| Popis: |
The knowledge of a protein’s structure is foundational to understanding how it accomplishes its function. Protein interactions with other proteins and DNA govern critical cellular events. However, protein complexes are difficult to study using traditional methods in structural biology. Electron microscopy (EM) has provided a way to study protein complexes at a level that is unachievable by other structural methods. Here we have used EM combined with various image processing methods to determine the structure of two protein complexes that have been either stabilized by crosslinking, or destabilized by pH. First, we have used negative-stain EM with random conical tilt procedures to determine a low resolution structure of the crosslinked Ccq1-Tpz1-Poz1 complex in fission yeast. This complex is intimately involved in events associated with telomere homeostasis, specifically regulating whether the telomere is in an extendable or a non-extendable state. Our results to the EM study were corroborated by Mapping Interfaces via Crosslinking Mass Spectrometry (MICro-MS), both of which indicated a C-terminal dimerization event in the Ccq1 component. Second, we used Cryo-EM and computational modeling to study the cascade of events that are involved in pore formation associated with the anthrax protective antigen (PA) heptamer. Using a D425A mutant which does not form the pore, we exposed the mutant heptamer to low pH, which initiates pore formation in the WT protein. Analysis of two dimensional (2D) class averages and fluorescence profiles across a pH range indicated a conformational change in the mutant at low pH, which suggested the presence of an intermediate state in pore formation. Analysis of the mutant three dimensional (3D) structure using molecular dynamics with flexible fitting showed that protonation of histidines is likely the pH-dependent step in anthrax pore formation and that D425 and K397 likely form an electrostatic interaction which is required for pore formation to occur. |
