PROTEIN ENGINEERING WITH MONOMERIC TRIOSEPHOSPHATE ISOMERASE (MONOTIM) - THE MODELING AND STRUCTURE VERIFICATION OF A 7-RESIDUE LOOP

Protein engineering experiments have been carried out with loop-1 of m onomeric triosephosphate isomerase (monoTIM). Loop-1 of monoTIM is dis ordered in every crystal structure of liganded monoTIM, but in the wil dtype TIM it is a very rigid dimer interface loop. This loop connects the first beta-strand with the first alpha-helix of the TIM-barrel sca ffold. The first residue of this loop, Lys13, is a conserved catalytic residue. The protein design studies with loop-1 were aimed at rigidif ying this loop such that the Lys13 side chain points in the same direc tion as seen in wild type. The modelling suggested that the loop shoul d be made one residue shorter. With the modelling package ICM the opti mal sequence of a new seven-residue loop-1 was determined and its stru cture was predicted. The new variant could be expressed and purified a nd has been characterized. The catalytic activity and stability are ve ry similar to those of monoTIM. The crystal structure (at 2.6 Angstrom resolution) shows that the experimental loop-1 structure agrees well with the modelled loop-1 structure. The direct superposition of the se ven loop residues of the modelled and experimental structures results in an r.m.s. difference of 0.5 Angstrom for the 28 main chain atoms. T he good agreement between the predicted structure and the crystal stru cture shows that the described modelling protocol can be used successf ully for the reliable prediction of loop structures.