THE SECOND CYCLE OF A PROTEIN ENGINEERING PROJECT: THE REPLACEMENT OF A LOOP IN MONOMERIC TRIOSEPHOSPHATE ISOMERASE (MONOTIM). R.K. Wierenga, N. Thanki, W. Schliebs, J. Ph. Zeelen, R. Abagyan¹, R. Jaenicke², EMBL, Postfach 102209, D69012 Heidelberg, Germany, ¹Skirball Institute, NYU, New York, USA, ²University of Regensburg, Regensburg, Germany

The disorderd loop-1 of monoTIM, including the catalytic lysine-13, has been redesigned with the modelling package ICM, aimed at rigidifying this loop. The new variant is as active as monoTIM and its crystal structure has been determined at 2.6Å resolution.

Protein loops play an important role in molecular recognition. In proteins with the TIM-barrel framework eight loops determine the shape of the active site pocket. In triosephosphate isomerase (TIM), some of these loops (in particular loop-3) are also crucial for formation of very stable dimers. In a first protein design experiment with TIM we designed a new loop-3 which converted the protein into a stable monomeric TIM. The protein, referred to as monoTIM, has 1/1000 fold less catalytic activity than wild type TIM, possibly due to an increased flexibility of some active site loops. In particular loop-1 is highly disordered in different crystal structures of monoTIM(1). Nevertheless, the importance of the catalytic Lys13 of loop-1 for the catalytic activity of monoTIM has been demonstrated by site directed mutagenesis (2). We started a second cycle of protein engineering aimed to rigidify this loop. The actual loop modelling was done with ICM on a seven residue loop. The coordinates of the modelled structure have been deposited at the PDB (1MTM). The variant was made, purified, characterised and crystallised. First insight into the crystal structure of this new variant (at 2.6Å resolution) suggests that loop-1 has indeed been rigidified, whereas Lys13 is still pointing into the active site.

(1). Structure (1995),3,669-679.

(2). Protein Science (1996), 5, 229-239.