Far-ultraviolet difference absorption and circular dichroism studies on partially synthetic ribonucleases S′

Abstract

Far-ultraviolet difference absorption and circular dichroism (CD) spectra were recorded upon recombination of synthetic S-peptide analogs, i.e. 1 ∈,7 ∈-diguanidino-[Tyr 8]-, 1 ∈,7 ∈-diguanidino-[Asn 14]-, [Phe(F) 8,Orn 10]-, [Cha 8,Orn 10]- and 1 ∈,7 ∈-diguanidino-S-peptide, with S-protein. The aromatic chromophores contributions to the absorption spectra in the 220–250 nm wavelengths interval strongly exceed the hyperchromism due to the random coil to right handled α-helix transition of the S-peptide, accompanying the association process. Contributions resulting from peptide transitions constitute a large portion of the total dichroism, nevertheless substitution of the non aromatic cycloexylalanine residue in position 8 of the S-peptide with phenylalanine, p-fluorophenylalanine and tyrosine leads to CD negative maxima located at 215, 215 and 212,5 nm, respectively. The strongest ellipticity increment (28%), relative to that of the Cha-derivative, was observed at 212,5 nm for the [Tyr 8]-S-peptide analog, while in the narrow 222 nm range minimal differences were found upon insertion into the position 8 of the S-peptide of the above aromatic residues. Information derived from above data and from a comparison of RNAase A, S and S-protein CD spectra enabled us to assign the positive CD band at 240 nm in RNAase A spectrum to transitions of phenylalanines and inaccessible tyrosines.