title: A structural double-mutant cycle: estimating the strength of a buried salt bridge in barnase.
abstract: Double-mutant cycles are widely used in the field of protein engineering to measure intermolecular and intramolecular interactions. Ideally, there should be no structural rearrangement of the protein on making the two single mutations and the double mutation within the cycle. However, structural pertubation on mutation does not preclude the use of this method, providing the sum of the changes in the single mutants equals the change in the double mutant. In this way, the energy associated with any structural rearrangement cancels in the double-mutant cycle. Previously, the contribution of a buried salt bridge between Arg69 and Asp93 in barnase to the stability of the folded protein has been determined by double-mutant cycle analysis. In order to determine whether the measured interaction of -14.0 kJ mol(-1) represents the true interaction energy, the crystal structure of each mutant within the double-mutant cycle was solved. Although mutation results in structural shifts, the majority of those in the single mutants are also found in the double mutant; their energetic effects in the double-mutant cycle are therefore cancelled. This study highlights the robust nature of the double-mutant cycle analysis.
authors.affiliation: Section of Structural Biology, The Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, England.
authors.forename: Cara K
authors.initials: CK
authors.lastname: Vaughan
authors.forename: Pia
authors.initials: P
authors.lastname: Harryson
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Buckle
authors.forename: Alan R
authors.initials: AR
authors.lastname: Fersht
mesh_terms: D001120:Arginine / Q000737:chemistry; D001426:Bacterial Proteins; D008958:Models, Molecular; D009154:Mutation; D012260:Ribonucleases / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D001120:Arginine; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease