Preparation and Studies of 19F-Labeled and Enriched 13C-Labeled Semisynthetic Ribonuclease-s' Analogues

Abstract

Abstract Isotopically normal and both 19F- and enriched 13C-containing analogue forms of semisynthetic ribonuclease-S' (SRNase-S') have been prepared. This noncovalent complex contains the polypeptide fragments synthetic-(1–15) (the solid phase synthesized peptide corresponding to residues 1 through 15 of bovine pancreatic ribonuclease A (RNase-A)) and RNase-S-(21–124) (the native fragment of RNase A containing residues 21 through 124). The normal SRNase-S' complex, obtained after mixing crude synthetic-(1–15) with the corresponding native fragment and subsequently fractionating on Sulfoethyl-Sephadex, was equally as active as native ribonuclease-S' (RNase-S') (which contains the NH2-terminal eicosapeptide fragment of RNase A instead of the pentadecapeptide). The analogues, containing either carbon 13-enriched phenylalanine or p-fluorophenylalanine (fluorine as naturally occurring 19F) at position 8 (the [Phe8-13C] and [pFPhe8] analogues, respectively), were purified by similar procedures to a state about equally as active enzymically as the normal complex. The normal and analogue complexes, as well as the component synthetic-(1–15) peptides and native RNase-S', were characterized, where appropriate, by either 13C or 19F proton noise-decoupled Fourier transform nuclear magnetic resonance spectroscopy. There are several significant changes in the 13C NMR chemical shift values of the enriched carbon atoms between [Phe8-13C] synthetic-(1–15) and [Phe8-13C]SRNase-S', indicating the occurrence of changes in environment at specific phenylalanine 8 loci when SRNase-S' complex is formed from isolated fragments. Chemical shift changes for some of the enriched carbon atoms are also observed when 2'-cytidine monophosphate (2'-CMP) is added to [Phe8-13C]SRNase-S'. Additionally, the spin-lattice relaxation times (T1) for some of the en riched ring carbons have been monitored and found to decrease progressively in going through the transition of (1–15) peptide to SRNase-S' complex to complex plus 2'-CMP. The above changes in chemical shift and T1 values have been interpreted in terms of (a) formation of the phenylalanine 8-containing NH2-terminal α-helix, and concomitant introduction of the phenylalanine 8 side chain into a nonpolar, sterically rigid environment, which occur when SRNase-S' folds, and (b) localized structural changes around the phenylalanine side chain when 2'-CMP binds to the folded complex. The transitions of (1–15) peptide to SRNase-S' complex and of this complex upon addition of 2'-CMP were also observed by 19F NMR using [pFPhe8]synthetic-(1–15) and [pFPhe8]SRNase-S'.