| Protein activity (enzymatic or binding) is responsible for most biological processes. These processes are highly regulated at multiple points, including at the transcriptional, translational, and post-translational levels. Proteins are therefore designed with inherent controls, or switches, built in. Of growing interest are relatively unstructured proteins and regions of proteins known as intrinsically disordered proteins. It is believed that the lack of secondary structure allows these regions to rapidly bind to targets and provide efficient dynamic control of protein activity. Before these technologies can be fully utilized, we need to be able to be able to (1) design intrinsically disordered proteins that can form secondary and tertiary structures in response to stimuli, and (2) utilize intrinsically disordered proteins as switchable scaffolds to perform biomolecular recognition.;This thesis describes attempts to use a single-chain antibody (scFv) as a peptide conformational change sensor. The structure of a peptide linker fused between IgG heavy- and light-variable regions affects the binding properties of the scFv. In this way, the scFv can report the structure of a peptide. Furthermore, it may be possible to use a similar conformational change sensor to sort random peptide libraries for stimulus-responsive peptide structure formation. Advances towards this goal and limitations are discussed.;Next, an intrinsically disordered protein, the RTX-motif from Bordetalla pertussis, is characterized using Forster Resonance Energy Transfer (FRET). This method offers a rapid way to determine the extent to which a protein is disordered and can be used to monitor conformational dynamics.;Further characterization of the RTX-motif has demonstrated the importance of entropic capping for the calcium-responsive folding of this intrinsically disordered protein. Using CD spectroscopy, we show that flanking regions fused to the C-terminal end of the RTX-motif are critical for folding that appears to initiate at the C-terminal end. The natural capping group, a yellow fluorescent protein, and a maltose binding protein can all be used to enable calcium-responsive folding of the RTX-motif. The successful employment of diverse set of fused protein domains underscores the generality of the capping effect strongly suggests a largely entropic effect. Insight into the potential function of the RTX-motif as a molecular ratchet is discussed in light of the entropic stabilization of the C-terminal repeats necessary for calcium-responsive folding.;Last, the calcium-responsive RTX-motif and two RTX libraries are expressed as fusions to the eCPX outer membrane protein. All three RTX-motifs maintain calcium-responsive behavior on the bacterial cell surface. The intrinsic disorder of the RTX-motif permits the efficient transport and folding of the eCPX-RTX-motif fusion that is not typically possible with fusions of this size. When combined with preliminary magnetic activated cell sorting, the design of allosterically controlled biomolecular recognition is close to fruition. The generality of this technique may be utilized in the design of myriad calcium-controlled biomolecular recognition events for use in "catch and release" chromatography systems and biosensing applications. |
