ARCHITECTURE AND DESIGN OF PROTEIN-PROTEIN BINDING SITES USING THE
PROTEON™ XPR36 PROTEIN INTERACTION ARRAY SYSTEM
Gideon Schreiber (1), Dana Reichmann (1), Mati Cohen (1), Laila Roisman (1), Vladimir Potapov (2),
Vladimir Sobolev (2), Marvin Edelman (2), and Orly Dym (3).
Departments of (1) Biological Chemistry, (2) Plant Sciences, (3) Structural Biology, Weizmann
Institute of Science, Rehovot, Israel
ABSTRACT
The energy landscape of protein-binding sites is a complex network of inter-residue interactions not easily explained using
single mutation analysis. We analyzed the TEM1-BLIP interface using multiple mutations to decode its architecture and the
factors contributing to binding affinity and specificity. The ProteOn XPR36 system enabled parallel interaction measurements
in a 6x6 array. The resulting contact map showed a modular build of the binding site; each module comprised closely
interacting residues with cooperative intra-modular mutations yet additive inter-modular mutations. We suggest modules, not
single residues, be viewed as interface building blocks. A modular approach was used to engineer a new TEM-BLIP complex
of altered binding sites with high specificity & affinity above WT. We demonstrate that energetically interacting residues are
spatially close and thus can serve as distance constraints for structure calculations. Scanning the residue-residue contact map of
interferon-IFNAR2 provided 5 constraints used to calculate the structure of this complex. Decoding the 2nd interferonIFNAR1 interaction showed the basic difference in the architecture of a weak binding site with no hotspots, displaying very
low cooperativity.
ACTIN DEFICIENCY INDUCES COFILIN PHOSPHORYLATION: PROTEOME ANALYSIS OF
HELA CELLS AFTER β-ACTIN GENE SILENCING
Ning Liu*†, Katrina Academia*, Teresa Rubio*, Tim Wehr, Todd Yeck, Liz Jordan, Keith Hamby and Aran
Paulus
Bio-Rad Laboratories, Inc., Hercules, CA 94547
ABSTRACT
Actin-binding proteins regulate the dynamic structure and function of actin filaments in the cell. Much is known about
how manipulation of the actin-binding proteins affects the structure and function of actin filaments; however, little is
known about how manipulation of actin in the cell affects actin-binding proteins. We addressed this question by
utilizing two technologies: RNA interference and 2-dimensional gel electrophoresis. We knocked down β-actin
expression in HeLa cells using short interfering RNA and applied 2-DGE to examine alterations in the HeLa cell
proteome. We revealed a 2-5 fold increases of four protein spots on 2-D gels and identified these proteins by mass
spectrometry. Three of the four proteins were actin-binding proteins, including cofilin, which promotes both
disassembly and assembly of actin filaments but becomes inactivated when phosphorylated. Further examination
revealed that the cofilin total protein level barely increased, but the phosphorylated cofilin level increased dramatically
in HeLa cells after β-actin siRNA treatment. These results suggest that in response to siRNA-induced β-actin
deficiency HeLa cells inactivate cofilin by phosphorylation rather than down-regulate its protein expression level. This
study also demonstrates that the combination of RNA interference and 2-dimensional gel electrophoresis technologies
provides a valuable method to study protein interactions in a specific cellular pathway.
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