Thus. to improve predictions. we must explicitly consider contributions from rhe??o??stat posi-tions. To that end. the following objectives must be addressed: (i) develop a library of rheo??stat positions to guide and benchmark future computational studies; (ii) develop ge??neral me??thods to identi??fy?? rheostat positions; (iii) compare and contrast the contributions of rheo??stat posi??tions in pro??teins subject to different structural con??straints; and (iv) gener??ate the bio??phys??i??cal data requir-ed to for??mu??late new sub??sti??tution rules for rheostat positions. Work is ongoing to study rheostat posi??tions in globular pro??teins (described in section 2. below).To enhance the impact of our studies and to expand the range of objectives addressed. we will use high risk/high reward protein targets described below.Aims 1 and 2 will generate a database of experimentally iden-tified rheostat positions and their mutational outcomes in proteins that evolved under varied structural constraints. Aim 3 will evaluate whether the locations of rheostat positions correlate with patterns of change found in sequence alignments.