The effect of core destabilization on the mechanical resistance of I27.
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.
Brockwell
David J
DJ
School of Biochemistry and Molecular Biology, University of Leeds, United Kingdom.
Beddard
Godfrey S
GS
Clarkson
John
J
Zinober
Rebecca C
RC
Blake
Anthony W
AW
Trinick
John
J
Olmsted
Peter D
PD
Smith
D Alastair
DA
Radford
Sheena E
SE
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
D064211:Connectin; D009124:Muscle Proteins; D010446:Peptide Fragments; D011506:Proteins; D011494:Protein Kinases