There is a class of proteins known as intrinsically disordered proteins (IDPs). These typically do not fold into a well-defined structure until they are bound by a partner, which can be a small molecule, another protein or a nucleic acid. These have been shown to be unstructured in vitro and, in some cases, in vivo. It has recently been hypothesized that many of the binding targets of calmodulin (CaM), an (mostly) ordered protein, are intrinsically disordered. Among these is the important phosphotase calcineurin. Calcineurin is activated when CaM binds to part of a 95 residue disordered regulatory domain (see figure below). We are studying the conformational changes this regulatory domain undergoes when CaM binds. We are also studying other CaM targets known to be disordered. This work uses a variety of techniques, including CD, NMR and fluorescence, coupled with site-directed mutagenesis.
Protein folding has been studied extensively for decades. There are two general goals: protein structure prediction and understanding the protein folding process. Although these two goals are clearly not mutually exclusive, our focus is on the folding process rather than on prediction. The question is, just what steps do proteins go through to fold? To answer this, we must first understand where the folding process starts - protein unfolded states. By this we mean unfolded states under folding conditions. This is distinct from denatured states. A number of groups, including ours, have shown that denaturants perturb the ensemble of states adopted by an unfolded protein. In other words,
We are primarily interested in the conformations adopted by short stretches of sequence in unfolded proteins. Is there any local structure present? To address this we are studying the following:
Peptide models for unfolded states
We are studying short (~13 residue) peptides as models for unfolded proteins. These are useful because we can design sequences with understandable conformational properties and then see what various perturbations do to the ensemble of states. We have initially studied a series of alanine-based peptides that have a tendency towards α-helix formation, but are too short to form stable helices. This system was chosen because a lot is known about α-helix formation. Perturbations include:
We employ circular dichroism (CD) and fluorescence spectroscopy, plus some computer simulations, to measure and deconvolute the ensemble of conformations adopted by each peptide under each set of conditions.