title: The PGK-TIM fusion protein from Thermotoga maritima and its constituent parts are intrinsically stable and fold independently.
abstract: 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.
authors.affiliation: Institut für Biophysik und Physikalische Biochemie, Universität Regensburg, Germany.
authors.forename: N
authors.initials: N
authors.lastname: Beaucamp
authors.forename: H
authors.initials: H
authors.lastname: Schurig
authors.forename: R
authors.initials: R
authors.lastname: Jaenicke
mesh_terms: 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
chemical_list: D001426:Bacterial Proteins; D009097:Multienzyme Complexes; C109485:PGK-TIM fusion protein, Thermotoga maritima; D010735:Phosphoglycerate Kinase; D014305:Triose-Phosphate Isomerase; D019791:Guanidine