Abstract:
:The equilibrium and kinetic folding properties of a small oncogene product, P13(MTCP1), of novel topology have been investigated using perturbation by guanidine hydrochloride and observation by fluorescence, circular dichroism and two-dimensional heteronuclear NMR spectroscopy. The structure of P13(MTCP1) is comprised of a canonical filled beta-barrel, although the topology of the structure is absolutely unique, rendering the folding properties of this protein of great interest. Equilibrium measurements of the intrinsic fluorescence emission spectrum, the fluorescence decay, the circular dichroism spectrum and the (15)N-(1)H heteronuclear single quantum coherence (HSQC) correlation spectrum as a function of increasing concentrations of denaturant showed no evidence for the population of any equilibrium intermediates, although negative amplitudes on the blue edge of the tryptophan emission and loss of intensity of the native HSQC correlation peaks were indicative of increased conformational dynamics at low denaturant concentrations. The free energy and cooperativity of unfolding as observed by fluorescence and circular dichroism were in relatively good agreement, also consistent with a two-state transition. Kinetics measurements of the fluorescence emission as a function of denaturant concentration revealed that P13(MTCP1) is the slowest folding beta-structure protein reported to date. Comparison of the activation cooperativity values (m(f) and m(u)) indicates that the structure of the transition state is quite close to the folded state in terms of exposed surface area. The calculated contact order of P13(MTCP1) is relatively low and does not appear to explain its slow rate of folding. We suggest that the complex topology of this protein, which would require the ordering of the beta-barrel through a long loop joining the two L-shaped components of the barrel, could provide an explanation for this slow folding.
journal_name
J Mol Bioljournal_title
Journal of molecular biologyauthors
Roumestand C,Boyer M,Guignard L,Barthe P,Royer CAdoi
10.1006/jmbi.2001.4928keywords:
subject
Has Abstractpub_date
2001-09-07 00:00:00pages
247-59issue
1eissn
0022-2836issn
1089-8638pii
S0022-2836(01)94928-9journal_volume
312pub_type
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