Nina C. Gonnella

STRUCTURAL DEFINITION OF THE C5A C TERMINUS BY TWO-DIMENSIONAL NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY

The serum glycoprotein C5a, which is derived from the proteolytic cleavage of complement protein C5, has been implicated in the pathogenesis of a number of inflammatory and allergic conditions. Because C5a induces an inflammatory response upon binding to a specific receptor, structural and mutagenesis studies were carried out to gain a better understanding of this binding interaction. These studies led to the first structural definition of the C terminus of recombinant human (rh)‐C5a, determined by two‐dimensional nuclear magnetic resonance (NMR) spectroscopy. Our results show that the C terminus adopts an α‐helical conformation spanning residues 69 to 74, while the core domain exists as an antiparallel α‐helical bundle. This C‐terminal helix is connected to the core by a short loop that orients Arg 74 adjacent to Arg 62. Point mutation analysis had already revealed that residues 62 and 74 significantly contribute to agonist activity and receptor binding. Correlation of the C5a tertiary structure with mutational analyses clarifies the significance of the functional and binding properties of Arg 62 and suggests that both Arg 62 and Arg 74 interact at the same binding site on the receptor. Proteins 28:261–267, 1997

Residues 21-30 within the extracellular N-terminal region of the C5a receptor represent a binding domain for the C5a anaphylatoxin.

The functions of the C5a anaphylatoxin are expressed through its interaction with a cell-surface receptor with seven transmembrane helices. The interaction of C5a with the receptor has been explained by a two-site model whereby recognition and effector sites on C5a bind, respectively, to recognition and effector domains on the receptor, leading to receptor activation (Chenoweth, D. E., and Hugli, T. E. (1980) Mol. Immunol. 17, 151–161. In addition, the extracellular N-terminal region of the C5a receptor has been implicated as the recognition domain for C5a, responsible for ∼50% of the binding energy of the C5a-receptor complex (Mery, L., and Boulay, F. (1994) J. Biol. Chem. 269, 3457–3463; DeMartino, J. A., Van Riper, G., Siciliano, S. J., Molineaux, C. J., Konteatis, Z. D., Rosen, H., and Springer, M. S. (1994) J. Biol. Chem. 269, 14446–14450). In this work, the interactions of C5a with the N-terminal domain of the C5a receptor were examined by use of recombinant human C5a molecules and peptide fragments M1NSFN5YTTPD10YGHYD15DKDTL20DLNTP25VDKTS30NTLR(hC5aRF-1–34), acetyl-HYD15DKDTL20DLNTP25VDKTS30NTLR (hC5aRF-13–34), and acetyl-TL20DLNTP25VDKTS30N-amide (hC5aRF-19–31) derived from human C5a receptor. Binding induced resonance perturbations in the NMR spectra of the receptor fragments and the C5a molecules indicated that the isolated Nterminal domain or residues 1–34 of the C5a receptor retain specific binding to C5a and to a C5a analog devoid of the agonistic C-terminal tail in the intact C5a. Residues of C5a perturbed by the binding of the receptor peptides are localized within the helical core of the C5a structure, in agreement with the results from functional studies employing mutated C5a and intact receptor molecules. All three receptor peptides, hC5aRF-1–34, hC5aRF-13–34, and hC5aRF-19–31, responded to the binding of C5a through the 21–30 region containing either hydrophobic, polar, or positively charged residues such as Thr24, Pro25, Val26, Lys28, Thr29, and Ser30. The 21–30 segment of all three receptor fragments was found to have a partially folded conformation in solution, independent of residues 1–18. These results indicate that a short peptide sequence, or residues 21–30, of the C5a receptor N terminus may constitute the binding domain for the recognition site on C5a.

Quantitative Evaluation of Aqueous Isopropanol Enhancement of Skin Flux of Terbutaline (Sulfate). I. Ion Associations and Species Equilibria in the Formulation

It has been reported previously that saturated terbutaline sulfate in aqueous isopropanol significantly enhances the terbutaline flux through human skin in vitro. This paper demonstrates that the effect of isopropanol on the permeant species in the formulation contributes to the flux enhancement. This demonstration is based on studies involving measurements of conductivity and pKa as well as NMR spectroscopy in isopropanol-water mixtures. Increasing isopropanol concentration inhibits the proton dissociation of terbutaline and results in the ion associations between the protonated terbutaline and its counterion, sulfate anion. The species present in the formulation include protonated terbutaline, the negatively charged terbutaline-sulfate (1:1) ion pair, and the neutral terbutaline-sulfate (2:1) ion triplet. The results of the studies provide the basis for a quantitative evaluation of the species equilibria in solutions of terbutaline sulfate. The saturated terbutaline sulfate in 60% isopropanol produces the maximum concentration of the neutral ion triplet. This result is almost parallel to the terbutaline skin flux, which maximized at 60–80% isopropanol.

Design and construction of a simple double-tuned, single-input surface-coil probe

Abstract A double-tuned surface-coil NMR probe which allows generation of phosphorus-31 and proton NMR spectra in vivo using only a single input is reported. This resonant circuit requires a minimum number of reactive elements which contributes to its simplicity of design and ease of construction. The probe was designed to accommodate an 89 mm i.d. vertical wide-bore Oxford magnet with a field strength of 9.4 T. A three-turn coil (1.35 cm diameter) with an additional lead attached across one turn was doubly tuned to 31 P at 161.9 MHz and 1 H at 400 MHz. The circuit was made such that the coil can easily be changed producing multinuclear capability without disturbing the remaining circuit. Phosphorus-31 NMR spectra of rat abdominal muscle and surgically exposed rat liver were obtained with good signal-to-noise and without the need for Faraday shielding.