Gramicidin A and its complexes with Cs + and Tl + ions in organic solvents : A study by steady state and time resolved emission spectroscopy

Abstract

Gramicidin A (gr A), a linear pentadecapeptide containing four trp residues has been studied using steady state and time resolved fluorescence (at 298 K) and phosphorescence (at 77 K) in methanol (CH 3OH), ethanol (C 2H 5OH), dimethyl sulfoxide (DMSO), 1,4-dioxane, 2-methyl tetrahydrofuran (2-MeTHF), ethanol/benzene (C 2H 5OH/C 6H 6) mixed solvent. Similar studies have also been carried out in CH 3OH containing monovalent cations K +, Cs +, Tl + and divalent cation Ca 2+. λ max of fluorescence is found to be a good signature of the different forms having double helical structure [dh (1) to dh (4)] (J. Struct. Biol. 121 (1998) 123–141). Steady state and time resolved quenching studies of gr A by KI in CH 3OH and DMSO and life time of the emitting singlet states of gr A support that gr A exists as a mixture of different forms of double helical (dh) structure [dh (1) to dh (4)] in CH 3OH and as a random coil structure in DMSO. This study further indicates that emitting trp residue in DMSO is better shielded than that in CH 3OH. Phosphorescence spectra of gr A at 77 K in CH 3OH glass suggests that gr A retains a particular conformation dh (3) in this matrix. The phosphorescence spectra of gr A [conformation dh (4)] in 2-MeTHF at 77 K is further red shifted indicating that among all the dh forms, dh (4) has the emitting trp residue in most hydrophobic environment. The hydrophobicity of the emitting tryptophan environment is thus found to be in the order: dh (1)<dh (3)<dh (4). Since 2-MeTHF forms a clear glass at low temperature, it is thus possible to study the side chain arrangement of gr A dh (4) as a function of temperature. The phosphorescence spectra in different alcohol glassy matrix are in conformity with the observation of different side chain arrangement of gr A as one changes the polarity of alcohol. Steady state and time resolved quenching studies of gr A using Cs + ion in CH 3OH at 298 K clearly demonstrate the two binding sites for the metal ions and provide the value of equilibrium constant of the ‘non-emitting’ complex of gr A with Cs + ion in the ground state. The observation of distinct red shift of the (0,0) band of the phosphorescence spectra of the complexes of gr A with K +, Cs + and Tl + ions at 77 K compared to that in CH 3OH glass confirms the metal ion induced change of conformation in dh (3). The result also suggests that the emitting trp residues in the complexes are in somewhat more hydrophobic environment compared with that in the free gr A in CH 3OH glass. The triplet state life time of these complexes indicate that the heavy metal ions Cs + and Tl + are within a Van der Waal’s distance of emitting trp residue in gr A in CH 3OH glass at 77 K so that they are capable of inducing increased spin–orbit coupling due to a heavy atom effect.