Vibrational spectroscopy for characterisation of a cyclic antimicrobial peptide

Pfukwa, Ngaatendwe Buhle Cathrine (2021-12)

Thesis (PhD)--Stellenbosch University, 2021.

Thesis

ENGLISH ABSTRACT: In order to understand antimicrobial peptide action towards membrane surfaces it is imperative to first understand the structure and conformational behaviour of the antimicrobial in solution environments representative of the target membrane. Information on conformational behaviour can be extracted in more detail from analysis of the peptide secondary structure using the structural sensitivity afforded by two dimensional infrared (2D-IR) spectroscopy with which subtle details of peptide structure, which may not be well resolved with one dimensional infrared (1D-IR) spectroscopy, can be obtained. With 2D-IR spectroscopy spectral content is spread over two frequency axes and the high time resolution on femtosecond and picosecond timescales for transient processes provides an advantage over conventional techniques such as X-ray diffraction, circular dichroism (CD) or Ultraviolet spectroscopy. In this study the aims were to evaluate the adaptability of two-dimensional infrared (2D-IR) spectroscopy, together with multivariate data analysis methods such as principal component analysis (PCA), to provide and extract spectral details which can be correlated to changes in structure of a cationic antimicrobial peptide (CAMP) gramicidin S (GS). GS was selected as prototype as it is a well characterised anti-parallel β-sheet peptide, active by disrupting bacterial membranes, consequently causing membrane penetration. Furthermore, a qualitative working relationship between 1D and 2D IR and Raman spectroscopy in corroboration with quantum mechanical (QM) simulations was established towards correlating GS spectral features to conformational secondary structural changes. The structural changes were used to elucidate the conformational behaviour of GS in selected solvent environments which are representative of GS target membrane lipid bilayers. The solvent environments were an aqueous ubiquitous environment (H2O/D2O), a membrane mimetic partially polar environment (1-octanol) and a membrane mimetic strongly H-bonded environment (TFE). Self-association of GS was promoted in H2O/D2O and 1-octanol forming aggregates which persisted in solution at increasing peptide concentration, as revealed from increase in the β-sheet content and loss in β-turns. While in TFE, a strong H-bonding solvent, the GS aggregates were molecularly solvated. These changes were correlated to occur in the residues which contribute greatly to the amphiphilic nature of GS. Further solvent effects on the structural factors contributing to GS bioactivity are discussed. The thermostability of GS was investigated for the temperature range 20-80 ℃. The dissociation of GS aggregates/small oligomers to monomeric structures in 1-octanol was favoured at elevated temperatures. Thermodynamic parameters were extracted and the dissociation process was described by a bimodal profile with two identified melting transitions at Tm1 = 45 oC and Tm2 = 57 oC. Results obtained confirmed that both IR and Raman can provide complementary results as evidenced by their sensitivity towards hydrophilic and hydrophobic structures is GS. Further providing insight towards the type of residue substituents which can be interchanged in future synthesis of GS derivatives, with the aim of increasing the bioactivity of GS whilst lowering its haemolytic effects. The significance of SERS using silver nanoparticles in detection of low peptide concentrations is reported and a GS concentration of 1x10-5 M was detected. Information in thesis demonstrates the novelty and broad use of 1D and 2D IR, RS and SERS techniques in understanding the solvent and temperature induced conformational changes which occur in GS, which provides information on GS structure towards generation of more bioactive GS derivatives for therapeutic purposes.

AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/123623
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