Henno W. van den Hooven

The race-specific elicitor AVR9 of the tomato pathogen Cladosporium fulvum: a cystine knot protein: Sequence-specific 1H NMR assignments, secondary structure and global fold of the protein

© 1997 Federation of European Biochemical Societies.

The structure of the lantibiotic lacticin 481 produced by Lactococcus lactis : location of the thioether bridges

The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis ssp. lactis. This polypeptide contains 27 amino acids, including the unusual residues dehydrobutyrine and the thioether‐bridging lanthionine and 3‐methyllanthionine. Lacticin 481 belongs to a structurally distinct group of lantibiotics, which also include streptococcin A‐FF22, salivaricin A and variacin. Here we report the first complete structure of this type of lantibiotic. The exact location of the thioether bridges in lacticin 481 was determined by a combination of peptide chemistry, mass spectrometry and NMR spectroscopy, showing connections between residues 9 and 14, 11 and 25, and 18 and 26.

NMR and circular dichroism studies of the lantibiotic nisin in non-aqueous environments

The lantibiotic, nisin, which is known to interact with membranes of certain Gram‐positive bacteria, was studied in three model systems which mimic a membrane‐like environment, i.e. a mixture of trifluoroethanol and water, or micelles of sodium dodecyl sulfate or dodecylphosphocholine. The 1H NMR spectra of nisin in the non‐aqueous environments, at 40°C and pH 3.5, have been assigned completely. The CD and NMR results indicate that the conformation of nisin in the three non‐aqueous environments differs from that in aqueous solution, and that the conformation in the two micellar systems is similar. The major conformational changes, relative to nisin in aqueous solution, occur in the N‐terminus.

Extreme differences in charge changes during protein evolution.

SummaryThe maintenance of a proper distribution of charged amino acid residues might be expected to be an important factor in protein evolution. We therefore compared the inferred changes in charge during the evolution of 43 protein families with the changes expected on the basis of random base substitutions. It was found that certain proteins, like the eye lens crystallins and most histones, display an extreme avoidance of changes in charge. Other proteins, like phospholipase A2 and ferredoxin, apparently have sustained more charged replacements than expected, suggesting a positive selection for changes in charge. Depending on function and structure of a protein, charged residues apparently can be important targets for selective forces in protein evolution. It appears that actual biased codon usage tends to decrease the proportion of charged amino acid replacements. The influence of nonrandomness of mutations is more equivocal. Genes that use the mitochondrial instead of the universal code lower the probability that charge changes will occur in the encoded proteins.

NMR Studies of Lantibiotics: The Three-Dimensional Structure of Nisin in Aqueous Solution

Nisin, a bacteriocin produced by Lactococcus lactis ssp., is a post-translationally modified pentacyclic polypeptide of 34 amino acids. It is a member of the class of bacteriocins, known as lantibiotics, that contain the unusual amino acid lanthionine. Its structure in aqueous solution has been determined on the basis of data obtained from Nuclear Magnetic Resonance Spectroscopy (NMR) studies. Translation of the interproton distance constraints, derived from Nuclear Overhauser Enhancement Spectroscopy (NOESY)l, and torsion angle constraints, derived from Double Quantum Filtered Correlated Spectroscopy (DQF-COSY), into a 3D structure was carried out with the distance geometry program DISMAN, followed by restrained energy minimization using CHARMm. Due to the internal mobility of the polypeptide chain a determination of the precise overal folding of the molecule was prohibited, but parts of the structure could be obtained albeit with sometimes low resolution. The structure of nisin can best be described as follows: the outermost N-and C-terminal regions appear quite flexible, the remainder of the molecule consists of an amphiphilic N-terminal fragment (residues 3–19), joined by a flexible ‘hinge’ region to a rigid doublering fragment formed by residues 23–28. The latter fragment has the appearance of a somewhat overwound α-helix. It is postulated that i) the coupling between residues 23 and 26 as well as between 25 and 28 by thioether bridges and ii) the inversion of the Cα chiralities at positions 23 and 25 occurs via an intermediate α-helical structure of the prenisin molecule