Unnatural Amino Acids with Abbreviations
4.2. Experimental Section
Peptide synthesis, purification, characterization (Appendix, Figure A8-A10, A32, A50-52), MIC studies, hemolytic assay, cytotoxicity assay done according to the protocol discussed in Chapter 2.
4.2.1. Hemolytic and Bactericidal Activity Assay of VV-14 in Presence of both RBCs and Bacteria:
This experimental method is the little modified version of previously reported protocol.520 Over-night grown culture of S. typhi TY2 cells were centrifuged at 6000 rpm for 5 min and fresh human blood cell suspension was centrifuged at 8000 rpm for 10 min at 277 K. The pellets were washed three times with 10mM PBS (pH 7.4) and suspended in the same buffer to
Peptide Code Peptide Sequences
LL-14 H-LKWLKKLLKWLKKL-NH2
VV-14 H-VKWVKKVVKWVKKV-NH2
ββ-14 H-LKWβalaKKβalaLKWβalaKKβala-NH2
obtain 105 CFU/ suspension in case of bacteria and 5×109 cells/ in case of RBCs. 50 µL bacterial cell suspension and 5 µL blood cell suspension together were incubated with different concentrations of peptide VV-14 (ranging from 1-50 µM) prepared from 1mM stock, made the reaction volume to 100 µL and incubated at 310 K with shaking for 4 h. After incubation, 5 µL was taken from reaction volume, diluted in the same buffer and spread onto the NB agar plates for CFU counting after overnight incubation at 310K. The remaining volume was centrifuged and the absorbance of supernatant was measured at 414 nm using a sterile 96 well plate. For comparison, experiments were performed also with S. typhi TY2 cells and RBCs only, under the same conditions. All experiments were performed in triplicate.
4.2.2. Time Course of Bactericidal Activity Assay of VV-14:
Overnight grown culture of S. typhi TY2 was centrifuged at 6000 rpm for 5 min and the pellet was washed three times with 10 mM phosphate buffer (pH 7.4) and resuspended to obtained 105 CFU/ suspension. 50 µL of bacterial cell suspension was incubated with minimum inhibitory concentration of peptide VV-14 and incubated at 310 K for different time intervals (5-120 min). After each incubation time, 5 µl aliquot was taken from each reaction volume, diluted in the same buffer and spread onto the NB agar plates for CFU counting after overnight incubation at 310 K. All experiments were performed in triplicate.
4.2.3. Membrane Permeabilization Assay of VV-14:
To determine the outer membrane permeabalization ability of VV-14, NPN uptake assay was performed. Overnight grown culture of S. typhi TY2 cells was washed and resuspended in 10 mM sodium phosphate buffer (pH 7.4) to a final cell concentration of 106 CFU/ml. The peptide was added with increasing concentrations to a cuvette containing 1 of cells and 10 µM NPN.
The fluorescence of the dye was monitored for 10 min at room temperature and after peptide addition, increased fluorescence was monitored for 10 min using Hitachi F-7000 FL
spectrometer at excitation wavelength of 350 nm (slit width: 5 nm) and emission wavelength of 410 nm (slit width: 5 nm) to determine the NPN uptake.
Next, inner membrane permeabilization ability of VV-14 in S. typhi TY2 was examined by performing PI uptake assay as described earlier in Chapter 3, section 3.2.7.
4.2.4. Calcein Leakage Assay:
Two kinds of calcein encapsulated LUVs, mimicking anionic bacterial cell membranes and zwitterionic cell membranes were used to study calcein leakage in the presence of AMPs.
Anionic bacterial membrane and eukaryotic cell membrane mimicking model LUVs were made with the composition of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoetanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) at 3:1 ratio and 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol lipid at 6:4 ratio respectively, with a final concentration of 2 mg/. The rest of the experiment was performed as described earlier in Chapter 2 Section 2.2.8.
4.2.5. Fluorescence Spectroscopic Studies:
The intrinsic tryptophan fluorescence property of VV-14 peptide was used to analyse interaction and conformational transition of VV-14 with bacterial and eukaryotic cell membrane mimics. Intrinsic tryptophan of VV-14 was exited at 295 nm and emission spectra was recorded from 300-400 nm in presence of 10 mM phosphate buffer (pH 7.4), SDS and DPC using Hitachi F-7000 FL spectrometer at 25°C temperature. The experiments were performed at 5 µM peptide concentration and added SDS and DPC, in increasing concentrations ranging from 5-40 µM.
4.2.6. Fluorescence Anisotropy:
The binding affinity of VV-14 to SDS was determined by steady-state fluorescence anisotropy on the basis of intrinsic tryptophan fluorescence property of VV-14 peptide. The experiments were carried out using Hitachi F-7000 FL spectrometer equipped with a polariser. Initially fluorescence measurement was performed at 5 µM peptide concentration in 10 mM phosphate buffer (pH 7.4) and then SDS was added with increasing concentrations ranging from 5-40 µM.
