title: Thermodynamic linkage in the GrpE nucleotide exchange factor, a molecular thermosensor.
abstract: GrpE is the nucleotide exchange factor for the Escherichia coli molecular chaperone DnaK, the bacterial homologue of Hsp70. In the temperature range of the bacterial heat shock response, the long helices of GrpE undergo a helix-to-coil transition, and GrpE exhibits non-Arrhenius behavior with respect to its nucleotide exchange function. It is hypothesized that GrpE acts as a thermosensor and that unwinding of the long helices of E. coli GrpE reduces its activity as a nucleotide exchange factor. In turn, it was proposed that temperature-dependent down-regulation of the activity of GrpE may increase the time in which DnaK binds its substrates at higher temperatures. A combination of thermodynamic and hydrodynamic techniques, in concert with the luciferase refolding assay, were used to characterize a molecular mechanism in which the long helices of GrpE are thermodynamically linked with the beta-domains via an intramolecular contact between Phe86 and Arg183. These "thermosensing" long helices were found to be necessary for full activity as a nucleotide exchange factor in the luciferase refolding assay. Point mutations in the beta-domains and in the long helices of GrpE destabilized the beta-domains. Engineered disulfide bonds in the long helices alternately stabilized the long helices and the four-helix bundle. This allowed the previously reported 75 degrees C thermal transition seen in the excess heat capacity function as monitored by differential scanning calorimetry to be further characterized. The observed thermal transition represents the unfolding of the four-helix bundle and the beta-domains. The thermal transitions for these two domains are superimposed but are not thermodynamically linked.
authors.affiliation: Boston Biomedical Research Institute, 64 Grove Street, Watertown, Massachusetts 02472, USA.
authors.forename: Amy D
authors.initials: AD
authors.lastname: Gelinas
authors.forename: Joseph
authors.initials: J
authors.lastname: Toth
authors.forename: Kelley A
authors.initials: KA
authors.lastname: Bethoney
authors.forename: Knut
authors.initials: K
authors.lastname: Langsetmo
authors.forename: Walter F
authors.initials: WF
authors.lastname: Stafford
authors.forename: Celia J
authors.initials: CJ
authors.lastname: Harrison
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D006360:Heat-Shock Proteins / Q000737:chemistry* / Q000235:genetics; D008156:Luciferases / Q000737:chemistry; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017510:Protein Folding; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014461:Ultracentrifugation
chemical_list: D001426:Bacterial Proteins; D004220:Disulfides; D029968:Escherichia coli Proteins; C068113:GrpE protein, Bacteria; C489400:GrpE protein, E coli; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D008156:Luciferases