Timothy S. Harvey

Hepcidin revisited, disulfide connectivity, dynamics, and structure.

Hepcidin is a tightly folded 25-residue peptide hormone containing four disulfide bonds, which has been shown to act as the principal regulator of iron homeostasis in vertebrates. We used multiple techniques to demonstrate a disulfide bonding pattern for hepcidin different from that previously published. All techniques confirmed the following disulfide bond connectivity: Cys1–Cys8, Cys3–Cys6, Cys2–Cys4, and Cys5–Cys7. NMR studies reveal a new model for hepcidin that, at ambient temperatures, interconverts between two different conformations, which could be individually resolved by temperature variation. Using these methods, the solution structure of hepcidin was determined at 325 and 253 K in supercooled water. X-ray analysis of a co-crystal with Fab appeared to stabilize a hepcidin conformation similar to the high temperature NMR structure.

Structural and thermodynamic effects of ANS binding to human interleukin-1 receptor antagonist

Although 8‐anilinonaphthalene‐1‐sulfonic acid (ANS) is frequently used in protein folding studies, the structural and thermodynamic effects of its binding to proteins are not well understood. Using high‐resolution two‐dimensional NMR and human interleukin‐1 receptor antagonist (IL‐1ra) as a model protein, we obtained detailed information on ANS–protein interactions in the absence and presence of urea. The effects of ambient to elevated temperatures on the affinity and specificity of ANS binding were assessed from experiments performed at 25°C and 37°C. Overall, the affinity of ANS was lower at 37°C compared to 25°C, but no significant change in the site specificity of binding was observed from the chemical shift perturbation data. The same site‐specific binding was evident in the presence of 5.2 M urea, well within the unfolding transition region, and resulted in selective stabilization of the folded state. Based on the two‐state denaturation mechanism, ANS‐dependent changes in the protein stability were estimated from relative intensities of two amide resonances specific to the folded and unfolded states of IL‐1ra. No evidence was found for any ANS‐induced partially denatured or aggregated forms of IL‐1ra throughout the experimental conditions, consistent with a cooperative and reversible denaturation process. The NMR results support earlier observations on the tendency of ANS to interact with solvent‐exposed positively charged sites on proteins. Under denaturing conditions, ANS binding appears to be selective to structured states rather than unfolded conformations. Interestingly, the binding occurs within a previously identified aggregation‐critical region in IL‐1ra, thus providing an insight into ligand‐dependent protein aggregation.

Discovery of Ligands for Nurr1 by Combined Use of NMR Screening with Different Isotopic and Spin-Labeling Strategies

A comprehensive approach to target screening, hit validation, and binding site determination by nuclear magnetic resonance (NMR) spectroscopy is presented. NMR 19F signal perturbation was used to screen a small compound library and identify candidate ligands to the target of interest. Ligand dissociation constants were measured using a pegylated form of the protein, which resulted in a 2-fold increase in the strength of the saturation transfer difference signal. The initial small-molecule hits were further optimized by combining a residue-specific labeling strategy, to identify the specific sites of interaction with the protein, with a second site screening approach based on relaxation enhancement using a paramagnetic probe. The advantages of this combination strategy in the identification and optimization of weak binding chemical entities early in a program are illustrated with the discovery of a low micromolar ligand (Kd = 20 µM) for Nurr1 and identification of the binding site location through residue-specific 15N isotope labeling and derivatization of Cys residues with 2-mercaptoethanol-1-13C. (Journal of Biomolecular Screening 2007:301-311)