title: Branched intermediate formation stimulates peptide bond cleavage in protein splicing.
abstract: Protein splicing is a post-translational modification in which an intein domain excises itself out of a host protein. Here, we investigate how the steps in the splicing process are coordinated so as to maximize the production of the final splice products and minimize the generation of undesired cleavage products. Our approach has been to prepare a branched intermediate (and analogs thereof) of the Mycobacterium xenopi DNA gyrase A (Mxe GyrA) intein using protein semisynthesis. Kinetic analysis of these molecules indicates that the high fidelity of this protein-splicing reaction results from the penultimate step in the process (intein-succinimide formation) being rate-limiting. NMR experiments indicate that formation of the branched intermediate affects the local structure around the amide bond that is cleaved during succinimide formation. We propose that this structural change reflects a reorganization of the catalytic apparatus to accelerate succinimide formation at the C-terminal splice junction.
authors.affiliation: Laboratory of Synthetic Protein Chemistry, The Rockefeller University, New York, New York, USA.
authors.forename: Silvia
authors.initials: S
authors.lastname: Frutos
authors.forename: Michael
authors.initials: M
authors.lastname: Goger
authors.forename: Baldissera
authors.initials: B
authors.lastname: Giovani
authors.forename: David
authors.initials: D
authors.lastname: Cowburn
authors.forename: Tom W
authors.initials: TW
authors.lastname: Muir
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D027081:DNA Gyrase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D019912:Mycobacterium xenopi / Q000378:metabolism; D011487:Protein Conformation; D019154:Protein Splicing / Q000502:physiology*
chemical_list: D001426:Bacterial Proteins; D027081:DNA Gyrase