H-1 AND N-15 NUCLEAR-MAGNETIC-RESONANCE ASSIGNMENT AND SECONDARY STRUCTURE OF THE CYTOTOXIC RIBONUCLEASE ALPHA-SARCIN

The ribosome-inactivating protein alpha-Sarcin (alpha S) is a 150-resi due fungal ribonuclease that, after entering sensitive cells, selectiv ely cleaves a single phosphodiester bond in an universally conserved s equence of the major rRNA to inactivate the ribosome and thus exert it s cytotoxic action. As a first step toward establishing the structure- dynamics-function relationships in this system, we have carried out th e assignment of the H-1 and N-15 NMR spectrum of alpha S on the basis of homonuclear (H-1-H-1) and heteronuclear (H-1-N-15) two-dimensional correlation spectra of a uniformly N-15-labeled sample, and two select ively N-15-labeled (Tyr and Phe) samples, as well as a single three-di mensional experiment. The secondary structure of alpha S, as derived f rom the characteristic patterns of dipolar connectivities between back bone protons, conformational chemical shifts, and the protection of ba ckbone amide protons against exchange, consists of a long N-terminal b eta-hairpin, a short alpha-helical segment, and a C-terminal beta-shee t of five short strands arranged in a +1,+1,+1,+1 topology, connected by long loops in which the 13 Pro residues are located.