Dissociation of yeast hexokinase by hydrostatic pressure

The pressure-induced dissociation of the isozymes P1 and P2 of hexokinase was investigated by studies of the spectral shift of the intrinsic protein fluorescence and by the fluorescence polarization of dansyl conjugates. The free energy of association of the monomers at atmospheric pressure, Katm, was -14.2 kcal mol-1 at 20 degrees C and -11.4 kcal mol-1 at 0 degrees C. The positive enthalpy indicates that the association of the monomers is entropy-driven, overcoming the negative enthalpy of hydration of the subunit interfaces. At 0 degrees C and 1 bar, glucose stabilizes the association by -1.1 kcal mol-1 and the binding of both adenosine 5'-(beta, gamma-methylenetriphosphate) (AMPPCP) and glucose by an even larger amount, -1.34 kcal mol-1. Paradoxically, adenosine 5'-triphosphate (ATP), or AMPPCP, in the absence of glucose destabilizes the association by +0.34 kcal mol-1, while adenosine 5'-diphosphate (ADP) stabilizes it by -0.6 kcal mol-1. Comparison of dV0, the apparent standard volume of association, at different pHs and temperatures indicates that its value (115-160 mL mol-1) is strongly dependent upon the ionization of a group at the subunit interface with a pK near neutrality. Under dissociating pressures, trypsin action results in permanent dissociation of the dimer, confirming earlier observations of Colowick by less direct methods. The P1 and P2 enzymes differ in Katm and dV0 and markedly so in the effects of salt upon the stability of the dimer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermodynamics of reversible monomer-dimer association of tubulin

The equilibrium between the rat brain tubulin alpha beta dimer and the dissociated alpha and beta monomers has been studied by analytical ultracentrifugation with use of a new method employing short solution columns, allowing rapid equilibration and hence short runs, minimizing tubulin decay. Simultaneous analysis of the equilibrium concentration distributions of three different initial concentrations of tubulin provides clear evidence of a single equilibrium characterized by an association constant, Ka, of 4.9 X 10(6) M-1 (Kd = 2 X 10(-7) M) at 5 degrees, corresponding to a standard free energy change on association delta G degrees = -8.5 kcal mol-1. Colchicine and GDP both stabilize the dimer against dissociation, increasing the Ka values (at 4.5 degrees C) to 20 X 10(6) and 16 X 10(6) M-1, respectively. Temperature dependence of association was examined with multiple three-concentration runs at temperatures from 2 to 30 degrees C. The van't Hoff plot was linear, yielding positive values for the enthalpy and entropy changes on association, delta S degrees = 38.1 +/- 2.4 cal deg-1 mol-1 and delta H degrees = 2.1 +/- 0.7 kcal mol-1, and a small or zero value for the heat capacity change on association, delta C p degrees. The entropically driven association of tubulin monomers is discussed in terms of the suggested importance of hydrophobic interactions to the stability of the monomer association and is compared to the thermodynamics of dimer polymerization.     

Hysteresis and conformational drift of pressure-dissociated glyceraldehydephosphate dehydrogenase

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.     

Dissociation and unfolding of cold-active alkaline phosphatase from atlantic cod in the presence of guanidinium chloride.

Cold-adaptation of enzymes involves improvements in catalytic efficiency. This paper describes studies on the conformational stability of a cold-active alkaline phosphatase (AP) from Atlantic cod, with the aim of understanding more clearly its structural stability in terms of subunit dissociation and unfolding of monomers. AP is a homodimeric enzyme that is only active in the dimeric state. Tryptophan fluorescence, size-exclusion chromatography and enzyme activity were used to monitor alterations in conformational state induced by guanidinium chloride or urea. In cod AP, a clear distinction could be made between dissociation of dimers into monomers and subsequent unfolding of monomers (fits a three-state model). In contrast, dimer dissociation of calf AP coincided with the monophasic unfolding curve observed by tryptophan fluorescence (fits a two-state model). The DeltaG for dimer dissociation of cod AP was 8.3 kcal.mol-1, and the monomer stabilization free energy was 2.2 kcal.mol-1, giving a total of 12.7 kcal.mol-1, whereas the total free energy of calf intestinal AP was 17.3 kcal.mol-1. Thus, dimer formation provided a major contribution to the overall stability of the cod enzyme. Phosphate, the reaction product, had the effect of promoting dimer dissociation and stabilizing the monomers. Cod AP has reduced affinity for inorganic phosphate, the release of which is the rate-limiting step of the reaction mechanism. More flexible links at the interface between the dimer subunits may ease structural rearrangements that facilitate more rapid release of phosphate, and thus catalytic turnover.     

