Abstract:Our efforts on the synthesis of peptides with well-characterized secondary structures, combined with detailed spectroscopic investigations, most of them performed in collaboration with internationally recognized experts, have allowed us to publish the electronic (electronic circular dichroism) and vibrational (FTIR absorption, vibrational circular dichroism, Raman, and Raman optical activity) signatures of the poorly studied peptide 3(10)-helix (and the related β-bend ribbon spiral conformation as well) in com… Show more
“…Furthermore, the ellipticity ratio of R = 0.59 in 20-50 mM SDS (Table I) observed in the CD spectra indicated unique secondary structural contributions that deviate from canonical a helices. This result suggested that the rSpTrf-E1 was not a fully classical a helix in SDS (see Materials and Methods) (32,38). Overall, the observed conformational changes in SpTrf-E1 as the concentration of SDS was increased from 0 to 30 mM indicated that it transformed from disordered to 79% a helical structure (Fig.…”
Section: Resultsmentioning
confidence: 85%
“…CD spectra were used to calculate the mean residue ellipticity, or u, with standard units of degrees (deg) 3 cm 2 3 dmol 21 . The helical character was calculated using the ellipticity ratio (R = u 222 /u 207 ) between the two negative peaks at 222 nm (n → p* electronic transition of carbonyl compound) and 207 nm (parallel component of the split p → p* electron transition of the protein chromophore) (32,38). For a protein of known concentration and amino acid sequence that is 100% a helical, the ellipticity ratio is R = 1 and this is interpreted as 3.6 residues per turn positioned at an angle of 100˚between sequential amino acids around the helical axis.…”
Section: Circular Dichroismmentioning
confidence: 99%
“…SDS is an anionic detergent commonly used to simulate the interactions between anionic lipid membranes and amphipathic cationic peptides, as well as conformational changes that the peptides may undergo as a result of these interactions (35). TFE is often used as a cosolvent with peptides or proteins in solution to promote secondary structures of a helices and b strands (36)(37)(38). LPS, an endotoxin from Gramnegative bacteria, is bound specifically and tightly by rSp0032 (22).…”
The purple sea urchin, , expresses a diverse immune response protein family called Sp185/333. A recombinant Sp185/333 protein, previously called rSp0032, shows multitasking antipathogen binding ability, suggesting that the protein family mediates a flexible and effective immune response to multiple foreign cells. Bioinformatic analysis predicts that rSp0032 is intrinsically disordered, and its multiple binding characteristic suggests structural flexibility to adopt different conformations depending on the characteristics of the target. To address the flexibility and structural shifting hypothesis, circular dichroism analysis of rSp0032 suggests that it transforms from disordered (random coil) to α helical structure. This structural transformation may be the basis for the strong affinity between rSp0032 and several pathogen-associated molecular patterns. The N-terminal Gly-rich fragment of rSp0032 and the C-terminal His-rich fragment show unique transformations by either intensifying the α helical structure or changing from α helical to β strand depending on the solvents and molecules added to the buffer. Based on these results, we propose a name change from rSp0032 to rSpTransformer-E1 to represent its flexible structural conformations and its E1 element pattern. Given that rSpTransformer-E1 shifts its conformation in the presence of solvents and binding targets and that all Sp185/333 proteins are predicted to be disordered, many or all of these proteins may undergo structural transformation to enable multitasking binding activity toward a wide range of targets. Consequently, we also propose an overarching name change for the entire family from Sp185/333 proteins to SpTransformer proteins.
“…Furthermore, the ellipticity ratio of R = 0.59 in 20-50 mM SDS (Table I) observed in the CD spectra indicated unique secondary structural contributions that deviate from canonical a helices. This result suggested that the rSpTrf-E1 was not a fully classical a helix in SDS (see Materials and Methods) (32,38). Overall, the observed conformational changes in SpTrf-E1 as the concentration of SDS was increased from 0 to 30 mM indicated that it transformed from disordered to 79% a helical structure (Fig.…”
Section: Resultsmentioning
confidence: 85%
“…CD spectra were used to calculate the mean residue ellipticity, or u, with standard units of degrees (deg) 3 cm 2 3 dmol 21 . The helical character was calculated using the ellipticity ratio (R = u 222 /u 207 ) between the two negative peaks at 222 nm (n → p* electronic transition of carbonyl compound) and 207 nm (parallel component of the split p → p* electron transition of the protein chromophore) (32,38). For a protein of known concentration and amino acid sequence that is 100% a helical, the ellipticity ratio is R = 1 and this is interpreted as 3.6 residues per turn positioned at an angle of 100˚between sequential amino acids around the helical axis.…”
Section: Circular Dichroismmentioning
confidence: 99%
“…SDS is an anionic detergent commonly used to simulate the interactions between anionic lipid membranes and amphipathic cationic peptides, as well as conformational changes that the peptides may undergo as a result of these interactions (35). TFE is often used as a cosolvent with peptides or proteins in solution to promote secondary structures of a helices and b strands (36)(37)(38). LPS, an endotoxin from Gramnegative bacteria, is bound specifically and tightly by rSp0032 (22).…”
The purple sea urchin, , expresses a diverse immune response protein family called Sp185/333. A recombinant Sp185/333 protein, previously called rSp0032, shows multitasking antipathogen binding ability, suggesting that the protein family mediates a flexible and effective immune response to multiple foreign cells. Bioinformatic analysis predicts that rSp0032 is intrinsically disordered, and its multiple binding characteristic suggests structural flexibility to adopt different conformations depending on the characteristics of the target. To address the flexibility and structural shifting hypothesis, circular dichroism analysis of rSp0032 suggests that it transforms from disordered (random coil) to α helical structure. This structural transformation may be the basis for the strong affinity between rSp0032 and several pathogen-associated molecular patterns. The N-terminal Gly-rich fragment of rSp0032 and the C-terminal His-rich fragment show unique transformations by either intensifying the α helical structure or changing from α helical to β strand depending on the solvents and molecules added to the buffer. Based on these results, we propose a name change from rSp0032 to rSpTransformer-E1 to represent its flexible structural conformations and its E1 element pattern. Given that rSpTransformer-E1 shifts its conformation in the presence of solvents and binding targets and that all Sp185/333 proteins are predicted to be disordered, many or all of these proteins may undergo structural transformation to enable multitasking binding activity toward a wide range of targets. Consequently, we also propose an overarching name change for the entire family from Sp185/333 proteins to SpTransformer proteins.
