MSI-103 was found to have a higher antimicrobial activity than the parent peptide, while hemolytic side effects were reduced 3, 7. The full-length peptide was optimized by simplifying the sequence to contain only four types of amino acid and by increasing the positive charge while maintaining the overall amphipathic character 3. This heptameric motif was based on the sequence of PGLa, a member of the magainin family of antimicrobial peptides from the African frog Xenopus laevis 6. One approach is to modify natural sequences, while another strategy is based on the design of amphipathic sequences from scratch, as in the case of MSI-103 with the regular repeat (KIAGKIA) 3-NH 2 3. Besides exploring natural AMPs from a wide variety of organisms, much effort has also been spent to obtain new peptides with improved activities. Linear cationic amphipathic α-helices are the most common AMPs and also have the widest antimicrobial activity spectrum, some well-known examples being magainins from frogs 4 and LL-37 from humans 5. Over 2000 AMPs are known 2 and can be classified according to origin, activity or structure 1, 2, 3. Membrane-active antimicrobial peptides (AMPs) are found in almost all types of organisms and constitute a host defense system against microorganisms 1. By correlating the threshold lengths for biological activity with the biophysical results on model vesicles, the peptides could be utilized as molecular rulers to measure the membrane thickness in different cells. These data show that the formation of transmembrane pores is only possible under the condition of hydrophobic matching: the peptides have to be long enough to span the hydrophobic bilayer core to be able to induce vesicle leakage, kill bacteria and cause hemolysis. Biological assays for antimicrobial activity and hemolysis, as well as fluorescence vesicle leakage and solid-state NMR spectroscopy, were used to correlate peptide length with membranolytic activity. Nine peptides with lengths between 14 and 28 amino acids were designed from repeated KIAGKIA motifs and their helical nature was confirmed by circular dichroism spectroscopy. This concept was extended here to amphipathic membranolytic α-helices. None of the several internal hydrophobic regions of cytochrome P450, previously proposed as membrane spanning, function as a stop-transfer signal.Hydrophobic mismatch is a well-recognized principle in the interaction of transmembrane proteins with lipid bilayers. These results demonstrate that cytochrome P450, which is normally localized on the cytoplasmic side of the membrane, can be entirely translocated to the luminal side when two basic amino acids precede the hydrophobic core of its NH2-terminal insertion/stop-transfer signal. Since cleavage in the hybrid protein occurred after glycine 25, which is derived from P450IIC2, cytochrome P450 sequences COOH terminal to the cleavage site must decrease cleavage efficiency. In contrast to the proteolytic processing observed previously in a hybrid P450IIC2/parathyroid hormone protein, little or no cleavage of the NH2-terminal peptide of P450IIC2 was observed in the presence of membranes.
Similar results were obtained for a truncated species, P450IIC2(1-55), except that only a single glycosylated species was observed, consistent with the single remaining glycosylation site. Both the glycosylated and nonglycosylated forms of P450IIC2 were resistant to proteolytic digestion and to extraction from the membranes by alkaline solutions. After treatment with endoglycosidase H, the more slowly migrating species comigrated with P450IIC2 synthesized in the absence of membranes, indicating that the proteins had been glycosylated. In contrast, when -P450IIC2 was synthesized in the presence of membranes, two new species migrating more slowly during gel electrophoresis were observed. Cytochrome P450IIC2, synthesized in the presence of chicken oviduct microsomal membranes, was resistant to extraction by alkaline solutions, but was sensitive to proteolytic digestion. Insertion of rabbit cytochrome P450IIC2 and its modified form, P450IIC2, into microsomal membranes was studied in an in vitro transcription/translation/translocation system.