Effect of tacticity-derived topological constraints in bactericidal peptides
5.3 Results and Discussion
5.3.1 Modeling, simulation and electrostatic profiling
As described earlier, a 12 amino acid long polypeptide can have 4096 stereochemical variants and poly-L is only one among them. Since it is impossible to evaluate all 4096 stereochemical variants and its sequence variants, we opted to exhaustively evaluate the next possible variant of chiral variation; an alternating LDLD sequence.
LDLD sequence naturally adopts a structure similar to gramicidin helix. Depending on the handedness (right or left) and sequence, eight LDLD variant sequences are theoretically possible (Figure 5.1). Handedness is a result of differential phi and psi angle combinations, a particular syndiotactic backbone adopts, while folding to a given structure. Since we don’t have any control on the handedness of the synthesized peptide, out of the eight topological variants, only four can be
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Topology and Bactericidal Potency synthetically achieved; LDLD and DLDL stereochemical sequences with a given sequence and sequence reversed (Table 5.1).
Table 5.1. Amino acid sequence and stereochemical sequence of designed and synthesized model molecular systems 1 to 6 for biophysical studies and bactericidal activity. Number of clusters resulted from 10 ns Molecular Dynamics simulations, indicating the number of microstates at approximate equilibrium. Syndiotactic sequences are far more rigid; largely retain the designed topology, whereas poly-L sequence can adopt a range of degenerate conformational states. Small letter in the sequence indicates D-amino acid.
Model System (MS) Amino Acid Sequence Stereochemical sequence Number of clusters
MS1 KrKiFlRtKiLv LDLDLDLDLDLD 5
MS2 kRkIfLrTkIlV DLDLDLDLDLDL 1
MS3 vLiKtRlFiKrK DLDLDLDLDLDL 1
MS4 VlIkTrLfIkRk LDLDLDLDLDLD 3
MS5 KRKIFLRTKILV LLLLLLLLLLLL 22
MS6 VLIKTRLFIKRK LLLLLLLLLLLL 74
More precisely, compared to model system 1 (MS1), model system 2 (MS2) has its stereochemistry reversed and model system 4 (MS4) is sequence-reversed, model system 3 (MS3) is both stereochemically- and sequence-reversed. Model systems 5 (MS5) and 6 (MS6) are poly L (isotactic) variants of MS1 and MS3 to estimate their activity against the bacterial species (Table 5.1). More precisely, compared to model system 1 (MS1), model system 2 (MS2) has its stereochemistry reversed and model system 4 (MS4) is sequence-reversed, model system 3 (MS3) is both stereochemically- and sequence-reversed. Model systems 5 (MS5) and 6 (MS6) are poly L (isotactic) variants of MS1 and MS3 to estimate their activity against the bacterial species (Table 5.1).
The relative difference between two molecular systems in any of these six peptides are different either in sequence or stereochemistry or both; with invariable cationicity and amphipathicity in order to investigate specific role that stereochemistry imparts, manifested through their topology during membrane
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59 Figure 5.1. Possible backbone structures for syndiotactic peptides. The left and right handed conformations of designed syndiotactic peptides MS1 (A, B), MS3 (C, D), MS2 (E, F) and MS4 (G, H).
activity. Several models have been proposed to mechanistically explain membranolytic activity that principally arise from amphipathicity and cationicity of interacting molecule. Pore forming and non-pore forming mechanistic models, suggested for peptide-based membrane activity is essentially manifested through electrostatic interactions of amphipathic cationic peptide and its oppositely charged membrane counterpart. We use the finite difference Poisson-Boltzmann method as implemented in DelPhi 283, 284, to compute the electrostatic potential of all designed sequences on all theoretical possibilities of an alternating LDLD sequence, both in terms of its handedness and diastereomeric sequence.
Electrostatic cum topology based molecular fingerprints of all eight variants, theoretically possible with our designed systems were examined (Figure 5.2).
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Figure 5.2. Electrostatic potential maps of left handed (LH) and right handed (RH) conformations of designed syndiotactic peptides respectively. MS1 (A, B) and MS2 (C, D) along with MS3 (E, F) and MS4 (G, H). Electrostatic potential was calculated by solving finite difference Poisson-Boltzmann equation using Delphi software, summed for each amino acid side-chain and represented collectively at the chromophoric center of the side-chain. The backbone corresponding to its electrostatic profile with amphiphilic character has been shown (color zone) at the top right of each electrostatic profile map.
The N and C terminus for all the figures of electrostatic profile as well as backbone has been shown in this figure. The color scheme represents the distribution of potential in KT/e units.
The spatial disposition with charged and neutral groups in a peptide system was found to be distinct and unique in each case, while the syndiotactic polypeptide chain assumes its most stable gramicidin helical conformation. Since handedness in design cannot be implemented on synthetic sequences, our sample set will be reduced to four LDLD, DLDL, LDLD-sequence reversed and DLDL-sequence reversed. Their potencies are compared with both isotactic poly-L sequences thus forming a complete set of designed test systems to study the combined effect of topology and electrostatics of peptidic chromophoric groups on Gram-positive, Gram-negative and antibiotic resistant bacteria. Syndiotactic peptides are
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61 Figure 5.3. Position wise electrostatic potential distribution. The electrostatic potential was calculated by solving finite distance Poisson-Boltzmann equation using DelPhi. All potentials are in kT/e units.
amphiphilic by design, as can be verified from their electrostatic potential maps (Figure 5.2 & 5.3).
We performed molecular dynamics simulations of syndiotactic and isotactic variants at 298 K for 10 ns under NVT conditions using GROMACS program suite
285 with gramicidin helical conformation as the starting structure for syndiotactic peptides. The number of clusters for each structure has been shown in Table 5.1 and Figure 5.4. Syndiotactic peptides are quite stable and insensitive to its environment and in general agreement with earlier reports 238. MD results
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Figure 5.4. Clustering analysis and electrostatic potential distribution of designed peptides. All peptides were investigated for their conformational stability by molecular dynamics simulations. A 10 ns run with a 0.2 fs time step was completed, generating 1001 structures per run. These 1001 structures were clustered using g_cluster program from GROMACS package. Mean structures of significant clusters (> 20 structures) were extracted from the trajectory and electrostatic potential at each position was calculated by solving finite distance Poisson-Boltzmann equation using Delphi software. These electrostatic potential distributions were summed for each amino acid side-chain and represented collectively at the chromophoric center of the side chain as per a colour map, with red and violet indicating highly positive and highly negative values. Mean structures for significant clusters of respective peptides and the electrostatic potential distribution are shown in the panels (A-O).
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63 point to two important observations; i) syndiotactic structures are exceptionally stable under ambient conditions in gramicidin helical conformation, whereas poly L peptides generate conformational ensemble forming a very rugged energy landscape and ii) this distinct topological manifestation may translate to different activity profiles, while they encounter membranes of different cell types.
5.3.2 Peptide synthesis and characterization: The designed peptide sequences were synthesized, characterized by reversed phase HPLC and MALDI-TOF mass spectrometry. All the peptide was further purified using semi-preparative reversed phase HPLC (Appendix).
5.3.3 Peptide-lipid interaction: Differential topology dependent electrostatic