Electrostatic effects on the free-energy balance in folding a ribosome-inactivating protein

Electrostatics of globular proteins provides structural integrity as well a s specificity of biological function. This dual role is particularly striki ng for ricin A-chain (RTA), an N-glycosidase which hydrolyzes a single aden ine base from a conserved region of rRNA. The reported X-ray crystallograph ic structure of the RTA mutant E177A demonstrated a remarkable rescue of ch arge balance in the active site, achieved by the rotation of a second gluta mic acid (Glu-208) into the vacated space. To understand this conformationa l reorganization, molecular-dynamics simulations were applied to estimate r elative free energies that govern the thermodynamic stability of E177A toge ther with mutants E177Q and E177D. The simulations anticipate that while E1 77A is a non-conservative substitution, the protein is more stable than the other two mutants. However, the structural plasticity of the RTA active si te is not obtained penalty-free, rather E177A among the mutants shows the l argest unfavorable net change in the electrostatic contribution to folding. Of the E177A folded state, reorganization of Glu-208 lowers the electrosta tic cost of the free-energy change, yet interestingly, protein interactions oppose the rotational shift, while solvent effects favor the transition. ( C) 2001 Elsevier Science B.V. All rights reserved.