“…22 The detergent SDS is able to form micelles that have a negatively charged surface, 23 which mimics the bacterial membrane and forces the CAMP into a more ordered conformation such as a helix. 24, 25 TFE is used in CD to promote a helical structure 26 and stabilize secondary structure. 27
Fig.…”
Cationic antimicrobial peptides are multifunctional molecules that have a high potential as therapeutic agents. We have identified a histone H1-derived peptide from the Komodo dragon (Varanus komodoensis), called VK25. Using this peptide as inspiration, we designed a synthetic peptide called DRGN-1. We evaluated the antimicrobial and anti-biofilm activity of both peptides against Pseudomonas aeruginosa and Staphylococcus aureus. DRGN-1, more than VK25, exhibited potent antimicrobial and anti-biofilm activity, and permeabilized bacterial membranes. Wound healing was significantly enhanced by DRGN-1 in both uninfected and mixed biofilm (Pseudomonas aeruginosa and Staphylococcus aureus)-infected murine wounds. In a scratch wound closure assay used to elucidate the wound healing mechanism, the peptide promoted the migration of HEKa keratinocyte cells, which was inhibited by mitomycin C (proliferation inhibitor) and AG1478 (epidermal growth factor receptor inhibitor). DRGN-1 also activated the EGFR-STAT1/3 pathway. Thus, DRGN-1 is a candidate for use as a topical wound treatment. Wound infections are a major concern; made increasingly complicated by the emerging, rapid spread of bacterial resistance. The novel synthetic peptide DRGN-1 (inspired by a peptide identified from Komodo dragon) exhibits pathogen-directed and host-directed activities in promoting the clearance and healing of polymicrobial (Pseudomonas aeruginosa & Staphylococcus aureus) biofilm infected wounds. The effectiveness of this peptide cannot be attributed solely to its ability to act upon the bacteria and disrupt the biofilm, but also reflects the peptide’s ability to promsote keratinocyte migration. When applied in a murine model, infected wounds treated with DRGN-1 healed significantly faster than did untreated wounds, or wounds treated with other peptides. The host-directed mechanism of action was determined to be via the EGFR-STAT1/3 pathway. The pathogen-directed mechanism of action was determined to be via anti-biofilm activity and antibacterial activity through membrane permeabilization. This novel peptide may have potential as a future therapeutic for treating infected wounds.
“…Characteristic changes in the differences in absorption of circularly polarized light by the backbone amide bonds, result in secondary structures, such as α‐helix, β‐sheet and random coil, being associated with unique CD spectral properties . For instance, α‐helices present CD spectra with strong negative band peaks at 222nm and 208nm, corresponding to the n → π* and the parallel component of the split π → π* electronic transitions, respectively . For a peptide with 100% α‐helix structure, the ratio of the signals at 222 nm and 208 nm, adjusted for concentration and the number of residues (mean residue ellipticity, [ θ ]), has been experimentally determined to be unity, R = 1 .…”
Cationic antimicrobial peptides (CAMPs) represent an ancient defense mechanism against invading bacteria, with peptides such as the cathelicidins being essential elements of vertebrate innate immunity. CAMPs are typically associated with broad-spectrum antimicrobial potency and limited bacterial resistance. The cathelicidin identified from the elapid snake Naja atra (NA-CATH) contains a semi-conserved repeated 11-residue motif (ATRA motif) with a sequence pattern consistent with formation of an amphipathic helical conformation. Short peptide amides (ATRA-1, -1A, -1P, and -2) generated based on the pair of ATRA motifs in NA-CATH exhibited varied antimicrobial potencies. The small size of the ATRA peptides, coupled with their varied antimicrobial performances, make them interesting models to study the impact various physico-chemical properties have on antimicrobial performance in helical CAMPs. Accordingly, the D- and L-enantiomers of the peptide ATRA-1A, which in earlier studies had shown both good antimicrobial performance and strong helical character, were investigated in order to assess the impact peptide stereochemistry has on antimicrobial performance and interaction with chiral membranes. The ATRA-1A isomers exhibit varied potencies against four bacterial strains, and their conformational properties in the presence of mixed zwitterionic/anionic liposomes are influenced by anionic lipid content. These studies reveal subtle differences in the properties of the peptide isomers. Differences are also seen in the abilities of the ATRA-1A isomers to induce liposome fusion/aggregation, bilayer rearrangement and lysing through turbidity studies and fluorescence microscopy. The similarities and differences in the properties of the ATRA-1A isomers could aid in efforts to develop D-peptide-based therapeutics using high-performing L-peptides as templates.
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