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The conformations of nisin and two major degradation products, nisin-(1-32)-peptide (nisin1-32) and des-delta Ala5-nisin1-32 (where delta Ala is alpha beta-didehydroalanine), in aqueous solution have been determined from n.m.r. data. Sequential assignments of the peptides using correlation spectroscopy ('COSY'), homonuclear Hartmann-Hahn spectroscopy ('HOHAHA'), nuclear Overhauser enhancement spectroscopy (NOESY), relayed NOESY and rotating-frame nuclear Overhauser spectroscopy (ROESY) experiments are presented, including stereospecific assignments of beta-methylene protons of the lanthionine residues. ROESY experiments are also used to detect flexible regions in the polypeptide chain. A dynamic-stimulated-annealing approach is used for structural determination. It can be concluded that all these peptides are flexible in aqueous solution, with no experimental evidence of preferred overall conformations; the only defined conformational features are imposed by the presence of the lanthionine residues. Low-temperature studies also reveal that des-delta Ala5-nisin1-32 adopts conformations similar to those when the ring is intact, suggesting that the loss of activity of this degradation product is due to the absence of the delta Ala5 residue rather than to the conformational consequences of ring-opening.

Bovine liver catalase has been immobilized at surfaces by three separate methods. Firstly, catalase molecules were sprayed onto mica and coated with a platinum/carbon film. Images of single molecules showing submolecular structure were obtained, exhibiting a close correlation with data obtained by X-ray crystallography and electron microscopy. Secondly, catalase molecules were thiolated using Traut's reagent (2-iminothiolane) and adsorbed onto gold surfaces. Images of monolayers of the adsorbed protein were obtained, and the effects on the adsorbate of decreasing the tunnel gap resistance were investigated. Thirdly, 3-mercaptopropanoic acid was chemisorbed onto gold surfaces, and coupled to catalase molecules using a water-soluble carbodiimide reagent. Island structures were observed which were thought to be composed of immobilized catalase molecules. This interpretation was supported by observations of the effect of reversing the polarity of the bias voltage. The application of a platinum/carbon film was found to produce rigidly immobilized protein molecules, and yielded the best resolution. However, covalent coupling techniques offer particular promise as generally applicable methods by which assemblies of proteins may be prepared, facilitating the investigation by scanning tunneling microscopy of their responses to physical and chemical stimuli.

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