title: Contribution of inter-subunit interactions to the thermostability of Pyrococcus furiosus citrate synthase.
abstract: Using citrate synthase from the hyperthermophile Pyrococcus furiosus (PfCS) as our test molecule, we show through guanidine hydrochloride-induced unfolding that the dimer separates into folded, but inactive, monomers before individual subunit unfolding takes place. Given that forces across the dimer interface are vital for thermostability, a robust computational method was derived that uses the University of Houston Brownian Dynamics (UHBD) program to calculate both the hydrophobic and electrostatic contribution to the dimerisation energy at 100°C. The results from computational and experimental determination of the lowered stability of interface mutants were correlated, being both of the same order of magnitude and placing the mutant proteins in the same order of stability. This computational method, optimised for hyperthermophilic molecules and tested in the laboratory, after further testing on other examples, could be of widespread use in the prediction of thermostabilising mutations in other oligomeric proteins for which dissociation is the first step in unfolding.
authors.affiliation: Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, Bath, BA2 7AY, UK.
authors.forename: Vicky
authors.initials: V
authors.lastname: Moore
authors.forename: Ami
authors.initials: A
authors.lastname: Kanu
authors.forename: Olwyn
authors.initials: O
authors.lastname: Byron
authors.forename: Gordon
authors.initials: G
authors.lastname: Campbell
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Danson
authors.forename: David W
authors.initials: DW
authors.lastname: Hough
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Crennell
mesh_terms: D019843:Archaeal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002950:Citrate (si)-Synthase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003198:Computer Simulation; D004795:Enzyme Stability; D019791:Guanidine / Q000737:chemistry; D006358:Hot Temperature; D006860:Hydrogen Bonding; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D009154:Mutation; D017510:Protein Folding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D055672:Static Electricity; D013329:Structure-Activity Relationship; D013499:Surface Properties; D014461:Ultracentrifugation
chemical_list: D019843:Archaeal Proteins; D021122:Protein Subunits; D002950:Citrate (si)-Synthase; D019791:Guanidine