期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2002
卷号：99
期号：3
页码：1253-1258
DOI：10.1073/pnas.032665299
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：A challenge in understanding the mechanism of protein function in biology is to establish the correlation between functional form in the intracellular environment and high-resolution structures obtained with in vitro techniques. Here we present a strategy to probe conformational changes of proteins inside cells. Our method involves: (i) engineering binding proteins to different conformations of a target protein, and (ii) using them to sense changes in the surface property of the target in cells. We probed ligand-induced conformational changes of the estrogen receptor (ER) ligand-binding domain (LBD). By using yeast two-hybrid techniques, we first performed combinatorial library screening of "monobodies" (small antibody mimics using the scaffold of a fibronectin type III domain) for clones that bind to ER and then characterized their interactions with ER in the nucleus, the native environment of ER, in the presence of various ligands. A library using a highly flexible loop yielded monobodies that specifically recognize a particular ligand complex of ER, and the pattern of monobody specificity was consistent with the structural differences found in known crystal structures of ER-LBD. A more restrained loop library yielded clones that bind both agonist- and antagonist-bound ER. Furthermore, we found that a deletion of the ER F domain that is C-terminally adjacent to the LBD increased the crossreactivity of monobodies to the apo-ER-LBD, suggesting a dynamic nature of the ER-LBD conformation and a role of the F domain in restraining the LBD in an inactive conformation.
