PhD

dissertationThe 20S proteasome is an intracellular protease found in all kingdoms of life. In eukaryotes, the ubiquitin-proteasome is a major proteolytic pathway involved in the turn-over of cytosolic and nuclear proteins and controls many cellular processes. The 20S recruits one of three classes of proteasome activators that modulate and enforces its function: the PA700, the PA28 and the PA200/Blml0 families. In contrast to PA700 and PA28 that function by binding to the 20S as oligomeric structures, PA200/Blml0 binds to the 20S as a single polypeptide chain of about 250kDa. This dissertation focuses on structural studies of the BlmlO proteasome activator from the budding yeast S. cerevisiae. The 18A cryo-EM reconstruction of Blml0:20S complex shows that BlmlO is a dome-shaped protein that binds to the ends of the proteasome and opens its gate. The similarity of the cryo-EM structures of BlmlO and its bovine homologue, PA200, suggested a similar biological function; however, the precise biological role of PA200/Blml0 remains to be determined. To further advance understanding of BlmlO function, we determined the crystal structure of Blml0:20S complex at 3.4A resolution. BlmlO is a HEAT-repeat containing protein that folds into a left-handed solenoid burying a large solvent cavity over the 20S a subunits. BlmlO contains a pore large enough to allow passage of small peptides explaining the biochemical activity of Blml0:20S complex. Binding to the 20S occurs through many loops from different HEAT repeats and also by BlmlO's single C-terminal carboxylate that forms a salt bridge with a conserved lysine of 20S. Intriguingly, the penultimate residue of BlmlO is a conserved tyrosine that opens the gate locally at the proline reverse turn of a-5. This repositioning at a-5 is propagated to the neighboring subunits: a-6, 7, and 1 triggering their open conformation while disordering the gate at a-2, 3, and 4. Though the partial disordering of the 20S gate seems to be unique to BlmlO, the penultimate tyrosine of BlmlO is shared by subunits of the PA700 activator and provides a model for proteasome activation by ATPase activators

Structural Evidence: A Single Charged Residue Affects Substrate Binding in Cytochrome P450 BM-3

Cytochrome P450 BM-3 is a bacterial enzyme with sequence similarity to mammalian P450s that catalyzes the hydroxylation of fatty acids with high efficiency. Enzyme-substrate binding and dynamics has been an important topic of study for cytochromes P450 because most of the crystal structures of substrate-bound structures show the complex in an inactive state. We have determined a new crystal structure for cytochrome P450 BM-3 in complex with N-palmitoylglycine (NPG), which unexpectedly showed a direct bidentate ion pair between NPG and arginine 47 (R47). We further explored the role of R47, the only charged residue in the binding pocket in cytochrome P450 BM-3, through mutagenesis and crystallographic studies. The mutations of R47 to glutamine (R47Q), glutamic acid (R47E), and lysine (R47K) were designed to investigate the role of its charge in binding and catalysis. The oppositely charged R47E mutation had the greatest effect on activity and binding. The crystal structure of R47E BMP shows that the glutamic acid side chain is blocking the entrance to the binding pocket, accounting for NPG\u27s low binding affinity and charge repulsion. For R47Q and R47K BM-3, the mutations caused only a slight change in kcat and a large change in Km and Kd, which suggests that R47 mostly is involved in binding and that our crystal structure, 4KPA, represents an initial binding step in the P450 cycle

Mobile learning : structures, agency, practices

The main physics program of the International Linear Collider requires a measurement of the beam energy with a relative precision of the order 10(-4) or better. A magnetic spectrometer using high resolution beam position monitors (BPMs) has been proposed to achieve this goal. A prototype spectrometer chicane employing four dipole magnets is currently under development at the End Station A in SLAC, intending to demonstrate the required resolution and stability of this method and investigate possible systematic effects and operational issues. This contribution reports on the successful commissioning of the beam position monitor system and the resolution and stability achieved. Also, the initial results from a run with a full spectrometer chicane are presented