Antimicrobial peptides, or AMPs, refer to a heterogeneous class of compounds present in a variety of organisms, including human beings. Over the years, the compounds have been isolated and successfully characterized. They have been described in a variety of ways, based on their characteristics, structures, and activities. Mostly, they have been described as natural microbicides with selective toxicity toward bacteria and minimal cytotoxicity towards the mammalian cells of the host organism.

Their relatively strong electrostatic attraction to negatively charged bacteria cells is believed to be their main mode of operation in conjunction with their relatively low interaction with eukaryote host cells. Peptides have the ability to gather at the sites of infection. When this ability is combined with the peptides’ host cytotoxicity, they have proven to be versatile and useful regarding different interactions with a variety of bacterial cells.

Antimicrobial peptides are known to have a generally conservative structure in a variety of microorganisms. Owing to their structures, it is possible to try to determine some of their modes of operation. Under physiological conditions, most of the antimicrobial peptides are generally cationic and amphipathic. This characteristic is believed to be one of the major reasons for their ability to target cells selectively.

The ideal microbial peptide should portray low host cell toxicity, while at the same time possessing a wide range of pathogenic microbes. Also, their corresponding antimicrobial determinants should be easy to access and prone to changes or alterations. Generally, the amphipathic structure of the antimicrobial peptides makes it possible for them to interact with phospholipid membranes – structures that are predominant in nearly all pathogens.

Characteristics such as hydrophobic movements, hydrophobicity, polar angle, charge, and amphipathicity are crucial when it comes to the functioning of antimicrobial peptides. Additionally, all of these determinants are related to one another and any modification to one will have an effect on the functioning of others. Below is a detailed look at each of these crucial determinants for the functioning of antimicrobial peptides.

Conformation

It is a fact that antimicrobial peptides are present in a wide variety of microorganisms and have varying amino acid sequences. However, it is still possible to classify the antimicrobial peptides according to their secondary structures. The two largest and most common groups of antimicrobial peptides are those that portray α-helical or β-sheet secondary structures. The bulk of the rest of the antimicrobial peptides comprises those that are characterized by an unusually high proportion of one or more amino acids such as arginine and proline or tryptophan. The α-helical peptides are mostly found in the intercellular fluids of organisms such as insects and various types of amphibians.

Generally, they are known to adopt an extended or unstructured conformation when they are in an aqueous state. Upon interaction with a phospholipid membrane, it has been observed that only then do they adopt their helical structure. One possible explanation for this behavior can be that the intramolecular hydrogen bond necessary for α-helical conformation is usually disrupted when the peptide is exposed to polar solvents such as water. When interacting with a membrane, the polar hydrogen groups are normally shielded from the lipophilic membrane environments through the α-helical formation. Through this conformation, it is also possible to expose the apolar side chains to the neutral lipid environment right inside the membrane. In as much as the primary structure of the β-sheet group of antimicrobial peptides are slightly different from the amino acid sequence structures, when it comes to the amphipathic structures, they tend to share common features including very clear hydrophobic and hydrophilic properties.

Charge (Q)

It is worth noting that most of the antimicrobial peptides are generally cationic, with charges ranging from +2 to +9. It is also worth pointing out that the majority of these cationic antimicrobial peptides have highly defined negatively charged domains. The positive charge is vital for the first initial attraction as well as interaction with the anionic cellular membrane of the bacteria as well as other pathogenic microorganisms. In the same manner, the membranes of the host are less anionic and this implies that it does not electrostatically attract the antimicrobial peptides. As such, it may also have some cell selectivity capabilities regarding the peptides.

Pathogenic bacteria are known to have a very high concentration of acidic phospholipids such as PS, PG, and CL.

Also, the teichuronic and teichoic of the cell walls of gram-positive bacteria as well as the LPS of gram-negative bacteria offer additional insights into the electronegative charge of the bacterial cell surface. Through several studies, it has been established that the net charge of most bacteria is normally 50% higher than the net charge of normal mammalian cells. Due to this, there is a general consensus that the antimicrobial peptides may have a relatively higher concentration on the surface of the pathogenic microbes in an electrophoretic nature. Although it is not yet possible through studies to correlate the cationicity of the antimicrobial peptides to their antimicrobial activities, there isn’t any strict linear relationship that exists between the two.

In a study conducted by Darth and coworkers using analogs of magainin, it was observed that when the cationicity was increased from +3 to +5, there was a corresponding increase in the level of antimicrobial activities against both the gram-negative and gram-positive species. The studies, however, noted that the cationicity was limited since any increase in the positive charge didn’t come with any corresponding increase in the level of the antibacterial activities. Through the studies, it was concluded that the decline in the antimicrobial activities with a corresponding increase in the charge was a result of the peptides binding so strongly to the negatively charged phospholipids head group which then makes it impossible for the peptides to translocate into the cells.

