QB3 Postdoc Seminar

Seminar | February 15 | 4:30-5:30 p.m. | 177 Stanley Hall

 QB3 - California Institute for Quantitative Biosciences

Ellen Goodall | Martin Lab
Four substrate-engaged 26S proteasome structures reveal mechanisms for ATP-hydrolysis-driven translocation of substrates during degradation

The 26S proteasome is a molecular machine responsible for the bulk of targeted degradation in the eukaryotic cell. A heterohexameric, AAA-type ATPase within the proteasome pulls on substrate polypeptide, disrupting folded domains so that the substrate can be threaded into an interior proteolytic chamber. In addition, ubiquitin, the small protein modifier which targets substrates to the proteasome for degradation, is removed at the entrance to the AAA-motor. Preventing ubiquitin removal allowed us to capture four distinct, substrate-engaged conformations of the proteasome by cryo-electron microscopy, while still maintaining an active ATPase motor. These structures allow us to determine how ATP binding and hydrolysis are coordinated between the six subunits of the motor to cause the conformational changes that translocate the substrate through the proteasome for degradation.

Alan Marmelstein | Francis Lab
Selective chemical modification of protein phosphoryl groups to study a unique signaling mechanism of the inositol pyrophosphates

Intracellular inositol pyrophosphates (PP-InsPs) represent the most highly phosphorylated carbon scaffolds in nature and are conserved from plants and yeast to mammals. These highly charged molecules have been found to regulate a diverse array of cellular processes including phosphate metabolism, adipogenesis, and insulin secretion. Yet the signaling pathways that connect the PP-InsPs to many of their associated phenotypes remain to be elucidated. In vitro radiolabeling studies indicate that one way PP-InsPs transduce a signal is via a novel post-translational modification termed protein pyrophosphorylation, in which a high-energy phosphoryl group from a PP-InsP is transferred to an existing phosphorylated serine or threonine residue on a protein substrate. In order to study this novel PTM, we sought to develop affinity purification and mass spectrometry techniques for detecting pyrophosphorylated proteins in vivo. To this end, I developed a series of phosphorimidazolide reagents for the synthesis of pyrophosphorylated peptides necessary for method validation and characterization of the stability of these species. These reagents displayed a surprising proclivity for reacting with phosphoryl groups preferentially over other nucleophilic amino acid side chains, and this was demonstrated by selectively pyrophosphorylating a pre-phosphorylated version of full-length myoglobin. Synthetic pyrophosphorylation of peptides and proteins has enabled the identification of previously unknown pyrophosphorylated proteins in vivo and is being applied in new areas of chemical biology research.