1H and 15N nuclear magnetic resonance assignment and secondary structure of the cytotoxic ribonuclease α-Sarcin

✍ Scribed by Ramon Campos-Olivas; Marta Bruix; Jorge Santoro; Manuel Rico; Alvaro Martinez Del Pozo; Javier Lacadena; José G. Gavilanes

• Publisher: Cold Spring Harbor Laboratory Press
• Year: 2008
• Tongue: English
• Weight: 453 KB
• Volume: 5
• Category: Article
• ISSN: 0961-8368
• DOI: 10.1002/pro.5560050519

✦ Synopsis

Abstract

The ribosome‐inactivating protein α‐Sarcin (αS) is a 150‐residue fungal ribonuclease that, after entering sensitive cells, selectively cleaves a single phosphodiester bond in an universally conserved sequence of the major rRNA to inactivate the ribosome and thus exert its cytotoxic action. As a first step toward establishing the structure‐dynamics‐function relationships in this system, we have carried out the assignment of the ^1^H and ^15^N NMR spectrum of αS on the basis of homonuclear (^1^H‐^1^H) and heteronuclear (^1^H‐^15^N) two‐dimensional correlation spectra of a uniformly ^15^N‐labeled sample, and two selectively ^15^N‐labeled (Tyr and Phe) samples, as well as a single three‐dimensional experiment. The secondary structure of αS, as derived from the characteristic patterns of dipolar connectivities between backbone protons, conformational chemical shifts, and the protection of backbone amide protons against exchange, consists of a long N‐terminal β‐hairpin, a short α‐helical segment, and a C‐terminal β‐sheet of five short strands arranged in a+1,+1,+ 1, + 1 topology, connected by long loops in which the 13 Pro residues are located.