Ligand-induced conformational perturbations in androgen receptor (AR) are important in coactivator recruitment and transactivation. However, molecular rearrangements in AR ligand binding domain (AR-LBD) associated with agonist binding and their kinetic and thermodynamic parameters are poorly understood. We utilized steady state second derivative absorption and emission spectroscopy, pressure and temperature perturbations, and bis-ANS partitioning to determine the kinetics and thermodynamics of the conformational changes in AR-LBD after DHT binding.

In presence of DHT, the second derivative absorption spectrum showed a red shift and a change in peak-to-peak distance. Emission intensity increased upon DHT binding and center of spectral mass was blue shifted denoting conformational changes resulting in more hydrophobic environment for tyrosines and tryptophans within a more compact DHT-bound receptor. In pressure perturbation calorimetry, DHT-induced energetic stabilization increased the Gibbs Free Energy of unfolding to 8.4 ± 1.3 kcal/mol from 3.5 ± 1.6 kcal/mol. Bis-ANS partitioning studies revealed that upon DHT binding, AR-LBD underwent biphasic rearrangement with a high activation energy (13.4 kcal/mol). An initial, molten globule-like burst phase (k 30s^-1) with greater solvent accessibility was followed by rearrangement (k0.01s^-1) leading to a more compact conformation than apo AR-LBD. Molecular simulations demonstrated unique sensitivity of tyrosine and tryptophan residues during pressure unfolding with rearrangement of residues in the co-activator recruitment surfaces distant from ligand binding pocket. Conclusions: DHT binding leads to energetic stabilization of AR-LBD domain and substantial rearrangement of residues distant from the ligand binding pocket. DHT binding to AR-LBD involves biphasic receptor rearrangement including population of a molten globule-like intermediate state.