Gordon S. Rule

Expression and Secondary Structure Determination by NMR Methods of the Major House Dust Mite Allergen Der p 2

There exists a strong correlation between asthma and sensitization to indoor allergens. This study reports on the secondary structure of the major house dust mite allergen Deru2009pu20092, determined using heteronuclear NMR methods. The DNA was subcloned from the yeast expression vector pSAY1 into the high yield bacterial expression vector pET21a, resulting in yields of 50 mg/liter. The recombinant protein was shown to have immunoreactivity comparable with that of the natural mite protein using competitive inhibition enzyme-linked immunosorbent assay (ELISA) and a modified monoclonal radioallergosorbent test (RAST). The secondary structure was determined by examining chemical shifts, short and long range NOESYs, JHN-HA coupling constants, and amide exchange rates. From these data, it is clear that Deru2009pu20092 is composed of β-sheets and random coil. Based on long range distance constraints, a number of β-strands were aligned into two three-stranded, anti-parallel β-sheets.

Hydrogen Exchange Nuclear Magnetic Resonance Spectroscopy Mapping of Antibody Epitopes on the House Dust Mite Allergen Der p 2

New strategies for allergen-specific immunotherapy have focused on reducing IgE reactivity of purified recombinant allergens while maintaining T-cell epitopes. Previously, we showed that disrupting the disulfide bonds of the major house dust mite allergen Der p 2 resulted in 10–100-fold less skin test reactivity in mite-allergic subjects but did not change in vitro T-cell proliferative responses. To provide a more complete picture of the antigenic surface of Der p 2, we report here the identification of three epitopes using hydrogen protection nuclear magnetic resonance spectroscopy. The epitopes are defined by monoclonal antibodies that are able to inhibit IgE antibody binding to the allergen. Each monoclonal antibody affected the amide exchange rate of 2–3 continuous residues in different regions of Der p 2. Based on these data, a number of other residues were predicted to belong to each epitope, and this prediction was tested for monoclonal antibody 7A1 by generating alanine point mutants. The results indicate that only a small number of residues within the predicted epitope are functionally important for antibody binding. The molecular definition of these three epitopes will enable us to target limited positions for mutagenesis and to expand our studies of hypoallergenic variants for immunotherapy.

Structural and kinetic studies of the Fab fragment of a monoclonal anti-spin label antibody by nuclear magnetic resonance.

Nuclear magnetic resonance has been used to study the structure of the anti-spin label antibody AN02 combining site and kinetic rates for the hapten-antibody reaction. The association reaction for the hapten dinitrophenyl-diglycine (DNP-diGly) is diffusion-limited. The activation enthalpy for association, 5.1 kcal/mol, is close to the activation enthalpy for diffusion in water. Several reliable resonance assignments have been made with the aid of recently reported crystal structure. Structural data deduced from the nuclear magnetic resonance (n.m.r.) spectra compare favorably with the crystal structure in terms of the combining site amino acid composition, distances of tyrosine residues from the unpaired electron of the hapten, and residues in direct contact with the hapten. Evidence is presented that a single binding site region tyrosine residue can assume two distinct conformations on binding of DNP-diGly. The AN02 antibody is an autoantibody. Dimerization of the Fab fragments is blocked by the hapten DNP-diGly. The n.m.r. spectra suggests that some of the amino acid residues involved in the binding of the DNP-hapten are also involved in the Fab dimerization.

1H, 15N and 13C resonance assignments and secondary structure determination of the RNA-binding domain of E. coli rho protein

SummaryProtein fragments containing the RNA-binding domain of Escherichia coli rho protein have been over-expressed in E. coli. NMR spectra of the fragment containing residues 1–116 of rho protein (Rho116) show that a region of this protein is unfolded in solution. Addition of (dC)10 to this fragment stabilizes the folded form of the protein. The fragment comprising residues 1–130 of rho protein (Rho130) is found to be stably folded, both in the absence and presence of nucleic acid. NMR studies of the complex of Rho 130 with RNA and DNA oligonucleotides indicate that the binding-site size, affinity, and specificity of Rho 130 are similar to those of intact rho protein; therefore, Rho 130 is a suitable model of the RNA-binding domain of rho protein. NMR line widths as well as titration experiments of Rho130 complexed with oligonucleotides of various lengths suggest that Rho130 forms oligomers in the presence of longer oligonucleotides. 1H, 15N and 13C resonance assignments were facilitated by the utilization of two pulse sequences, CN-NOESY and CCH-TOCSY. The secondary structure of unliganded Rho130 has been determined by NMR techniques, and it is clear that the RNA-binding domain of rho is more structurally similar to the cold shock domain than to the RNA recognition motif.

