Conformations,Dynamics and Interactions of Intrinsically Disordered Proteins (IDPs)

Since regulatory biological function requires some degree of structural flexibility, proteins are inherently dynamic and sample several different conformations, although the degree of flexibility and the time scale of protein motions vary considerably. An extreme case of protein polymorphism is represented by IDPs, which lack a stable tertiary fold and rapidly sample highly heterogeneous conformations. About 65% of the signaling and 75% of the 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.

Conformations,Dynamics and Interactions of Intrinscially Disordered Proteins (IDPs)

We study two IDP systems using SMF techniques: Sic1, a kinase inhibitor in yeast that plays a role in the cell cycle, and 4E-BP2, a translation inhibitor in the nervous system involved in regulating synaptic plasticity. Sic1 forms a dynamic "fuzzy" complex with the WD40 domain of the Cdc4 protein, where the binding affinity shows an intriguing non-linear dependence on the number of Sic1 phosphorylations. The physical nature of this interaction and the factors that govern the dimensions of Sic1 remain unknown and they constitute some our main research goals. 4E-BP2 acts like a regulatory switch: in the non-phosphorylated state it is largely disordered and bound to the initiation factor eIF4E, whereas in the multi-phosphorylated state it folds into a beta-sheet structure and detaches, allowing another protein to dock on eIF4E and initiate translation. For this IDP, the main question concerns the role of phosphate groups, whether they stabilize the folding domain of 4E-BP2, or destabilize the interaction with eIF4E, or both. Other important questions are whether transient interactions of the C-terminal disordered region stabilize the beta fold and whether electrostatic interactions modulate the folding and binding.


G Protein Coupled Receptors (GPCRs): Ligands, Allostery, Signaling and Oligomers

GPCRs constitute the largest superfamily of proteins encoded by mammalian genomes. They serve as transducers of the signal between extracellular ligands such as hormones or drugs and intracellular mediators such as G proteins and arrestins. The physiological processes that involve GPCRs include the visual sense, the gustatory sense, the olfactory sense, behavioral and mood regulation, immune system regulation, and nervous system regulation. This versatility in function allows GPCRs to be the targets of more than 30% of all modern drugs, with the potential to treat many diseases and health conditions, such as diabetes, Parkinson's disease, cardiovascular disease, depression, drug addiction and obesity.

G Protein Coupled Receptors (GPCRs): Ligands, Allostery, Signaling and Oligomers

The M2 muscarinic cholinergic receptor modulates airway, eye and intestinal smooth muscle contraction, heart rate and glandular secretions. The A2A adenosine receptor is involved in regulating myocardial blood flow and dopamine release in the brain. Our SMF studies have shed light on the role of GPCR and G protein oligomers in activation and signalling, in particular the dynamic and ligand-dependent nature of the M2 oligomerization and of receptor-G protein coupling. In A2A we look at the conformational states, the fast segmental motions within the receptor, and the modulation induced by various ligands, with the aim to resolve receptor activation pathways. The coupling between M2 receptors and Gi1 proteins at the membrane of live cells is visualized at single-molecule level with (sub)millisecond resolution. Multicolor single-particle tracking, inter-molecular smFRET and dual-color FCS are corroborated with NMR to provide insights into allosteric modulation, partial and inverse agonism and cooperativity of GPCRs.


Integrating Computational Approaches to Interpret SMF Experiments on Heterogeneous Ensembles

The description of disordered proteins as mere random coils turned out to be oversimplified, and excluded volume effects, long-range contacts and electrostatics have to be considered when reconciling different types of structural data, e.g., NMR, SAXS and smFRET. Hybrid computational-experimental approaches in which sets of protein conformers are selected to fit NMR and SAXS data were previously developed (e.g., ENSEMBLE by the Forman-Kay lab), and we are currently working on incorporating constraints derived from SMF data.

Integrating Computational Approaches to Interpret SMF Experiments on Heterogeneous Ensembles

We collaborate with computational biophysics colleagues (Chan, Rauscher) to integrate course-grained models and molecular dynamics towards a more rigorous SMF analysis. We developed an excluded-volume based polymer model and used it to derive the radius of gyration (Rg) and the shape (asphericity) of conformational sub-ensembles of Sic1 and of the partially unfolded drkN SH3 domain. Based on this computational work with the Chan group, we have built a open webserver application, DICE (Dimensional Inferences from Coarse-grained Ensembles). DICE computes the distribution of Rg values that is consistent with experimental FRET, or the expected FRET and the distribution of Rg from a distribution of end-to-end distances, e.g., derived from coarse-grained simulations or from Protein Ensemble Database (pE-DB).