Mapping Conformations, Folding Pathways and Interactions in Unstructured Proteins

Intrinsically disordered proteins (IDPs) lack stable globular tertiary folded structure under physiological conditions. Roughly 65% of the signaling and 75% of the human cancer-associated proteins are predicted to have significant disordered regions, thus implying a role for disorder in mediating regulatory protein interactions in complex biological processes. Whether specific recognition can occur despite conformational disorder or disordered protein states may provide advantages in recognition over well-folded proteins is still an open question.

Mapping Conformations, Folding Pathways and Interactions in Unstructured Proteins

Single-molecule fluorescence spectroscopy applied to IDPs can provide important new insights into the conformational properties of the disordered ensemble and how these features are altered by solution properties (ionic strength, pH, osmolytes) and by binding to other proteins. We are currently developing single-molecule assays such as Förster Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS) to investigate two unstructured proteins, the yeast cyclin inhibitor Sic1 and the N-terminal SH3 domain of the Drosophila adapter protein Drk.



Allostery at Single Molecule Level

G-protein coupled receptors (GPCRs) are a superfamily of membrane proteins which represent both the most common hormone and neurotransmitter receptors. Upon binding of ligands, GPCRs undergo conformational changes that lead to activation of cellular signaling. These changes may occur within each receptor and also between receptors within oligomers, inducing cooperative behaviors.

Allostery at Single Molecule Level

The M2 muscarinic cholinergic receptor possesses an orthosteric ligand (agonist) binding site as well as an allosteric ligand binding site. When agonists bind to the orthosteric site, the M2 receptor transduces the signal to the intracellular G-protein. The common view of this process treats the receptor as a monomer that has two states formed by the transient interaction with the G-protein. However, recent evidence indicates that GPCRs function as oligomers, which could display multiple states as a result of transduced conformational changes. Furthermore, the mechanism by which allosteric ligands modulate efficacy of the receptor and their role in an oligomer complex remains elusive. Using quantitative single-molecule fluorescence techniques, such as smFRET, PCH and single-particle tracking, we aim to obtain a detailed picture of the ligand binding, aggregation and signaling processes of the M2 muscarinic cholinergic receptor.


Ultrasensitive Fluorescence Detection in Bioanalytical Applications

We design and build ultrasensitive fluorescence microscopes that can be customized for numerous spectroscopy and imaging applications. In addition to new optical layouts, lasers and detectors, we are continuously adding new data acquisition and analysis routines to our arsenal of experimental capabilities. A new and efficient sample preparation protocol for extended, unperturbed recordings of single-molecule fluorescence signals was developed in our group. Work in our lab also focused on finding the optimal conditions for accurate FCS measurements of local concentrations and diffusion coefficients on samples ranging from highly diluted (~10 pM), which is required for bioanalytical applications, to concentrated (~1 pM), which is similar to the protein/DNA concentration inside live cells.

Ultrasensitive Fluorescence Detection in Bioanalytical Applications

For the purpose of direct quantitative analysis of multiple miRNAs from a single cell lysate without enrichment or modification, we efficiently coupled on-column capillary electrophoresis with our multiparameter single-molecule microscopes. The detection sensitivity of our setup is approximately 500 times better and the separation time is around 3 times faster compared to protocols on commercial instruments.