A compact monomeric intermediate identified by NMR in the denaturation of dimeric triose phosphate isomerase

The denaturation of triose phosphate isomerase (TIM) from Saccharomyces cerevisiae by guanidine hydrochlorids at pH 7.2 has been monitored by NMR spectroscopy in conjunction with optical spectroscopy. In the absence of denaturant, the hydrodynamic radius of 29.6(±0.25) Å and the substantial chemical...

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Veröffentlicht in:Journal of molecular biology 2000-06, Vol.300 (1), p.11-16
Hauptverfasser: Morgan, Charles J., Wilkins, Deborah K., Smith, Lorna J., Kawata, Yasushi, Dobson, Christopher M.
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container_end_page 16
container_issue 1
container_start_page 11
container_title Journal of molecular biology
container_volume 300
creator Morgan, Charles J.
Wilkins, Deborah K.
Smith, Lorna J.
Kawata, Yasushi
Dobson, Christopher M.
description The denaturation of triose phosphate isomerase (TIM) from Saccharomyces cerevisiae by guanidine hydrochlorids at pH 7.2 has been monitored by NMR spectroscopy in conjunction with optical spectroscopy. In the absence of denaturant, the hydrodynamic radius of 29.6(±0.25) Å and the substantial chemical shift dispersion evident in the NMR spectrum are consistent with the highly structured dimeric native state of the protein. On the addition of 2.2 M guanidine hydrochloride the effective hydrodynamic radius increases to 51.4(±0.43) Å, consistent with that anticipated for the polypeptide chain in a highly unstructured random coil state. In 1.1 M guanidine hydrochloride, however, the effective hydrodynamic radius is 24.0(±0.25) Å, a value substantially decreased relative to that of the native dimeric state but very close to that anticipated for a monomeric species with native-like compaction (23.5 Å). The lack of chemical shift dispersion indicates, however, that few tertiary interactions persist within this species. Far UV CD and intrinsic fluorescence measurements show that this compact intermediate retains significant secondary structure and that on average the fluorophores are partially excluded from solvent. Such a species could be important in the formation of dimeric TIM from its unfolded state.
doi_str_mv 10.1006/jmbi.2000.3834
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subjects Circular Dichroism
Dimerization
Guanidine - pharmacology
guanidine hydrochloride
hydrodynamic radius
Magnetic Resonance Spectroscopy
NMR
protein denaturation
Protein Denaturation - drug effects
protein dimerisation
Protein Folding
Protein Structure, Secondary - drug effects
Protein Structure, Tertiary - drug effects
Saccharomyces cerevisiae
Saccharomyces cerevisiae - enzymology
Spectrometry, Fluorescence
TIM barrel
Triose-Phosphate Isomerase - chemistry
Triose-Phosphate Isomerase - metabolism
title A compact monomeric intermediate identified by NMR in the denaturation of dimeric triose phosphate isomerase
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