The fluorescence anisotropy values were calculated from the equation (2)
Anisotropy (r) = (IVV-G×IVH)/(IVV+2×G×IVH) (2)
Where, IVV and IVH are the emission intensity with fixed excitation polariser at vertical orientation, when the orientation of emission polariser varied vertically and horizontally. G is the sensitivity parameter for the spectrometer (Hitachi F-7000 FL spectrometer), which is represented as G = IHV/IHH. To determine the equilibrium dissociation constant (KD) of peptide and SDS micelle, the anisotropy values were plotted against SDS concentration (µM) and fitted using the Hill equation.
4.2.7. Flied Emission Scanning Electron Microscopy (FESEM):
The effect of VV-14 on the S. typhi TY2 cells was visualized by performing FESEM as described earlier in Chapter 2, Section 2.2.13.
4.2.8. Field Emission Transmission Electron Microscopy (FETEM):
Overnight grown culture of S. typhi TY2 cells were collected by centrifugation at 6000 rpm for 5 min, washed three times and resuspended in 10 mM sodium phosphate buffer at pH 7.4 to final number of 105 cells/. The cell suspensions were incubated with VV-14 at MIC for 1 hrs.
at 310 K and untreated cells were set as the control. After incubation, 2 µL aliquot was placed on a copper grid, stained with 1% uranyl acetate and air dried.
4.2.9. CD experiment:
CD spectroscopy of all the designed AMPs were recorded in water, TFE, and different membrane mimetic environments (SDS (30 mM) and DPC (2.5 mM)) as described in Chapter 2 section 2.2.10.
4.2.10. NMR Experiments:
All NMR spectra were recorded at 37°C using either a Bruker Avance III 500 MHz spectrometer equipped with a 5 mm SMART probe or a 700 MHz spectrometer equipped with a RT probe. For SDS bound structure calculation of VV-14, 1mM peptide dissolved in 10 mM sodium phosphate buffer (pH -4.5) was used for two-dimensional (2D) 1H−1H total correlation spectroscopy (2D TOCSY) and 2D 1H−1H nuclear Overhauser effect spectroscopy (2D NOESY) with mixing times of 80 and 150 ms, respectively. The experiments were performed in absence and presence of 200 mM deuterated SDS (SDS-D25, Cambridge Isotope Laboratories, USA) to compare free and bound structures. 10% D2O was added into the tube for locking purposes and 3-(trimethylsilyl)propanoic acid (TSP) was used as an internal standard (0.0 ppm).521 The recycle delay was set to 1.5 sec and the experiments were performed with 2K (t2) x 456 (t1) increments with excitation sculpting for water suppression. States TPPI was used for quadrature detection in t1 dimension. Number of scans were 20 and 40 for TOCSY and NOESY, respectively.
Live cell NMR experiment was recorded on a Bruker Avance III 500 MHz spectrometer as described in chapter 3. Briefly, S. typhi TY2 cells were grown in Nutrient Broth containing 50 µg/ streptomycin and maintained at 310 K with shaking conditions to reach logarithmic phase (OD600=0.4 – 0.6). Then the cells were washed three times by using 10mM phosphate buffer (pH-6.5) and re-suspended in the same buffer to a final concentration of approximately 1 × 108 cells/. The suspension was then divided into two aliquots of the same volume and centrifuged for 5 min at 7000 rpm to remove the supernatant and obtain two pellets containing approximately the same number of living cells. The first pellet was re-suspended in 10 mM sodium phosphate buffer (pH 6.5) and subjected to NMR analysis in presence of 1 mM VV-14 peptide, while the second was re-suspended in the same buffer and subjected to time kinetics using agar plate method. The duration of each experiment was set around 5 min keeping the recycle delay at 1 s.
All NMR data processing and analysis were carried out using the Topspin v3.1 (Bruker Biospin, Switzerland) and SPARKY (Goddard, T. D., and Kneller, D. G. University of California, San Francisco) programs, respectively.
4.2.11. Calculation of NMR Derived Structures:
For calculation of the SDS-bound three dimensional structure of the VV-14 peptide, the volume integrals of their respective NOE cross-peaks were qualitatively differentiated into strong, medium and weak, depending on their intensities in the NOESY spectra. This information was further transformed to inter-proton upper bound distances of 3.0, 4.0 and 5.0 Å for strong, medium and weak NOEs respectively, while the lower bound distance was fixed to 2.0 Å. The dihedral angle restraint for phi (φ) angles were obtained from PREDITOR web server with the
help of Hα and 13CαH chemical shifts and the predicted torsional angles were further relaxed with ± 20° as upper and lower limits for further structure calculation while the values for psi (ψ) angles were kept flexible (120 º to -120°) for all non-glycine residues to limit the conformational space. CYANA program v2.1 was used for all structure calculations with iterative refinement of the structure based on distance violation. Hydrogen bonding constraints were excluded from structure calculation. The NMR-derived ensemble structures were analysed using PyMolTM (2.0.4, student version), chimera (1.13.1)522 and their stereochemistry was checked using Procheck.523
4.3. Results and Discussions