Protein folding, stability, structure and function in the cell

Project description

Protein folding in vitro has been extensively characterized in dilute conditions. However, the total macromolecule concentration in a cell may be as high as 400 mg/ml. This reduction of free volume is predicted to dramatically affect protein conformational and binding equilibria by favoring the states that exclude the least volume, which normally involves the compact ‘native’ states. In this project, we will systematically investigate, by mutagenesis, biophysical, and computational methods, the in vitro folding and binding reactions of three strategic proteins (i.e., azurin, flavodoxin and myoglobin) that have distinct tertiary folds (i.e., -barrel,  and -helical) and chemically-different cofactors (i.e., copper ion, flavin moiety and heme) at conditions mimicking the space confined in vivo milieu using synthetic macromolecular crowding agents of different chemistry, shape, size and concentration. Our goals are to obtain mechanisms for how volume-restricted environments, such as those found in living organisms, regulate protein stability, folding mechanisms, the final folded structure, and physiological function. The proposed project is pioneering since there are no reported studies of the effects of macromolecular crowding on folding and function of cofactor-dependent proteins.
Aim 1. What are the effects of macromolecular crowding on equilibrium stability of proteins with different folds, cofactors and mechanisms? We predict that crowding has stabilizing effects on all proteins; the magnitude will depend on intrinsic stability and protein fold. Thermal and chemical unfolding of apo- and holo-forms of azurin, flavodoxin and myoglobin will be carried out using spectroscopic methods as a function of increasing crowding levels using inert polymers (i.e., Ficoll, dextran, and polyethylene glycol; PEG) in biologically relevant amounts.
Aim 2. What are the structural effects on folded, intermediate, and unfolded states due to macromolecular crowding? We predict that the proteins become more compact and, for the native states, more secondary structure is induced in the presence of crowding agents. The magnitude of structural changes in apo- and holo-proteins due to macromolecular crowding will be assessed by spectroscopy. Mapping of crowding-induced structural changes to specific residues in each protein will be revealed by NMR and computational methods.
Aim 3. Do macromolecular crowding effects originate in altered unfolding and/or folding kinetics and how are the folding-transition states affected? We predict that macromolecular crowding speeds up folding to a certain point; upon further additions of crowders, folding will be retarded. Protein folding and unfolding kinetics will be obtained as a function of chemical denaturants in the presence of fixed amounts of crowding agents. The phi-value approach (i.e., Ala variants) will provide residue-specific resolution of effects on intermediate and transition-state structures.
Aim 4. How does macromolecular crowding affect cofactor binding and protein-functional properties? We hypothesize that macromolecular crowding makes the cofactors bind tighter to the proteins and that redox potentials and ligand-binding kinetics are modulated. Rate and equilibrium constants for cofactor binding to apo-proteins will be tested as a function of crowding agents; functional properties (i.e., cofactor-redox potentials, electron-transfer and ligand binding) will be determined at conditions that mimic the in vivo milieu.