Hysteresis and conformational drift of pressure-dissociated glyceraldehydephosphate dehydrogenase

Abstract

Pressure dissociation of yeast glyceraldehydephosphate dehydrogenase (GAPDH) was studied by fluorescence spectroscopy. Observations in the range of -5 to 30 degrees C indicate that monomer association into the tetramer proceeds with an enthalpy change of -14 kcal mol-1 and a large increase in entropy which at 25 degrees C amounts to 18 kcal mol-1. The large conformational drift and the low-temperature stability of the tetramer recovered after decompression facilitated a comparison of its properties with those of the native tetramer. Significant differences in absorption and fluorescence-excitation polarization spectra, yield of tryptophan fluorescence, and binding of anilinonaphthalenesulfonate and NADH were observed. At 0 degree C the standard free energies of association of the monomers into the native and drifted tetramers were respectively -32 and -29 kcal mol-1. The volume change upon association measured from the pressure span of the compression curves was 200-230 mL mol-1 but four times as large when derived from the displacement of the compression curves with total protein concentration. This large discrepancy can be explained by the existence in the native tetramer population of a distribution of free energies of association with a dispersion from the mean of about 6 kcal mol-1. At 0 degree C and 1 bar ATP and ADP decreased the stability of the GAPDH tetramer by changes in free energy of association of +3.7 and +4.1 kcal mol-1, respectively. NAD and c-AMP stabilized it by -2.3 and -1.3 kcal mol-1. The variation in sign and magnitude of the ligand-induced changes in free energy of association observed in this case, and previously in hexokinase [Ruan, K., & Weber, G. (1988) Biochemistry 27, 3295], and the heterogeneity of the free energy of association of GAPDH, revealed as indicated above, lead to the conclusion that oligomeric aggregates exist in a variety of conformations that depend upon the protein concentration, temperature, pressure, and the presence of specific ligands. The multiplicity of species revealed by the energetics raises questions about the significance of the structures of oligomeric proteins determined by X-ray crystallography.

Effects of a High-Pressure Treatment on Bovine Gamma Globulin and Its Reduction in Allergenicity

“…A red shift was apparent, with a decrease in the intrinsic fluorescence intensity, thus suggesting a considerable increase in the polarity of the tyrosine and tryptophan environment accompanying the dissociation of subunits under high pressure. 40) These results indicate that the environmental polarity of the tyrosine and tryptophan residues became stronger due to the pressureinduced change in the tertiary structure of the BGG molecule. As shown in Fig.…”

Subunit interactions in hemoglobin probed by fluorescence and high-pressure techniques

“…Alcala and co-workers (Alcala et al, 1987) have interpreted the distributed fluorescence decay in a number of proteins as arising from gound-state conformational heterogeneity. Recently, Ruan and Weber (1989) have concluded from their high-pressure dissociation studies that the subunit affinity in yeast glyceraldehydephosphate dehydrogenase tetramers presents considerable heterogeneity. These observations concerning the heterogeneity of the physical states of macromolecules in vitro raise additional questions as to the existence and eventual role of such heterogeneity in vivo.…”

Modulation of the stability of a gene-regulatory protein dimer by DNA and cAMP.

“…Weber and colleagues (20)(21)(22)(23)(24) and others (25) have studied the dissociation of various oligomeric proteins by high pressure and subsequent reassembly at atmospheric pressure. The association constants reported for dimeric proteins are similar in magnitude to the value we report for CAP.…”