title: Folding kinetics of the human prion protein probed by temperature jump.
abstract: Temperature-jump perturbation was used to examine the relaxation kinetics of folding of the human prion protein. Measured rates were very fast (approximately 3,000 s(-1)), with the extrapolated folding rate constant at approximately 20 degrees C in physiological conditions reaching 20,000 s(-1). By a mutational analysis of core residues, we found that only 2, on the interface of helices 2 and 3, have significant phi-values in the transition state. Interestingly, a mutation sandwiched between the above 2 residues on the helix-helix contact interface had very little effect on the overall free energy of folding but led to the formation of a monomeric misfolded state, which had to unfold to acquire the native PrP(C) conformation. Another mutation that led to a marked destabilization of the native fold also formed a misfolded intermediate, but this was aggregation-prone despite the native state of this mutant being soluble. Taken together, the data imply that this fast-folding protein has a transition state that is not compact (m value analysis gives a beta(t) value of only 0.3) but contains a developing nucleus between helices 2 and 3. The fact that a mutation in this nucleus had a negligible effect on stability but still led to formation of aberrant conformations during folding implies an easily perturbed folding mechanism. It is notable that in inherited forms of human prion disease, where point mutations produce a lethal dominant condition, 20 of the 33 amino acid replacements occur in the helix-2/3 sequence.
authors.affiliation: Medical Research Council Prion Unit, Institute of Neurology, Queen Square, London WC1N 3BG, United Kingdom.
authors.forename: Tanya
authors.initials: T
authors.lastname: Hart
authors.forename: Laszlo L P
authors.initials: LL
authors.lastname: Hosszu
authors.forename: Clare R
authors.initials: CR
authors.lastname: Trevitt
authors.forename: Graham S
authors.initials: GS
authors.lastname: Jackson
authors.forename: Jonathan P
authors.initials: JP
authors.lastname: Waltho
authors.forename: John
authors.initials: J
authors.lastname: Collinge
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Clarke