Single-Molecule Force Spectroscopy on Unfolded and Intrinsically Disordered Proteins

Transcription factors and cell-signaling proteins contain a hyper abundance of domains or segments that are intrinsically disordered (ID) under native conditions. In many cases, intrinsically disordered proteins (IDPs) or ID segments undergo coupled folding and binding and this transition is associated to its biological role. Many techniques for dissecting folding are not readily amenable to IDPs because they are aggregation prone and insoluble. Single-molecule methods provide an avenue to mitigate these issues. We have performed single-molecule force spectroscopy experiments on a Trimethylamine N-oxide (TMAO) refolded IDP and found that this IDP unfolds cooperatively under mechanical force. The data are rich in information and reveal that what appears to be a two-state transition in bulk is actually multi-state at the single-molecule level. We found that the analysis methods typically employed in such experiments may not be applicable because of the high concentrations of TMAO. To address these issues, we have designed unfolded mutants of T4 lysozyme using a random mutagenesis phenotypic screen. We are actively performing both bulk and single-molecule experiments to characterize unfolded mutants of T4 Lysozyme and TMAO refolded IDPs.