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A new low-molecular-mass (6767.8 Da) serine proteinase isoinhibitor has been isolated from oil-rape (Brassica napus var. oleifera) seed, designated 5-oxoPro1-Gly62-RTI-III. The 5-oxoPro1-Gly62-RTI-III isoinhibitor is longer than the Asp2-Pro61-RTI-III and the Ser3-Pro61-RTI-III forms, all the other amino acid residues being identical. In RTI-III isoinhibitors, the P1-P1' reactive site bond (where residues forming the reactive site have been identified as PnellipsisP1 and P1'ellipsisPn', where P1-P1' is the inhibitor scissile bond) has been identified at position Arg21-Ile22. The inhibitor disulphide bridges pattern has been determined as Cys5-Cys27, Cys18-Cys31, Cys42-Cys52 and Cys54-Cys57. The disulphide bridge arrangement observed in the RTI-III isoinhibitors is reminiscent of that found in a number of toxins (e.g. erabutoxin b). Moreover, the organization of the three disulphide bridges subset Cys5-Cys27, Cys18-Cys31 and Cys42-Cys52 is reminiscent of that found in epidermal growth factor domains. Preliminary 1H-NMR data indicates the presence of alphaalphaNOEs and 3JalphaNH coupling constants, typical of the beta-structure(s). These data suggest that the three-dimensional structure of the RTI-III isoinhibitors may be reminiscent of that of toxins and epidermal growth factor domains, consisting of three-finger shaped loops extending from the crossover region. Values of the apparent association equilibrium constant for RTI-III isoinhibitors binding to bovine beta-trypsin and bovine alpha-chymotrypsin are 3.3 x 109 m-1 and 2.4 x 106 m-1, respectively, at pH 8.0 and 21.0 degrees C. The serine proteinase : inhibitor complex formation is a pH-dependent entropy-driven process. RTI-III isoinhibitors do not show any similarity to other serine proteinase inhibitors except the low molecular mass white mustard trypsin isoinhibitor, isolated from Sinapis alba L. seed (MTI-2). Therefore, RTI-III and MTI-2 isoinhibitors could be members of a new class of plant serine proteinase inhibitors.

We have suggested elsewhere the use of surface mapping by spin label probes (Esposito et al., 1992). According to this approach, soluble nitroxides are added to a protein solution. Resonances of protons that are accessible to the nitroxide are broadened and bleached out of the spectrum, while resonances in the protein interior remain unaffected. This approach is, in principle, complementary to another technique, photochemically induced dynamic nuclear polarization, which maps the position of aromatic protons on the protein surface. A detailed comparison between the two techniques is necessary for a confident use of the more recent suggested nitroxide perturbation approach. In the present study, we show that the results obtained by the two techniques for the native state of bovine alpha-lactalbumin are fully consistent and may therefore be combined for the study of protein surfaces.

We have characterized the high-temperature molten globule state of bovine alpha-lactalbumin by a combined use of photochemically induced dynamic nuclear polarization and nitroxide surface perturbation. Both techniques are extremely well suited to follow the progressive increase of exposed surfaces in the native state and the appearance of partially or completely unfolded species. Our results suggest that the molten globule state obtained at high temperature and pH 7, and the state obtained at pH 2 are not only thermodynamically but also structurally very similar.

1H spectra at 270 MHz of zwitterionic Met-enkephalin pentapeptide (Tyr-Gly-Gly-Phe-Met) in (C2H3)2SO/water mixtures are reported and discussed in terms of solvent-induced conformational transitions. The analysis of the chemical shifts, line widths, coupling constants and rotamer populations around chi 1 and chi 2 suggests that the conformational properties of Met-enkephalin in the two solvents are quite different. In aqueous solution, the preferred structure, characterized by the absence of intramolecularly hydrogen-bonded NH groups and head-to-tail interactions, very likely is an equilibrium of unfolded conformations with approximately equal energy. In (C2H3)2SO, the preferred structure is folded, with the Met-5 NH intramolecularly bonded and the Gly-3 NH protected from the solvent, while the Gly-2 and Phe-4 amide protons are solvent exposed. A conformational transition of Met-enkephalin from the intramolecularly bonded to the unbonded one takes place at about 40 mol-% water in (C2H3)2SO, involving the Met-5 NH proton and the Tyr-Gly-Gly fragment. The Phe-4 and Met-5 phi angles do not change appreciably, which suggest that an inversion at the Gly-3 residue of the folded form, responsible for the conformational transition, does not affect the C-terminal moiety. At about 70 mol-% water in (C2H3)2SO a change in the solvent mixture properties affects the chi 1 rotamer populations and the ring dynamics of the aromatic side chains. The line broadening of the Tyr-1 delta and epsilon proton resonances indicates a specific interaction of the N-terminal ring with the solvent.

1H-NMR spectra at 600 MHz and 270 MHz and photo-chemically induced dynamic nuclear polarization (photo-CIDNP) spectra at 360 MHz of beta-endorphin in the presence of sodium dodecyl sulfate (SDS) micelles are reported and discussed in terms of structural changes and immobilization upon binding. On addition of micelles several NH protons show slow H-D exchange rate in 2H2O at p2H 4.6, 25 degrees C, which indicates that some regions of the polypeptide are buried and shielded from the direct interaction with the solvent. Moreover, in the methyl region of the spectrum strong changes are detected both in chemical shifts and line-widths, suggesting an appreciable interaction between the hydrophobic residues of beta-endorphin and the detergent micelles. All aromatic residues are strongly affected by the presence of SDS, supporting the notion that beta-endorphin can interact with both the hydrophobic and hydrophilic portion of the micelles. At physiological pH photo-CIDNP experiments indicate that SDS has about the same immobilizing effect on Tyr-1 and Tyr-27 as n-dodecylphosphorylcholine.

The photochemically-induced dynamic nuclear polarization technique has been used to investigate the access of a photoexcited flavin dye to tyrosyl and histidyl residues in [Met]enkephalin and human and camel beta-endorphins, both alone and in the presence of n-dodecylphosphorylcholine micelles. The results indicate that the mode of binding of Tyr-1, but not of residue 27, is similar in the two endorphins and differs from that of Tyr-1 in [Met]enkephalin. In human beta-endorphin, accessibility and mobility of Tyr-27 are strongly reduced in the presence of lipid at physiological pH, whereas in camel beta-endorphin His-27 becomes immobilized only at high pH. Moreover, nuclear Overhauser enhancement experiments suggest a rigidifying influence of the peptide on the polar head groups of the micelles.