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Conformation and Function of Gramicidin S, A Peptide Antibiotic Which Mediates Phase Transfer of Nucleotides and Nucleic Acids


Krauss, Eric Martin (1983) Conformation and Function of Gramicidin S, A Peptide Antibiotic Which Mediates Phase Transfer of Nucleotides and Nucleic Acids. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/d8tf-3k19.


Gramicidin S (GrS; cyclo-(Val1-Orn2-Leu3-D-Phe4-Pro5)2) is a cyclosymmetric decapeptide antibiotic synthesized by mature cultures of the gram-positive spore former, Bacillus brevis. Its amphiphilic character and antiparallel β-pleated conformation in solution are experimentally established, but the extent of internal hydrogen bonding, the structural basis of its cytotoxicity and the physiological role in the producer organism remain unelucidated. These issues are addressed in the present investigation.

The secondary structure of GrS is studied by a combination of proton exchange kinetics and vibrational spectroscopy. Dispersion in amide proton exchange rates provides a means of synthesizing variably N-deuterated GrS congeners, the isotopic composition of which are determined quantitatively by NMR. Difference IR of Me2SO solutions then affords isolation of residue-specific peptide group vibrations. Two pairs of equivalent hydrogen bonds with donor groups Leu NH and Val NH are identified, and their geometry and energetics characterized. The β-pleated conformation is thus shown to be stabilized by the maximum of four transannular hydrogen bonds.

The solution conformation of the functionally essential ornithine side chains is then investigated by 1H and 15N NMR spectroscopy at 11.7 Tesla. Rotational averaging of the chemical shifts of the Orn CδH2 protons is incomplete, the degree to which the apparent motility of the side chain is limited varying inversely with the ability of the solvent to compete for hydrogen bonding donor or acceptor sites. Methylation of GrS to give [Nδ-trimethylornithyl2,2']-GrS results in an upfield shift of 3.5 ppm in the 15N resonance of Pro in MeOH and abolishes the correlation of the Orn CδH2 splitting with solvent basicity. The data are consistent with the presence of intramolecular Orn NδH3+--O=C D-Phe hydrogen bonds, each with formation constant ~1.1 in MeOH at 23°C, and exerting a substantial charge relay effect on the Pro 15N chemical shift. Proton exchange kinetics and nuclear Overhauser enhancement measurements indicate that these hydrogen bonds are formed in the i → i + 2 sense. Estimates for the Orn side chain torsional angles in the intramolecularly hydrogen bonded configuration are given, and the possible origin of the Orn CδH2 chemical shift inequivalence discussed.

The studies of the solution conformation raise the possibility that the biological actions of GrS involve complexation of polyvalent anions; Orn NδH3+--O=C D-Phe hydrogen bonding might then ensure a critical intercationic distance (6-8 Å) which is complementary to the architecture of the anionic species. Indeed, it is shown that GrS binds nucleotides in water to yield a complex which partitions into organic solvents. The observed phase transfer efficiencies at a given pH increase in the order AMP < ADP < ATP. The lipophilic complexes have well-defined stoichiometries, which are determined to be 1:1 for ADP-GrS at pH 7 and ATP-GrS at pH 3 and 1:2 for ATP-GrS at pH 7. The interaction is primarily ionic, involving coordination of the Orn NδH3+ groups of the peptide and the phosphoryl groups of the nucleotide, with little contribution from the nucleoside moiety. The nucleotide complexes are sparingly soluble in water, and self-associate extensively in CHCl3, most likely by cross-β aggregation, to yield large, ribbonlike aggregates which give rise to broad NMR, resonances. structures for the 1:1 and 1:2 complexes are proposed. In the latter, two GrS molecules envelop the nucleotide, orienting their apolar faces externally in opposite directions, while the lateral faces retain considerable polar character and direct aggregation in organic media. The 1:1 complex possesses a single apolar face and is less lipophilic. Binding constants are estimated by simulation of the extraction data.

A novel interaction between GrS and nucleic acids is subsequently characterized which, like that between GrS and nucleotides, exploits both the dicationic and amphiphilic properties of the peptide. Complex formation between calf thymus DNA and GrS is demonstrated by (i) phase transfer to CHCl3 of ultrasonically irradiated DNA and (ii) inhibition of phase transfer to CHCl3 of ATP by either native or ultrasonically irradiated DNA. The stoichiometry of the interaction is 2:1 (DNA-P/GrS) as expected for a predominantly electrostatic mode of binding. The extraction-competition data suggest that the affinity of GrS for DNA is considerably higher than it is for free nucleotides. It is proposed that during dormancy stoichiometric binding by GrS ensheaths the bacterial chromosome in a dense peptide matrix possessing a highly apolar external surface, which should constitute an effective barrier against chemical degradation.

In order to elucidate the molecular basis for GrS toxicity in vivo microbiological studies of GrS and analogues derivatized at ornithine are undertaken. The minimum lethal concentrations of GrS, [Nδ-trimethylornithyl2,2']-GrS, and [N-acetylornithyl2]-GrS in a standard bioassay employing Staphylococcus aureus in nutrient broth are compared. In addition, the time course of attenuation of the culture by GrS and the dependence of the minimum lethal concentration on initial cell concentration are determined. A model for GrS action is proposed involving internalization and formation of cytotoxic lipophilic complexes with polyanionic substrates , possibly nucleotides. The limiting antibiotic potencies at zero initial cell concentration and apparent extraction constants for nucleotides estimated in vitro are shown to be consistent with this model.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Chemistry
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Dervan, Peter B.
Thesis Committee:
  • Dervan, Peter B. (chair)
  • Chan, Sunney I.
  • Marsh, Richard Edward
  • Hopfield, John J.
Defense Date:11 February 1983
Funding AgencyGrant Number
Earle C. Anthony FellowshipUNSPECIFIED
Record Number:CaltechTHESIS:10172019-173615812
Persistent URL:
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:11825
Deposited By: Mel Ray
Deposited On:18 Oct 2019 17:25
Last Modified:16 Apr 2021 22:21

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