Hydrophobicity and Amphipathicity

The amphipathicity of antimicrobial peptides is considered almost a universal feature and it is made possible through the presence of various peptide structures. The amphipathic α-helical structure of the peptides is believed to be one of the most common and simplest forms of these features. When the cationic and anionic amino residues are changed at every three to four positions of the peptides, then it becomes possible to create a secondary structure that can allow for optimal electrostatic interactions if the antimicrobial peptides are exposed to amphipathic phospholipid membranes. Through these features, it becomes possible for the antimicrobial peptides to display cytotoxic activities against negatively charged cell membranes and neutral membranes or membranes that are amphipathic in nature.

Amphipathicity

Amphipathicity of a peptide refers to its hydrophobic moment which, in turn, refers to the vectorial sum of individual amino acid hydrophobicities, then normalized to an ideal helix. Whenever there is an increase in the hydrophobic moments, there is a corresponding increase in the permeabilization of the membranes of the target cells.

This is an immensely important factor when it comes to the interactions with the lipid membranes that are usually neutrally charged. The reason for this is that the charge factors are likely to bring any significant changes necessary for the desired attraction and interaction with the target cell membranes. Just like the α-helical structures of the antimicrobial peptides, the β-sheet host defenses peptides also show very strong indications of amphipathicity. This has been confirmed with the presence of the varying β-sheet which has been carefully organized to form both hydrophilic and hydrophobic surfaces.

The β-sheets, which are normally arranged in an anti-parallel manner, get their stabilization through the regularly spaced disulfide bonds or through the cyclization process of the peptide backbone. Through this intramolecular bonding, it becomes possible for the β-sheet antimicrobial peptides to adopt a rigid conformation even when they are in contact with aqueous fluid. It also makes it possible for the β-sheet antimicrobial peptides to undergo multimerization since the hydrophobic surfaces will come together so that they are not exposed to the aqueous environment.

The mechanism through which these antimicrobial peptides bring about cell membrane disruption is not yet understood primarily because it is not yet known how exactly they conform to the cell membranes, however there are several studies to suggest that segregated amphipathicity of the antimicrobial peptides have a huge effect on the specific mechanisms used by the peptides to disrupt the natural biomembranes.

Hydrophobicity

The hydrophobicity of a peptide refers to the percentage of the hydrophobic amino acid residues that are present in the primary structure of the entire peptide. Most antimicrobial peptides usually have a hydrophobicity of about 50%. This is vital for their normal functioning since it is the feature that makes it possible for the peptides to interact with and also penetrate the phospholipid bilayer. Although antimicrobial peptides need to have a certain amount of hydrophobicity, too much or excessive hydrophobicity comes with an increased likelihood of destruction of the host’s cells in addition to reducing the specificity of the microbial cells.

In a study conducted by Wieprecht et al. regarding the relationship that exists between hydrophobicity of the peptides and the peptide’s ability or inability to permeate biomembranes, it was concluded that the hydrophobicity had very little effect on the peptide’s ability to bind or permeate the cell membranes when the membranes consisted of exclusively PG. The study was done using magainin analogs as the model antimicrobial peptides. During the studies, the model antimicrobial peptides were able to keep factors such as helicity, the charge, and the hydrophobic moments nearly constant. However, when the membranes had a ratio of 3:1 for PC: PG, it was observed that the peptides that had the highest hydrophobicity had permeability approximately sixty times higher than the least hydrophobic peptide. Also, the study showed that for membranes that had only PC, the difference in the permeability was 300 times.

Polar Angle

The polar angle of an antimicrobial peptide refers to the relative proportion of the polar to the non-polar facets of the peptide conformed to an amphipathic helix. A helical peptide comprising just a single facet that is entirely composed of polar amino acid residues and the other facets comprising entirely a non-polar amino acid residue would portray a polar angle of 1800. In instances where there is less segregation between the domains or there exists an oversupply of hydrophobic residues, there is a higher likelihood that a lower polar angle would be witnessed in that antimicrobial peptide structure.

In a study conducted by Matsuzaki and Uematsu on naturally occurring peptides and synthetic peptides, it was observed that both the peptides have a lower polar angle and as such, they had their more hydrophobic facet rendered more conductive to membrane permeabilization. Correlations have also been drawn between polar angle and the ability of antimicrobial peptides to induce pores in biomembranes. The same study also concluded that some of the antimicrobial peptides that generally have smaller polar angles had the ability to induce higher degrees of membrane permeabilization. Additionally, the peptides could allow for higher degrees of translocation compared to the peptides that had relatively bigger polar angles.

But, it should be noted that the pores which are formed by the peptides during the process of membrane permeabilization with peptides that have small polar angles tend to be less stable than the pores formed by antimicrobial peptides with bigger polar angles. It is also worth noting that both hydrophilic and hydrophobic properties of the antimicrobial peptides are also acutely evident in the interactions with and the permeabilization of phospholipid cell membranes.

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