Basta Anastasia- CD spectroscopy

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Applications of Circular Dichroism for Structural Analysis of Gelatin and Antimicrobial PeptidesRamamourthy Gopal , Jin Soon Park, Chang Ho Seo and Yoonkyung ParInt. J. Mol. Sci. 2012, 13, 3229-3244; doi:10.3390/ijms13033229

Abstract: Circular dichroism (CD) is a useful technique for monitoring changes in the conformation of antimicrobial peptides or gelatin. In this study, interactions between cationic peptides and gelatin were observed without affecting the triple helical content of the gelatin, which was more strongly affected by anionic surfactant. The peptides did not adopt a secondary structure in the presence of aqueous solution or Tween 80, but a peptide secondary structure formed upon the addition of sodium dodecyl sulfate (SDS). The peptides bound to the phosphate group of lipopolysaccharide (LPS) and displayed an alpha-helical conformation while (KW)4 adopted a folded conformation. Further, the peptides did not specifically interact with the fungal cell wall components of mannan or laminarin. Tryptophan blue shift assay indicated that these peptides interacted with SDS, LPS, and gelatin but not with Tween 80, mannan, or laminarin. The peptides also displayed antibacterial activity against P. aeruginosa without cytotoxicity against HaCaT cells at MIC, except for HPA3NT3-analog peptide. In this study, we used a CD spectroscopic method to demonstrate the feasibility of peptide characterization in numerous environments. The CD method can thus be used as a screening method of gelatin-peptide interactions for use in wound healing applications. , ... : 49 1

Figure 1. Effects of peptides on Circular dichroism (CD) spectra of gelatin in the wavelength region from 250190 nm. Gelatin solution was treated with peptides at an increasing molar ratio of gelatin to peptide from 11:1 to 11:3 and incubated for 10 min at 25 C. (A) (KW)4; (B) HPA3NT3-analog; (C) NRC-16; (D) Magainin-II; and (E) Reduced glutathione (GSH). CD spectra of 0.1% gelatin in the absence of peptides (solid line). Peptide concentrations were as follows: 50 M (dotted line), 100 M (dashed line), and 200 M (dashed-dotted line). (F) CD spectra of gelatin in the absence (solid line) or presence of 1 mM SDS (dotted line), 5 mM SDS (dashed line), and 10 mM SDS (dashed dotted line). , ... : 50

Figure 2. CD spectra of peptides in aqueous solution (dashed line) as well as in the presence of 0.1% Tween 80 (dotted line) and 30 mM sodium dodecyl sulfate (SDS) (solid line). (A) (KW)4; (B) HPA3NT3-analog; (C) NRC-16; and (D) Magainin-II. , ... : 51

Figure 3. CD spectra of peptides in the presence of 0.1% mannan (dashed line), 0.1% laminarin (dotted line), and 0.1% lipopolysaccharide (LPS) (solid line). (A) (KW)4; (B) HPA3NT3-analog; (C) NRC-16; and (D) Magainin-II. , ... : 52

ConclusionThe native structure of gelatin was not altered upon treatment with peptides. Therefore, changes in the structural properties of gelatin upon interaction with peptides assist the preparation of gelatin-peptide sheets. In this study, the tested peptides showed antimicrobial activity, which suggests that they might be useful in the development of a topical application for wound sites. Among them, (KW)4 possesses a simple composition with microbial selective properties, making it economically viable for many applications, including inhibition of bacterial infection at wound sites, ... : 53

, ... : 54Monitoring protein aggregation during thermal unfolding in circular dichroism experimentsSangeeta Benjwal,1,3 Shikha Verma,2,3 Klaus-Heinrich Rhm,2 and Olga GurskyProtein Sci. 2006 Mar; 15(3): 635639. doi: 10.1110/ps.051917406Abstract: Thermal unfolding monitored by spectroscopy or calorimetry is widely used to determine protein stability. Equilibrium thermodynamic analysis of such unfolding is often hampered by its irreversibility, which usually results from aggregation of thermally denatured protein. In addition, heat-induced protein misfolding and aggregation often lead to formation of amyloid-like structures. We propose a convenient method to monitor in real time protein aggregation during thermal folding/ unfolding transition by recording turbidity or 90 light scattering data in circular dichro