No abstract

The fast intrinsic time scale of infrared absorption and the sensitivity of molecular vibrational frequencies to their environments can be applied with site-specificity by introducing the artificial amino acid β-thiocyanatoalanine, or cyanylated cysteine, into chosen sites within intrinsically disordered proteins. This amino acid can be inserted through native chemical ligation at single cysteines introduced via site-directed mutagenesis. The CN stretching band of cyanylated cysteine is sensitive to local changes in both structural content and solvent exposure. This dual sensitivity makes cyanylated cysteine an especially useful probe of binding-induced structural transitions in IDPs. The general strategy of creating single-site cysteine mutations and chemically modifying them to create the vibrational chromophore, as well as observation, processing and analysis of the CN stretching band, is presented.

Intrinsically disordered proteins (IDPs) that undergo a coupled folding and binding event are found to be important in many recognition and signaling processes. However, exactly how this mechanism is linked to these functions is not completely understood. This is primarily because the structural flexibility of IDPs limits the number of suitable characterization techniques. We are using small-angle neutron scattering (SANS) to investigate the structure and binding interaction properties between NCBD, an IDP region of CREB binding protein (CBP), and its binding partner, ACTR, which also is an IDP. CBP is a transcription co-activator that is essential in embryonic development, growth control, and homeostasis, and its dysfunction is implicated in neurological disorders such as Huntington's disease and some cancers. SANS indicates the NCBD/ ACTR complex is a globular, folded structure with a smaller radius of gyration compared to ACTR alone, which is mostly unfolded. Using ab initio shape reconstruction programs to gain further insight into structural flexibility, we find good agreement between the shape reconstruction and NMR structure for the NCBD/ACTR complex. In contrast, SANS reveals the nature of how ACTR is more expanded when in its unbound, unfolded state. This research should provide new possibilities for the study of disordered protein regions and yield unique perspectives into the mechanism of IDP function.