The PGK-TIM fusion protein from Thermotoga maritima and its constituent parts are intrinsically stable and fold independently.
In the hyperthermophilic bacterium Thermotoga maritima, the two glycolytic enzymes phosphoglycerate kinase (PGK) and triosephosphate isomerase (TIM) are covalently connected forming a tetrameric single-chain PGK-TIM fusion protein. A frameshift allows the translation of PGK alone, whereas TIM activity exclusively resides in the fusion protein (Schurig et al., 1995). Cloning the pgk-tim gene from Thermotoga maritima in Escherichia coli, yields monomeric PGK and tetrameric PGK-TIM fusion protein as authentic recombinant proteins (Beaucamp et al., 1995). Both exhibit high intrinsic stability. The thermal transitions at approximately 80 degrees C are irreversible, rendering determination of thermodynamic data impossible. The half-concentrations, (cGdmCl)1/2, of the guanidinium-chloride induced unfolding transitions are 3.0 and 3.9 M GdmCl for PGK and the PGK-TIM fusion protein, respectively. Monitoring denaturation by activity, fluorescence emission and circular dichroism, deactivation and unfolding of the two-domain PGK is found to precede the transitions of the TIM domain. With increasing temperature, (cGdmCl)1/2 is shifted to lower denaturant concentrations; at the same time, the transitions change from bimodal to unimodal. As indicated by the incomplete reversibility of the deactivation/unfolding/dissociation transitions, misfolding, as well as wrong domain interactions seem to interfere with the correct folding and association of the bienzyme complex.
Beaucamp
N
N
Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
Schurig
H
H
Jaenicke
R
R
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D002942:Circular Dichroism; D004795:Enzyme Stability; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D019791:Guanidine; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014305:Triose-Phosphate Isomerase / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D009097:Multienzyme Complexes; C109485:PGK-TIM fusion protein, Thermotoga maritima; D010735:Phosphoglycerate Kinase; D014305:Triose-Phosphate Isomerase; D019791:Guanidine