Structures of highly flexible intracellular domain of human α7 nicotinic acetylcholine receptor.

The intracellular domain (ICD) of Cys-loop receptors mediates diverse functions. To date, no structure of a full-length ICD is available due to challenges stemming from its dynamic nature. Here, combining nuclear magnetic resonance (NMR) and electron spin resonance experiments with Rosetta computations, we determine full-length ICD structures of the human α7 nicotinic acetylcholine receptor in a resting state. We show that ~57% of the ICD residues are in highly flexible regions, primarily in a large loop (loop L) with the most mobile segment spanning ~50 Å from the central channel axis. Loop L is anchored onto the MA helix and virtually forms two smaller loops, thereby increasing its stability. Previously known motifs for cytoplasmic binding, regulation, and signaling are found in both the helices and disordered flexible regions, supporting the essential role of the ICD conformational plasticity in orchestrating a broad range of biological processes.

Cu2+-based distance measurements by pulsed EPR provide distance constraints for DNA backbone conformations in solution.

Electron paramagnetic resonance (EPR) has become an important tool to probe conformational changes in nucleic acids. An array of EPR labels for nucleic acids are available, but they often come at the cost of long tethers, are dependent on the presence of a particular nucleotide or can be placed only at the termini. Site directed incorporation of Cu2+-chelated to a ligand, 2,2'dipicolylamine (DPA) is potentially an attractive strategy for site-specific, nucleotide independent Cu2+-labelling in DNA. To fully understand the potential of this label, we undertook a systematic and detailed analysis of the Cu2+-DPA motif using EPR and molecular dynamics (MD) simulations. We used continuous wave EPR experiments to characterize Cu2+ binding to DPA as well as optimize Cu2+ loading conditions. We performed double electron-electron resonance (DEER) experiments at two frequencies to elucidate orientational selectivity effects. Furthermore, comparison of DEER and MD simulated distance distributions reveal a remarkable agreement in the most probable distances. The results illustrate the efficacy of the Cu2+-DPA in reporting on DNA backbone conformations for sufficiently long base pair separations. This labelling strategy can serve as an important tool for probing conformational changes in DNA upon interaction with other macromolecules.

Understanding and controlling the metal-directed assembly of terpyridine-functionalized coiled-coil peptides.

Metal-binding peptides are versatile building blocks in supramolecular chemistry. We recently reported a class of crystalline materials formed through a combination of coiled-coil peptide self-association and metal coordination. Here, we probe the serendipitously discovered metal binding motif that drives the assembly and apply these insights to exert rational control over structure and morphology in the materials.

Increasing nitroxide lifetime in cells to enable in-cell protein structure and dynamics measurements by electron spin resonance spectroscopy.

There is increasing evidence that the stability, structure, dynamics, and function of many proteins differ in cells versus in vitro. The determination of protein structure and dynamics within the native cellular environment may lead to better understanding of protein behavior. Electron spin resonance (ESR) has emerged as a technique that can report on protein structure and dynamics within cells. Nitroxide based spin labels are capable of reporting on protein dynamics, structure, and backbone flexibility but are limited due to nitroxide reduction occurring in cells. In order to overcome this limitation, we used the oxidizing agent potassium ferricyanide (K3Fe(CN)6) as well as the cleavage resistant spin label 3-malemido-PROXYL (5-MSL). Furthermore, we hypothesized that injection concentration is an important parameter regarding nitroxide reduction kinetics. By increasing the injection concentration of doubly 5-MSL labeled protein into Xenopus laevis oocytes, we found an increased nitroxide lifetime. Our work demonstrates unprecedented incubation times of 3-h in-cell and 5-h in-cytosol for double electron-electron resonance (DEER) experiments using nitroxide spin labels. This allows for more meaningful measurements of larger protein systems which may require longer incubation times for equilibration in the cellular milieu. Even longer incubation times are possible by combining our approach with more shielded nitroxides and Q-band.

ESR Resolves the C Terminus Structure of the Ligand-free Human Glutathione S-Transferase A1-1.

Nitroxide- and Cu2+-based electron spin resonance (ESR) are combined to provide insight into the conformational states of the functionally important α-helix of the human glutathione S-transferase A1. Distance measurements on various spin-labeled dimeric human glutathione S-transferase A1-1 all result in bimodal distance distributions, indicating that the C-terminus exists in two distinct conformations in solution, one of which closely matches that found in the crystal structure of the ligand-bound enzyme. These measurements permit the generation of a model of the unliganded conformation. Room temperature ESR indicates that the second conformation has high mobility, potentially enabling the enzyme's high degree of substrate promiscuity. This model is then validated using computational modeling and further Cu2+-based ESR distance measurements. Cu2+-based ESR also provides evidence that the secondary structure of the second conformation is of helical nature. Addition of S-hexyl glutathione results in a shift in relative populations, favoring the state that is similar to the previously known structure of the ligand-bound enzyme.

The Cu2+-nitrilotriacetic acid complex improves loading of α-helical double histidine site for precise distance measurements by pulsed ESR.