Expression of glutathione S-transferase-cardiotoxin fusion protein in Escherichia coli

We report here the construction of cardiotoxin V gene, from cobra snake venom (Naja naja atra), by chemically synthesized oligonucleotides and its expression as a glutathione S-transferase-cardiotoxin fusion protein in the inclusion bodies of Escherichia coli. The expression of cardiotoxin fusion protein in protein with a yield of about 35 mg/liter culture was confirmed by highly specific anti-peptide antibodies generated against the unique amino acid residues located at the tip of loop II of cardiotoxin V. Since the fusion protein can be easily treated by CNBr to free the toxin moiety, as revealed by immunoblotting of the cleaved protein, the results provide an avenue for future structural and functional studies of cardiotoxin molecules.

1H, 15N and 13C resonance assignments and secondary structure of group II phospholipase A2 from Agkistrodon piscivorus piscivorus: Presence of an amino-terminal helix in solution

Summary1H, 15N and 13C resonance assignments are presented for the group II phospholipase A2 (PLA2) from Agkistrodon piscivorus piscivorus. The secondary structure of the enzyme has been inferred from an analysis of coupling constants, interproton distances, chemical shifts, and kinetics of amide exchange. Overall, the secondary structure of this PLA2 is similar to the crystal structure of the homologous group II human nonpancreatic secretory phospholipase [Scott, D.L., White, S.P., Browning, J.L., Rosa, J.J., Gelb, M.H. and Sigler, P.B. (1991) Science, 254, 1007–1010]. In the group I enzyme from porcine pancreas, the amino-terminal helix becomes fully ordered in the ternary complex of enzyme, lipid micelles and inhibitor. The formation of this helix is thought to be important for the increase in activity of phospholipases on aggregated substrates [Van den Berg, B., Tessari, M., Boelens, R., Dijkman, R., De Haas, G.H., Kaptein, R. and Verheij, H.M. (1995) Nature Struct. Biol., 2, 402–406]. However, the group II enzyme from Agkistrodon piscivorus piscivorus possesses a defined and well-positioned aminoterminal helix in the absence of substrate. Therefore, there is a clear difference between the conformations of group I and group II enzymes in solution. These conformational differences suggest that formation of the amino-terminal helix is a necessary, but not sufficient, step in interfacial activation of phospholipases.

Specificities of Germ Line Antibodies

The past few years have seen remarkable progress in research on the structure and function of antibodies. In our own work we have shown that it is possible to obtain extensive significant information on the composition and structure of antibody combining sites using NMR, together with nitroxide spin-label haptens (Anglister et al, 1984a, 1985 and 1987; Frey et al, 1984). This derived information includes the amino acid composition of the combining site region, that is, the number of tyrosines, alanines, etc. that are within ~ 20 A of the odd electron on the paramagnetic hapten. In antibody molecules there are typically 40–50 amino acids in this combining site region. We have shown that NMR titration data can be used to estimate distances between individual protons on amino acid side chains and the odd electron (Anglister et.al., 1984b; Frey et. al., 1988). These measured distances extend out to about 20 A, and in to distances of the order of 3–5 A. Shorter distances can sometimes be estimated from nuclear magnetization transfer experiments. The NMR data also provide a powerful and convenient means of obtaining the on-off kinetics of hapten-antibody reactions, using resonance signals from the hapten as well as from the protein.

NMR confirms the presence of the aminoterminal helix of group II phospholipase A2 in solution

Publisher Summary In humans, the group I phospholipases A 2 (PLA 2 ) is present in the pancreas and the group II extracellular PLA 2 are found in platelets and arthritic synovial joints. The tertiary structure and disulfide bond pattern are similar among the two groups, with the following main differences: group II PLA 2 have an extension of approximately seven residues on the carboxy terminal part, which is also connected by a disulfide bond, group I PLA 2 have an insertion among the residues 54–56, and in group I the amino terminal helix is connected by a disulfide bond at residue 11. Recent nuclear magnetic resonance (NMR) determination of the solution structure of the pancreatic group I PLA 2 showed that, in contrast to the crystal structures, the beginning of the amino terminal helix is partly disordered. Moreover, the solution structure of the same PLA 2 complexed with the inhibitor and micelle displayed ordering of this region, which became more similar to the crystal structure. In this chapter, a NMR study has been performed on a group II PLA 2 from snake Agkistrodon piscivorus piscivorus , which has previously been extensively used in the biophysical studies on the membrane–protein interactions. In the present study, different techniques from the repertoire of the NMR methods have been used. Those techniques have provided independent data that are consistent and permitted reaching conclusion on the secondary structure of the protein. Improvement in production and refolding procedure for recombinant PLA 2 has allowed preparation of 13C and 15N labeled protein, allowing its assignment and measurement of several structural parameters that describe the conformation of the amino terminal helix. These parameters include NOE signal, main chain dihedral angles, coupling constants, amide hydrogen exchange rates and the deviation of the chemical shift, respectively. These data provide reliable conclusions about the secondary structure of the protein even before building the complete tertiary structure.