Beyond structure: Deciphering site-specific dynamics in proteins from double histidine-based EPR measurements.

Site-specific dynamics in proteins are at the heart of protein function. While electron paramagnetic resonance (EPR) has potential to measure dynamics in large protein complexes, the reliance on flexible nitroxide labels is limitating especially for the accurate measurement of site-specific ß-sheet dynamics. Here, we employed EPR spectroscopy to measure site-specific dynamics across the surface of a protein, GB1. Through the use of the double Histidine (dHis) motif, which enables labeling with a Cu(II) - nitrilotriacetic acid (NTA) complex, dynamics information was obtained for both α-helical and ß-sheet sites. Spectral simulations of the resulting CW-EPR report unique site-specific fluctuations across the surface of GB1. Additionally, we performed molecular dynamics (MD) simulations to complement the EPR data. The dynamics observed from MD agree with the EPR results. Furthermore, we observe small changes in gǁ values for different sites, which may be due to small differences in coordination geometry and/or local electrostatics of the site. Taken together, this work expands the utility of Cu(II)NTA-based EPR measurements to probe information beyond distance constraints.

Copper Based Site-directed Spin Labeling of Proteins for Use in Pulsed and Continuous Wave EPR Spectroscopy.

Site-directed spin labeling in conjunction with electron paramagnetic resonance (EPR) is an attractive approach to measure residue specific dynamics and point-to-point distance distributions in a biomolecule. Here, we focus on the labeling of proteins with a Cu(II)-nitrilotriacetic acid (NTA) complex, by exploiting two strategically placed histidine residues (called the dHis motif). This labeling strategy has emerged as a means to overcome key limitations of many spin labels. Through utilizing the dHis motif, Cu(II)NTA rigidly binds to a protein without depending on cysteine residues. This protocol outlines three major points: the synthesis of the Cu(II)NTA complex; the measurement of continuous wave and pulsed EPR spectra, to verify a successful synthesis, as well as successful protein labeling; and utilizing Cu(II)NTA labeled proteins, to measure distance constraints and backbone dynamics. In doing so, EPR measurements are less influenced by sidechain motion, which influences the breadth of the measured distance distributions between two spins, as well as the measured residue-specific dynamics. More broadly, such EPR-based distance measurements provide unique structural constraints for integrative structural biophysics and complement traditional biophysical techniques, such as NMR, cryo-EM, FRET, and crystallography. Graphic abstract: Monitoring the success of Cu(II)NTA labeling.

A novel role for small molecule glycomimetics in the protection against lipid-induced endothelial dysfunction: Involvement of Akt/eNOS and Nrf2/ARE signaling.

BACKGROUND: Glycomimetics are a diverse array of saccharide-inspired compounds, designed to mimic the bioactive functions of glycosaminoglycans. Therefore, glycomimetics represent a unique source of novel therapies to target aberrant signaling and protein interactions in a wide range of diseases. We investigated the protective effects of four newly synthesized small molecule glycomimetics against lipid-induced endothelial dysfunction, with an emphasis on nitric oxide (NO) and oxidative stress. METHODS: Four aromatic sugar mimetics were synthesized by the stepwise transformation of 2,5-dihydroxybenzoic acid to derivatives (C1-C4) incorporating sulfate groups to mimic the structure of heparan sulfate. RESULTS: Glycomimetic-treated human umbilical vein endothelial cells (HUVECs) were exposed to palmitic acid to model lipid-induced oxidative stress. Palmitate-induced impairment of NO production was restored by the glycomimetics, through activation of Akt/eNOS signaling. Furthermore, C1-C4 significantly inhibited palmitate-induced reactive oxygen species (ROS) production, lipid peroxidation, and activity and expression of NADPH oxidase. These effects were attributed to activation of the Nrf2/ARE pathway and downstream activation of cellular antioxidant and cytoprotective proteins. In ex vivo vascular reactivity studies, the glycomimetics (C1-C4) also demonstrated a significant improvement in endothelium-dependent relaxation and decreased ROS production and NADPH oxidase activity in isolated mouse thoracic aortic rings exposed to palmitate. CONCLUSIONS: The small molecule glycomimetics, C1-C4, protect against lipid-induced endothelial dysfunction through up-regulation of Akt/eNOS and Nrf2/ARE signaling pathways. Thus, carbohydrate-derived therapeutics are a new class of glycomimetic drugs targeting endothelial dysfunction, regarded as the first line of defense against vascular complications in cardiovascular disease.

Novel heparin/heparan sulfate mimics as inhibitors of HGF/SF-induced MET activation.

The synthesis of simple, non-sugar glycosaminoglycan (GAG) mimics has been achieved and the analogues evaluated for their ability to inhibit the activation of the MET receptor by hepatocyte growth factor/scatter factor (HGF/SF).

Generation of superoxide and singlet oxygen from alpha-tocopherolquinone and analogues.

Three potential routes to generation of reactive oxygen species (ROS) from alpha-tocopherolquinone (alpha-TQ) have been identified. The quinone of the water-soluble vitamin E analogue Trolox C (Trol-Q) is reduced by hydrated electron and isopropanol alpha-hydroxyalkyl radical, and the resulting semiquinone reacts with molecular oxygen to form superoxide with a second order rate constant of 1.3 x 10(8) dm(3)/mol/s, illustrating the potential for redox cycling. Illumination (UV-A, 355 nm) of the quinone of 2,2,5,7,8-pentamethyl-6-hydroxychromanol (PMHC-Q) leads to a reactive short-lived (ca. 10(- 6) s) triplet state, able to oxidise tryptophan with a second order rate constant greater than 10(9) dm(3)/mol/s. The triplet states of these quinones sensitize singlet oxygen formation with quantum yields of about 0.8. Such potentially damaging reactions of alpha-TQ may in part account for the recent findings that high levels of dietary vitamin E supplementation lack any beneficial effect and may lead to slightly enhanced levels of overall mortality.