Abstract:Cationic amphiphilic peptides have been engineered to target both Gram-positive and Gram-negative bacteria while avoiding damage to other cell types. However, the exact mechanism of how these peptides target, bind, and disrupt bacterial cell membranes is not understood. One specific peptide that has been engineered to selectively capture bacteria is WLBU2 (sequence: RRWVRRVRRWVRRVVRVVRRWVRR). It has been suggested that WLBU2 activity stems from the fact that when interacting with bacterial cell membranes the p… Show more
“…As a comparison to literature studies, Golbek et al added WLBU2 to the subphase of a condensed phase of DPPC monolayers at room temperature and used sum frequency generation spectroscopy to find an increase in chain order due to WLBU2. 70 However, given the lack of cholesterol and PE and different experimental conditions, this comparison may not be meaningful.…”
“…As a comparison to literature studies, Golbek et al added WLBU2 to the subphase of a condensed phase of DPPC monolayers at room temperature and used sum frequency generation spectroscopy to find an increase in chain order due to WLBU2. 70 However, given the lack of cholesterol and PE and different experimental conditions, this comparison may not be meaningful.…”
“…Recently, nonlinear surface spectroscopy techniques including SFG have emerged as a methodology for directly probing protein-membrane interactions in a label-free manner (14)(15)(16)(17). SFG is a second order nonlinear optical technique, involving a visible beam that is pulsed in temporal and spatial synchronicity with an infrared laser.…”
Proteins that contain C2 domains are involved in a variety of biological processes, including encoding of sound, cell signaling, and cell membrane repair. Of particular importance is the interface activity of the C-terminal C2F domain of otoferlin due to the pathological mutations known to significantly disrupt the protein's lipid membrane interface binding activity, resulting in hearing loss. Therefore, there is a critical need to define the geometry and positions of functionally important sites and structures at the otoferlin-lipid membrane interface. Here, we describe the first in situ probe of the protein orientation of otoferlin's C2F domain interacting with a cell membrane surface. To identify this protein's orientation at the lipid interface, we applied sum frequency generation (SFG) vibrational spectroscopy and coupled it with simulated SFG spectra to observe and quantify the otoferlin C2F domain interacting with model lipid membranes. A model cell membrane was built with equal amounts of phosphatidylserine and phosphatidylcholine. SFG measurements of the lipids that make up the model membrane indicate a 62% increase in amplitude from the SFG signal near 2075 cm À1 upon protein interaction, suggesting domain-induced changes in the orientation of the lipids and possible membrane curvature. This increase is related to lipid ordering caused by the docking interaction of the otoferlin C2F domain. SFG spectra taken from the amide-I region contain features near 1630 and 1670 cm À1 related to the C2F domains beta-sandwich secondary structure, thus indicating that the domain binds in a specific orientation. By mapping the simulated SFG spectra to the experimentally collected SFG spectra, we found the C2F domain of otoferlin orients 22 normal to the lipid surface. This information allows us to map what portion of the domain directly interacts with the lipid membrane.
“…The vibrational spectrum of C〓O groups from the amide backbone group can provide the secondary structure of the peptides. Consequently, vibrations from acyl chains of the lipid bilayer can provide important information about the interaction of peptide with the membrane as well as information about the molecular structure of the peptide, without the use of vesicles and labels to complicate the process of analysis [50].…”
Defensins are naturally occurring antimicrobial peptides secreted in the human body. Mammalian defensins are small, cysteine-rich, cationic peptides, generally consisting of 18-45 amino acids. The antimicrobial activity of defensins arises from their unique amino acid sequence, showing activity against both Gram-positive and Gram-negative bacteria, fungi and enveloped viruses. The use of antimicrobial peptides is rising due to their potential to control biofilm formation and kill microorganisms that are highly tolerant to antibiotics. In free-form, defensins are capable of destroying such microorganisms through numerous mechanisms mainly the carpet, the toroidal and the Barrel-Stave models. However, immobilization of antimicrobial peptides (AMPs) on surfaces with the help of coupling agents and spacers can improve the AMPs' lifespan and stability in the physiological environment leading to applications for medical devices and implants. Fundamental understanding of both free-form and surface-immobilized defensins is important to design more effective antimicrobial peptides and improve their performance in future developments.
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