneration. Ctr1, a high-affinity copper transporter, is a homotrimeric integral membrane protein that provides the main route for cellular copper uptake. Together with a sophisticated copper transport system, Ctr1 regulates Cu(I) metabolism in eukaryotes. Despite its pivotal role in normal cell function, the molecular mechanism of copper uptake and transport via Ctr1 remains elusive. In this study, electron paramagnetic resonance (EPR), UV-visible spectroscopy, and all-atom simulations were employed to explore Cu(I) binding to full-length human Ctr1 (hCtr1), thereby elucidating how metal binding at multiple distinct sites affects the hCtr1 conformational dynamics. We demonstrate that each hCtr1 monomer binds up to five Cu(I) ions and that progressive Cu(I) binding triggers a marked structural rearrangement in the hCtr1 C-terminal region. The observed Cu(I)-induced conformational remodeling suggests that the C-terminal region may play a dual role, serving both as a channel gate and as a shuttle mediating the delivery of copper ions from the extracellular hCtr1 selectivity filter to intracellular metallochaperones. Our findings thus contribute to a more complete understanding of the mechanism of hCtr1-mediated Cu(I) uptake and provide a conceptual basis for developing mechanism-based therapeutics for treating pathological conditions linked to de-regulated copper metabolism.

Cellular Uptake of the ATSM-Cu(II) Complex under Hypoxic Conditions.

The Cu(II)-diacetyl-bis (N4-methylthiosemicarbazone) complex (ATSM-Cu(II)) has been suggested as a promising positron emission tomography (PET) agent for hypoxia imaging. Several in-vivo studies have shown its potential to detect hypoxic tumors. However, its uptake mechanism and its specificity to various cancer cell lines have been less studied. Herein, we tested ATSM-Cu(II) toxicity, uptake, and reduction, using four different cell types: (1) mouse breast cancer cells (DA-3), (2) human embryonic kidney cells (HEK-293), (3) breast cancer cells (MCF-7), and (4) cervical cancer cells (Hela) under normoxic and hypoxic conditions. We showed that ATSM-Cu(II) is toxic to breast cancer cells under normoxic and hypoxic conditions; however, it is not toxic to normal HEK-293 non-cancer cells. We showed that the Cu(I) content in breast cancer cell after treatment with ATSM-Cu(II) under hypoxic conditions is higher than in normal cells, despite that the uptake of ATSM-Cu(II) is a bit higher in normal cells than in breast cancer cells. This study suggests that the redox potential of ATSM-Cu(II) is higher in breast cancer cells than in normal cells; thus, its toxicity to cancer cells is increased.

EPR spectroscopy shows that the blood carrier protein, human serum albumin, closely interacts with the N-terminal domain of the copper transporter, Ctr1.

Copper is an essential metal whose localization within the cells must be carefully controlled to avoid copper dependent redox cycling. Although most of the key proteins involved in cellular copper transfer have been identified, fundamental questions regarding the copper transfer mechanism have yet to be resolved. One of the blood carrier proteins believed to be involved in copper transfer to the cell is human serum albumin (HSA). However, direct evidence for close interaction between HSA and the extracellular domain of the copper transporter Ctr1 has not yet been found. By utilizing EPR spectroscopy, we show here that HSA closely interacts with the first 14 amino acids of the Ctr1, even without the presence of copper ions.

EPR and NMR spectroscopies provide input on the coordination of Cu(I) and Ag(I) to a disordered methionine segment.

Methionine motifs are methionine-rich metal-binding segments found in many human, yeast, and bacterial proteins involved in the transportation of copper ion to other cellular pathways, and in protecting copper from oxidation. Methionine motifs are found to bind Ag(I) and Cu(I) ions. Proteins or peptides that can bind different metal ions should have the ability to differentiate between them, to be able to shuttle them to various pathways in the cell. This study utilizes electron paramagnetic resonance spectroscopy together with circular dichroism and nuclear magnetic resonance to probe structural changes in the methionine segment upon coordinating Cu(I) and Ag(I) metal ions. The data collected here indicate that methionine segments experience structural changes while coordinating Cu(I) and Ag(I), however, the differences between the coordination of Cu(I) vs. Ag(I) to the methionine segment are mild. Since Cu(I) and Ag(I) metal ions are pretty similar in their nature and charge, the minor structural changes reported here are significant towards the understanding of the differences in the transport mechanism of these two metal ions in prokaryotic and eukaryotic cells.

Exploring the radical nature of a carbon surface by electron paramagnetic resonance and a calibrated gas flow.

While the first Electron Paramagnetic Resonance (EPR) studies regarding the effects of oxidation on the structure and stability of carbon radicals date back to the early 1980s the focus of these early papers primarily characterized the changes to the structures under extremely harsh conditions (pH or temperature)(1-3). It is also known that paramagnetic molecular oxygen undergoes a Heisenberg spin exchange interaction with stable radicals that extremely broadens the EPR signal(4-6). Recently, we reported interesting results where this interaction of molecular oxygen with a certain part of the existing stable radical structure can be reversibly affected simply by flowing a diamagnetic gas through the carbon samples at STP(7). As flows of He, CO2, and N2 had a similar effect these interactions occur at the surface area of the macropore system. This manuscript highlights the experimental techniques, work-up, and analysis towards affecting the existing stable radical nature in the carbon structures. It is hoped that it will help towards further development and understanding of these interactions in the community at large.