Water Requirements in Monomer Folding and Dimerization of Triosephosphate Isomerase in Reverse Micelles. Intrinsic Fluorescence of Conformers Related to Reactivation

Abstract

The possibility of using reverse micelles to stabilize monomers prior to formation of dimeric triosephosphate isomerase (TPI) from rabbit muscle was studied. TPI denatured with guanidine hydrochloride undergoes reactivation in reverse micelles formed with n-octane, hexanol, cetyltrimethylammonium bromide, and water. Reactivation of around 80% is observed at TPI concentrations of about 2 micrograms/mL of reverse micelles and water concentrations above 4.0%. With 3.0% water, reactivation is about 10%. If denatured TPI is incubated for a few seconds in reverse micelles with 5.0% water (or higher) followed by incubation in 3.0% water, reactivation is between 35% and 50%. That is, a brief exposure of denatured TPI to reverse micelles with a relatively high water concentration yielded a significant amount of structures competent for formation of catalytically active dimers. As evidenced by kinetic data, these structures correspond to monomers of TPI [Garza-Ramos, G. Tuena de Gómez-Puyou, M., Gómez-Puyou, A., & Gracy R. W. (1992) Eur. J. Biochem. 208, 389-395]. After a 5-2.0% water transition, competent monomers were stabilized for at least 30 min; a subsequent rise in water concentration led to dimerization and appearance of activity. By changes in the amount of water, it was possible to determine in reverse micelles the amount of water required for monomer folding and dimerization; i.e., less water was required in the dimerization step. Experiments with a model system, trypsin and the soybean inhibitor, showed that, in reverse micelles with 2.0% water, protein-protein interactions readily take place.(ABSTRACT TRUNCATED AT 250 WORDS)

Equilibrium and Kinetic Folding of Rabbit Muscle Triosephosphate Isomerase by Hydrogen Exchange Mass Spectrometry

“…18 Another study of rabbit TIM folding found no evidence for an intermediate when the protein was folded in water, but did find evidence for an intermediate when the protein was folded in reverse micelles. 19 Neither the structure nor the affects of the micellar environment on the folding process were determined. Results of another study on rabbit muscle TIM folding in reverse micelles, but using FRET measurements, suggested that the intermediate may be an inactive dimer.…”

Oxidative refolding of reduced, denatured lysozyme in AOT reverse micelles

“…Water requirements in monomer folding and dimerization of triosephosphate isomerase in reverse micelles have been uncovered by Fernandez-Velasco et al [3], and these studies have led to the concept that water is fundamental in the function and stability of a protein [3,26,27]. However, the precise contribution of water and particularly how much water is needed for protein folding are not well understood so far.…”

“…So far several groups have already investigated protein refolding and unfolding in reverse micelles [3][4][5][6][7]. FernandezVelasco and colleagues have found that water plays important roles in monomer folding and dimerization of triosephosphate isomerase in reverse micelles [3]. Shastry and Eftink have observed a reversible transition of thermal unfolding of RNase T1 in reverse micelles [4].…”

Preliminary X-ray diffraction studies of rabbit muscle triose phosphate isomerase (TIM)

“…However, despite being one of the ®rst glycolytic enzymes to be puri-®ed (Krietsch et al, 1970;Scopes, 1977), the structure of rabbit muscle TIM has not yet been reported. The amino-acid sequence of this protein is known (Corran & Ley, 1975) and its kinetic properties have been extensively studied (Garza-Ramos et al, 1992;Fernandez-Velasco et al, 1995) and compared with those of T. brucei brucei and yeast (Saccharomyces cerevisiae) TIMs (Lambeir et al, 1987). Biophysical studies of the enzyme have been carried out by infrared spectroscopy (Castresana et al, 1988) and electron microscopy (Tanaka et al, 1994).…”