Reading Conformational Changes in Proteins with a New Colloidal-Based Interfacial Biosensing System.

Many biological events such as mutations or aberrant post-translational modifications can alter protein conformation and/or folding stability and their subsequent biological function, which may trigger the onset of diseases like cancer. Evaluating protein folding is hence crucial for the diagnosis of these diseases. Yet, it is still challenging to detect changes in protein folding, especially if they are subtle, in a simple and highly sensitive manner with the current assays. Herein, we report a new colloidal-based interfacial biosensing approach for qualitative and quantitative profiling of various types of changes in protein folding, from denaturation to variant conformations in native proteins, such as protein activation by underlying mutations or phosphorylation. The approach is based on the direct interfacial interaction of proteins freely available in solution with added tannic acid-capped gold nanoparticles (AuNPs), enabling interrogating protein's folding in their solubilized form. We found that under the optimized conditions, proteins can modulate the solvation of the colloids according to their folding or conformational status, which can be visualized in a single step, by the naked eye, with minimal protein input requirements (LOD of 1 ng/µL). Protein folding detection was achieved regardless of protein topology and size without using conformation-specific antibodies or mutational analysis, which are the most common assays for sensing malfunctioning proteins. The approach showed excellent sensitivity, superior to Circular Dichroism, for the detection of the very subtle conformational changes in EGFR and ERK proteins induced by activating mutations and phosphorylation, enabling their detection even in complex samples derived from lung cancer cells, which contained up to 95% excess of their wild-type forms. Broader clinical translation was showed via monitoring the action of conformation restoring drugs, such as Tyrosine Kinase Inhibitors on EGFR conformation and its downstream protein network, using ERK protein as a surrogate.