Design and Characterization of Modular Scaffolds for Tubulin Assembly

Abstract

Background: Stathmin-like domain (SLD) proteins from vertebrates bind two tubulin molecules. Results: SLDs that bind tubulin with a programmed stoichiometry are characterized. Conclusion: Rules are established to design (tubulin) x -SLD complexes, starting from a 1:1 stoichiometry. Significance: This work provides new insights into stathmin family member function. The SLDs produced will be useful tools to study interactions of microtubule regulators with tubulin.

Design, construction, and characterization of a second‐generation DARPin library with reduced hydrophobicity

“…[Fig. (A)], 18 crystal structures of DARPins in complex with their target proteins have been reported . We analyzed all DARPin‐target protein interfaces of crystals solved at resolutions of 3 Å and better using the EPPIC server (www.eppic-web.org) (Supporting Information File “DARPin_interfaces.xls”).…”

“…1(A)], 18 crystal structures of DARPins in complex with their target proteins have been reported. 12,21,22,28,36,[38][39][40][41][42][43][44][45][46] We analyzed all DARPin-target protein interfaces of crystals solved at resolutions of 3 Å and better using the EPPIC server (www.eppic-web.org) 47 (Supporting Information File "DARPin_interfaces.xls"). Twentytwo interfaces found in 13 crystal structures were taken into account for analysis (some interfaces were present twice in the asymmetric unit).…”

Biochemical and Structural Insights into Microtubule Perturbation by CopN from Chlamydia pneumoniae

“…This result strongly suggests that pCopN interacts with the ␤-tubulin longitudinal interface in the vicinity of the exchangeable nucleotide binding site. To confirm the identity of the tubulin surface contacted by pCopN, we performed a competition experiment for tubulin binding between FL_pCopN and the D2 DARPin, which binds to the longitudinal interface of the tubulin ␤ subunit (19). To this end, we labeled the natural cysteine of FL_ pCopN with acrylodan.…”

“…Affinity Measurement by Fluorescence Spectroscopy-pCopN was labeled with acrylodan following established protocols (19). In short, the natural cysteine of pCopN (Cys-217) was reduced and then reacted with acrylodan, following which the excess dye was removed by desalting on a PD-10 column (GE Healthcare).…”

“…To the preformed acrylodan-labeled FL_pCopN-tubulin complex we added an excess of the D2 DARPin (19), and the fluorescence decrease was recorded. As a positive chase control, unlabeled FL_pCopN was added to the fluorescent complex.…”