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␣) ligandbinding 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 antagonistbound 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.M any biological processes are regulated by proteins. Regulatory proteins undergo conformational changes to alter the surface properties that in turn modulate their interactions with partners and͞or alter their catalytic efficiency. Thus, it is essential to detect conformational changes of proteins to understand the molecular mechanism underlying their functions. It is generally accepted that the ''molecular crowding'' within the cellular environment can significantly affect ligand binding, catalysis, stability, and folding of macromolecules (1). Thus, the structures and relative populations of ''active'' and ''inactive'' conformations of a protein might be quite different from those determined by using in vitro biophysical methods. In vivo techniques such as fluorescence resonance energy transfer (FRET) (2) and enzyme fragment complementation (3) have been successfully used to detect gross conformational changes of proteins (e.g., folding, large structural rearrangement, and subunit association). The goal of this study has been to establish an in vivo technique to detect more subtle conformational changes of proteins (e.g., secondary structure rearrangement) that nevertheless lead to significant changes in surface property.An alternative approach to directly obtaining geometric information (e.g., FRET) is to characterize the binding of conformation-specific probes to a target of interest. A classic example is conformation-specific antibodies...