In order to test the influence of chemical modifications designed to allow covalent coupling of channel-forming peptide motifs into variable sized oligomers, a series of alamethicin derivatives was ...prepared. The building block encompassing the N-terminal 1–17 residues of alamethicin behaved normally in the conductance assay on planar lipid bilayers, albeit at higher concentration and with a slightly reduced voltage-dependence. A linker Ac-K-OCH
2C
6H
4CH
3
p attached via the epsilon amino group of lysine to the C-terminus of alamethicin(1–17) increased membrane affinity. The latter was further enhanced in a dimer and a tetramer in which alamethicin(1–17) chains were tethered to di- or tetra-lysine linkers, respectively, but macroscopic current–voltage curves displayed much reduced voltage-dependencies and reversed hysteresis. An usual behaviour with high voltage-dependence was restored with the modified dimer of alamethicin(1–17) in which alanine separated the two consecutive lysine residues in the linker. Of special interest was the development of a ‘negative resistance’ branch in macroscopic current–voltage curves for low concentrations of this dimer with the more flexible linker. Single channel events displayed only one single open state with fast kinetics and whose conductance matches that of the alamethicin heptamer or octamer.
A new spectroscopic technique, rotational-echo double-resonance (REDOR) NMR, for solids utilizes magic-angle spinning and measures directly the dipolar coupling between stable-isotope-labeled nuclei ...and, thus, interatomic distances. REDOR has been used to measure the {sup 13}C-{sup 15}N interatomic distance in a nine-residue fragment, Ac-Phe-MeA(1-{sup 13}C)-MeA(d{sub 6})-MeA-Val-Gly({sup 15}N)-Leu-MeA-MeA-OBzl (MeA = {alpha}-methylalanine or aminoisobutyric acid (Aib)), of the peptide antibiotic emerimicin. The crystal structure of the peptide emerimicin 1-9 benzyl ester was determined previously, and the measurement by REDOR of a known interatomic distance allows both validation and a practical demonstration of the precision of REDOR. The ability to map precisely intermolecular distances suggests applications of REDOR in the solid, or aggregated state, for determinations of the conformations of ligand molecules in drug-receptor, inhibitor-enzyme, and antigen-antibody complexes.
In many complexes formed by serine proteinases and their inhibitors, the hydroxyl group provided by water molecule or by the inhibitor Ser residue is located close to the inhibitor
P
1
–
P
1
′
...reactive site. In order to investigate the role of this group, we synthesized analogues of trypsin inhibitor SFTI-1 isolated from the seeds of sunflower modified in P
1 by α-hydroxymethylserine (HmSer) and both enantiomers of α-hydroxymethylvaline (HmVal). All the synthesized analogues inhibited bovine β-trypsin and human leukocyte elastase. SFTI-1 analogues with HmVal and HmSer appear to be potent inhibitors of bovine β-trypsin, whereas Val
5SFTI-1 is practically inactive. Also trypsin inhibitory activity of Ser
5SFTI-1 is significantly lower. Since the electrostatic interaction between protonated ε-NH
2 group of the inhibitor P
1 position and β-carboxylate of trypsin Asp
189 is the main driving force for interaction of both molecules, the results obtained are very interesting. We believe that these SFTI-1 analogues belong to a novel class of serine proteinase inhibitors.
In many complexes formed by serine proteinases and their inhibitors, the hydroxyl group provided by water molecule or by the inhibitor Ser residue is located close to the inhibitor P{sub 1}-P{sub ...1}{sup '} reactive site. In order to investigate the role of this group, we synthesized analogues of trypsin inhibitor SFTI-1 isolated from the seeds of sunflower modified in P{sub 1} by {alpha}-hydroxymethylserine (HmSer) and both enantiomers of {alpha}-hydroxymethylvaline (HmVal). All the synthesized analogues inhibited bovine {beta}-trypsin and human leukocyte elastase. SFTI-1 analogues with HmVal and HmSer appear to be potent inhibitors of bovine {beta}-trypsin, whereas Val{sup 5}SFTI-1 is practically inactive. Also trypsin inhibitory activity of Ser{sup 5}SFTI-1 is significantly lower. Since the electrostatic interaction between protonated {epsilon}-NH{sub 2} group of the inhibitor P{sub 1} position and {beta}-carboxylate of trypsin Asp{sup 189} is the main driving force for interaction of both molecules, the results obtained are very interesting. We believe that these SFTI-1 analogues belong to a novel class of serine proteinase inhibitors.
The presence of multiple alpha,alpha-dialkyl amino acids such as alpha-methylalanine (alpha-aminoisobutyric acid, Aib) leads to predominantly helical structures, either with alpha-helical or ...3(10)-helical hydrogen bonding patterns. The crystal structure of emerimicin-(1-9) benzyl ester (Ac-Phe-Aib-Aib-Aib-Val-Gly-Leu-Aib-Aib-OBzl) reported here shows essentially pure alpha-helical character, whereas other similar compounds show predominantly 3(10)-helical structures. The factors that govern helical preference include the inherent relative stability of the alpha-helix compared with the 3(10)-helix, the extra hydrogen bond seen with 3(10)-helices, and the enhanced electrostatic dipolar interaction of the 3(10)-helix when packed in a crystalline lattice. The balance of these forces, when combined with the steric requirements of the amino acid side chains, determines the relative stability of the two helical conformations under a given set of experimental conditions.
We have used a combined chemical-enzymatic approach to facilitate the total synthesis of the 20-residue peptaibol, alamethicin. The 1-11 segment of alamethicin, having a C-terminal Gly, and the 12-20 ...segment, having an N-terminal Leu, were prepared by well-established chemical methods, and then coupled using papain to afford a 54% yield of alamethicin in straightforward fashion. In contrast to the reported chemical syntheses of alamethicin requiring side-chain protection at Glu,18 the papain-catalyzed coupling proceeded readily and selectively using a C-terminal segment having a free gamma-carboxyl group at this position. Several alamethicin partial sequences were obtained via enzymatic formation of the Gly11-Leu12 bond. The high efficiency of this route is illustrated by the enzymatic assembly of the 1-17 alamethicin fragment on a 400-mg scale in 62% yield. An alternative route to alamethicin through enzymatic formation of the Ala6-Gln7 bond was less successful because of a low yield in the final coupling.
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