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

The denaturation of triose phosphate isomerase (TIM) from Saccharomyces cer evisiae by guanidine hydrochlorids at pH 7.2 has been monitored by NMR spec troscopy in conjunction with optical spectroscopy. In the absence of denatu rant, the hydrodynamic radius of 29.6(+/-0.25) Angstrom 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 increase s to 51.4(+/-0.43) Angstrom, consistent with that anticipated for the polyp eptide chain in a highly unstructured random coil state. In 1.1 M guanidine hydrochloride, however, the effective hydrodynamic radius is 24.0(+/-0.25) Angstrom, a value substantially decreased relative to that of the native d imeric state but very close to that anticipated for a monomeric species wit h native-like compaction (23.5 Angstrom). The lack of chemical shift disper sion 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 av erage the fluorophores are partially excluded from solvent. Such a species could be important in the formation of dimeric TIM from its unfolded state. (C) 2000 Academic Press.