Protein Engineering, Vol 10, 159-167, Copyright © 1997 by Oxford University Press
N Thanki, JP Zeelen, M Mathieu, R Jaenicke, RA Abagyan, RK Wierenga and W Schliebs
Protein engineering experiments have been carried out with loop-1 of
monomeric triosephosphate isomerase (monoTIM). Loop-1 of monoTIM is
disordered in every crystal structure of liganded monoTIM, but in the
wild-type 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
scaffold. The first residue of this loop, Lys13, is a conserved catalytic
residue. The protein design studies with loop-1 were aimed at rigidifying
this loop such that the Lys13 side chain points in the same direction as
seen in wild type. The modelling suggested that the loop should be made one
residue shorter. With the modelling package ICM the optimal sequence of a
new seven-residue loop-1 was determined and its structure was predicted.
The new variant could be expressed and purified and has been characterized.
The catalytic activity and stability are very similar to those of monoTIM.
The crystal structure (at 2.6 A resolution) shows that the experimental
loop-1 structure agrees well with the modelled loop-1 structure. The direct
superposition of the seven loop residues of the modelled and experimental
structures results in an r.m.s. difference of 0.5 A for the 28 main chain
atoms. The good agreement between the predicted structure and the crystal
structure shows that the described modelling protocol can be used
successfully for the reliable prediction of loop structures.
ARTICLES
Protein engineering with monomeric triosephosphate isomerase (monoTIM): the modelling and structure verification of a seven-residue loop
Institut fur Biophysik und Physikalische Biochemie, Universitt Regensburg, Germany.
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