Mutations that significantly change the stability, flexibility and quaternary structure of the l‐lactate dehydrogenase from Bacillus megaterium
Authors: KOTIK Michael , ZUBER Herbert 
Published: March 2005

In order to investigate the physical basis of protein stability, two mutant L‐lactate dehydrogenases (LDH) and the wild‐type enzyme from Bacillus megaterium were analyzed for differences in quaternary structure, global protein conformation, thermal stability, stability against guanidine hydrochloride, and polypeptide chain flexibility. One mutant enzyme, ([T29A, S39A]LDH), differing at two positions in the α‐B helix, exhibited a 20°C increase in thermostability. Hydrogen/deuterium exchange revealed a rigid structure of this enzyme at room temperature. The substitutions Ala37 to Val and Met40 to Leu destabilize the protein. This is observable in a greater susceptibility to thermal denaturation and in an unusual monomer/dimer/tetramer equilibrium in the absence of fructose 1,6‐bisphosphate Fru(1,6)P2. The stability, flexibility and protein‐conformation measurements were all performed in the presence of 5 mM Fru(1,6)P2, i.e. under conditions where the three investigated LDH species are stable tetramers. Tryptophan fluorescence was used to monitor the unfolding in guanidine HCl of two local structures in or very close to the β‐sheets at the protein surface. The LDHs form folding intermediates in guanidine HCl that aggregate at elevated temperatures. Pronounced differences between the three investigated enzymes are found in their ability to aggregate. The exchange of Thr29 and Ser39 for Ala leads to significantly less aggregation in guanidine HCl than is observed for wild‐type LDH. Using 8‐anilinonaphthalene‐1‐sulfonic acid, the folding intermediates were shown to be in accordance with molten‐globule‐like structures. We have found, by means of molecular sieve chromatography, that the [T29A, S39A]LDH with its increased thermostability has lower susceptibility to disintegrate into monomers in guanidine HCl at 25°C. Despite the differences in aggregation at low guanidine HCl concentrations and temperatures above 25°C, the molten‐globule‐like structures of the three investigated LDH species are structurally similar as shown by molecular‐sieve chromatography. Although the thermostabilities of the three LDH species are so different in aqueous buffers, their stabilities in guanidine HCl at 20°C are, surprisingly, almost identical. Some comments are made as to the origin of the observed difference between thermal and guanidine HCl stabilities of the LDH. Near‐ultraviolet and far‐ultraviolet circular dichroism measurements, as well as differences in the amount of activation by Fru(1,6)P2, point to small global structural rearrangements caused by the mutations. Conformational changes upon Fru(1,6)P2 binding or point mutations in the α‐B helix show that the Fru(1,6)P2‐binding site and the α‐B helix are structurally linked together. Binding studies with Nile red revealed temperature‐dependent differences in the surface hydrophobicity of the three investigated enzymes. The data suggest that α‐helices of structural importance (hot spots) prove to be good targets for engineering the thermal stability of a protein.