Identification of microtubule-binding domains on microtubule-associated proteins by major coat phage display technique.
Microtubule is an important structural and functional component in cells. Microtubule-associated proteins (MAPs) are a class of proteins that can bind to microtubules and stabilize them to maintain their functions. However, not all the specific microtubule-binding domains on MAPs are clear. Here we report the study of microtubule-binding domains on MAPs from a new angle by biopanning a new type of phage-displayed random peptide library (called landscape phage library) against purified alpha- and beta-tubulins. In the landscape phage library, billions of fd-tet phage clones are present and a unique 9-mer peptide is fused to each of the approximately 3900 copies of major coat protein (pVIII) in each clone. The affinity-selected peptides derived from the biopanning were analyzed by the receptor ligand contacts (RELIC) suite of programs, which is a bioinformatics tool for combinatorial peptide analysis and identification of protein-ligand interaction sites. By using RELIC, the affinity-selected peptides were shown to have similarity with the sequences of two MAP families (MAP1 and MAP2/tau), thereby identifying putative microtubule-binding domains on these MAPs. The tubulin-binding affinity was also confirmed by using transmission electron microscopy (TEM) to characterize the interaction between affinity-selected tubulin-binding phage and tubulins. Our results confirm some known microtubule-binding domains and identify some new microtubule-binding domains and thus shed light into the mechanism of microtubule-MAPs interactions.
Cao
Binrui
B
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 73019, USA.
Mao
Chuanbin
C
D003001:Cloning, Molecular; D020099:Coated Materials, Biocompatible / Q000737:chemistry*; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D046911:Macromolecular Substances / Q000737:chemistry*; D008869:Microtubule-Associated Proteins / Q000737:chemistry*; D008870:Microtubules / Q000737:chemistry*; D019151:Peptide Library; D014404:Tubulin / Q000737:chemistry
D020099:Coated Materials, Biocompatible; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D019151:Peptide Library; D014404:Tubulin