title: The effect of core destabilization on the mechanical resistance of I27.
abstract: It is still unclear whether mechanical unfolding probes the same pathways as chemical denaturation. To address this point, we have constructed a concatamer of five mutant I27 domains (denoted (I27)(5)*) and used it for mechanical unfolding studies. This protein consists of four copies of the mutant C47S, C63S I27 and a single copy of C63S I27. These mutations severely destabilize I27 (DeltaDeltaG(UN) = 8.7 and 17.9 kJ mol(-1) for C63S I27 and C47S, C63S I27, respectively). Both mutations maintain the hydrogen bond network between the A' and G strands postulated to be the major region of mechanical resistance for I27. Measuring the speed dependence of the force required to unfold (I27)(5)* in triplicate using the atomic force microscope allowed a reliable assessment of the intrinsic unfolding rate constant of the protein to be obtained (2.0 x 10(-3) s(-1)). The rate constant of unfolding measured by chemical denaturation is over fivefold faster (1.1 x 10(-2) s(-1)), suggesting that these techniques probe different unfolding pathways. Also, by comparing the parameters obtained from the mechanical unfolding of a wild-type I27 concatamer with that of (I27)(5)*, we show that although the observed forces are considerably lower, core destabilization has little effect on determining the mechanical sensitivity of this domain.
authors.affiliation: School of Biochemistry and Molecular Biology, University of Leeds, United Kingdom.
authors.forename: David J
authors.initials: DJ
authors.lastname: Brockwell
authors.forename: Godfrey S
authors.initials: GS
authors.lastname: Beddard
authors.forename: John
authors.initials: J
authors.lastname: Clarkson
authors.forename: Rebecca C
authors.initials: RC
authors.lastname: Zinober
authors.forename: Anthony W
authors.initials: AW
authors.lastname: Blake
authors.forename: John
authors.initials: J
authors.lastname: Trinick
authors.forename: Peter D
authors.initials: PD
authors.lastname: Olmsted
authors.forename: D Alastair
authors.initials: DA
authors.lastname: Smith
authors.forename: Sheena E
authors.initials: SE
authors.lastname: Radford
mesh_terms: D000595:Amino Acid Sequence; D064211:Connectin; D004305:Dose-Response Relationship, Drug; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009010:Monte Carlo Method; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D064211:Connectin; D009124:Muscle Proteins; D010446:Peptide Fragments; D011506:Proteins; D011494:Protein Kinases