Site-directed spin labeling using two strategically placed natural histidine residues allows for the rigid attachment of paramagnetic Cu2+. This double histidine (dHis) motif enables extremely precise, narrow distance distributions resolved by Cu2+-based pulsed ESR. Furthermore, the distance measurements are easily relatable to the protein backbone-structure. The Cu2+ ion has, till now, been introduced as a complex with the chelating agent iminodiacetic acid (IDA) to prevent unspecific binding. Recently, this method was found to have two limiting concerns that include poor selectivity towards α-helices and incomplete Cu2+-IDA complexation. Herein, we introduce an alternative method of dHis-Cu2+ loading using the nitrilotriacetic acid (NTA)-Cu2+ complex. We find that the Cu2+-NTA complex shows a four-fold increase in selectivity toward α-helical dHis sites. Furthermore, we show that 100% Cu2+-NTA complexation is achievable, enabling precise dHis loading and resulting in no free Cu2+ in solution. We analyze the optimum dHis loading conditions using both continuous wave and pulsed ESR. We implement these findings to show increased sensitivity of the Double Electron-Electron Resonance (DEER) experiment in two different protein systems. The DEER signal is increased within the immunoglobulin binding domain of protein G (called GB1). We measure distances between a dHis site on an α-helix and dHis site either on a mid-strand or a non-hydrogen bonded edge-strand ß-sheet. Finally, the DEER signal is increased twofold within two α-helix dHis sites in the enzymatic dimer glutathione S-transferase exemplifying the enhanced α-helical selectivity of Cu2+-NTA.

Analysis of Nitroxide-Based Distance Measurements in Cell Extracts and in Cells by Pulsed ESR Spectroscopy.

Measurements of distances in cells by pulsed ESR spectroscopy afford tremendous opportunities to study proteins in native environments that are irreproducible in vitro. However, the in-cell environment is harsh towards the typical nitroxide radicals used in double electron-electron resonance (DEER) experiments. A systematic examination is performed on the loss of the DEER signal, including contributions from nitroxide decay and nitroxide side-chain cleavage. In addition, the possibility of extending the lifetime of the nitroxide radical by use of an oxidizing agent is investigated. Using this oxidizing agent, DEER distance measurements are performed on doubly nitroxide-labeled GB1, the immunoglobulin-binding domain of protein G, at varying incubation times in the cellular environment. It is found that, by comparison of the loss of DEER signal to the loss of the CW spectrum, cleavage of the nitroxide side chain contributes to the loss of DEER signal, which is significantly greater in cells than in cell extracts. Finally, local spin concentrations are monitored at varying incubation times to show the time required for molecular diffusion of a small globular protein within the cellular milieu.

Nucleotide-Independent Copper(II)-Based Distance Measurements in DNA by Pulsed ESR Spectroscopy.

A site-specific Cu2+ binding motif within a DNA duplex for distance measurements by ESR spectroscopy is reported. This motif utilizes a commercially available 2,2'-dipicolylamine (DPA) phosphormadite easily incorporated into any DNA oligonucleotide during initial DNA synthesis. The method only requires the simple post-synthetic addition of Cu2+ without the need for further chemical modification. Notably, the label is positioned within the DNA duplex, as opposed to outside the helical perimeter, for an accurate measurement of duplex distance. A distance of 2.7 nm was measured on a doubly Cu2+ -labeled DNA sequence, which is in exact agreement with the expected distance from both DNA modeling and molecular dynamic simulations. This result suggests that with this labeling strategy the ESR measured distance directly reports on backbone DNA distance, without the need for further modeling. Furthermore, the labeling strategy is structure- and nucleotide-independent.

Cu(II)-Zn(II) Cross-Modulation in Amyloid-Beta Peptide Binding: An X-ray Absorption Spectroscopy Study.

In this work we analyze at a structural level the mechanism by which Cu(II) and Zn(II) ions compete for binding to the Aß peptides that is involved in the etiology of Alzheimer's disease. We collected X-ray absorption spectroscopy data on samples containing Aß with Cu and Zn at different concentration ratios. We show that the order in which metals are added to the peptide solution matters and that, when Zn is added first, it prevents Cu from binding. On the contrary, when Cu is added first, it does not (completely) prevent Zn binding to Aß peptides. Our analysis suggests that Cu and Zn ions are coordinated to different numbers of histidine residues depending on the [ion]:[peptide] concentration ratio.

Conformational Changes Underlying Desensitization of the Pentameric Ligand-Gated Ion Channel ELIC.

Structural rearrangements underlying functional transitions of pentameric ligand-gated ion channels (pLGICs) are not fully understood. Using (19)F nuclear magnetic resonance and electron spin resonance spectroscopy, we found that ELIC, a pLGIC from Erwinia chrysanthemi, expanded the extracellular end and contracted the intracellular end of its pore during transition from the resting to an apparent desensitized state. Importantly, the contraction at the intracellular end of the pore likely forms a gate to restrict ion transport in the desensitized state. This gate differs from the hydrophobic gate present in the resting state. Conformational changes of the TM2-TM3 loop were limited to the N-terminal end. The TM4 helices and the TM3-TM4 loop appeared relatively insensitive to agonist-mediated structural rearrangement. These results indicate that conformational changes accompanying functional transitions are not uniform among different ELIC regions. This work also revealed the co-existence of multiple conformations for a given state and suggested asymmetric conformational arrangements in a homomeric pLGIC.

Chemical and Electrochemical Manipulation of Mechanical Properties in Stimuli-Responsive Copper-Cross-Linked Hydrogels.

Inspiration for the design of new synthetic polymers can be found in the natural world, where materials often exhibit complex properties that change depending on external stimuli. A new synthetic electroplastic elastomer hydrogel (EPEH) that undergoes changes in mechanical properties in response to both chemical and electrochemical stimuli has been prepared based on these precedents. In addition to having the capability to switch between hard and soft states, the presence of both permanent covalent and dynamic copper-based cross links also allows this stimuli-responsive material to exhibit a striking shape memory capability. The density of temporary cross links and the mechanical properties are controlled by reversible switching between the +1 and +2 oxidation states.