Active-site copper and zinc ions modulate the quaternary structure of prokaryotic Cu,Zn superoxide dismutase

The influence of the constitutive metal ions on the equilibrium properties of dimeric Photobacterium leiognathi Cu,Zn superoxide dismutase has been studied for the wild-type and for two mutant protein forms bearing a negative charge in the amino acid clusters at the dimer association interface. Depletion of copper and zinc dissociates the two mutant proteins into monomers, which reassemble toward the dimeric state upon addition of stoichiometric amounts of zinc. Pressure-dependent dissociation is observed for the copper-depleted wild-type and mutated enzymes, as monitored by the fluorescence shift of a unique tryptophan residue located at the subunit association interface. The spectral shift occurs slowly, reaching a plateau after 15-20 minutes, and is fully reversible. The recovery of the original fluorescence properties, after decompression, is fast (less than four minutes), suggesting that the isolated subunit has a relatively stable structure, and excluding the presence of stable intermediates during the dimer-monomer transition. The dimer dissociation process is still incomplete at 6.5 kbar for the copper-depleted wild-type and mutated enzymes, at variance with what is generally observed for oligomeric proteins that dissociate below 3 kbar. Measurement of the degree of dissociation, at two different protein concentrations, allows us to calculate the standard volume variation upon association, Delta V, and the dissociation constant K(d0), at atmospheric pressure, (25 ml/mol and 3 x 10(-7)M, respectively). The holoprotein is fully dimeric even at 6.5 kbar, which allows us to evaluate a lower Delta G degrees limit of 11.5 kcal/mol, corresponding to a dissociation constant K(d0)<10(-9)M.     

Hydrostatic pressure and calcium-induced dissociation of calpains

The dissociation of mu- and m-calpains was studied by fluorescence spectroscopy under high hydrostatic pressure (up to 650 MPa). Increasing pressure induced a red shift of the tryptophan fluorescence of the calcium-free enzyme. The concentration dependence of the spectral transition was consistent with a pressure-induced dissociation of the subunits. Rising temperature increased the stability of calpain heterodimers and confirmed the predominance of hydrophobic interactions between monomers. At saturating calcium, the spectral transition was not observed for native or iodoacetamide-inactivated calpains, indicating that they were already dissociated by calcium. The reaction volume was about -150 ml mol-1 for both isoforms, and the dissociation constants at atmospheric pressure are approximately 10-12 M and 10-15 M for mu- and m-calpains, respectively. This result indicates a tighter interaction in the isoform that requires higher calcium concentration for activity.     

The monovalent cation-induced association of formyltetrahydrofolate synthetase subunits. Kinetic and thermodynamic aspects.

Formyltetrahydrofolate synthetase monomers are converted to catalytically active tetramers in the presence of monovalent cations. The stoichiometry of the reaction is 4M + 2C+ in equilibrium M4C2(2+). A positive deltaS compensates for an unfavorable positive deltaH so that the overall reaction is exergonic. Both deltaH and deltaS become more positive as the temperature is increased. Association of subunits of the enzyme prepared from Clostridium cylindrosporum is second order with respect to monomer concentration, consistent with a rate-determining dimerization step. Activation parameters for this step at 20 degrees are: deltaG, 12.6 kcal mol-1; deltaH, 12.5 kcal mol-1; deltaS, -05 e.u. The rate-limiting step for the cation-dependent association of Clostridium acidi-urici monomers is believed to be a conformational alteration since first order kinetics is observed. The Eyring plot of the kinetic data obtained for the C. acidi-urici system has a sharp break at 15 degrees. Activation parameters for cation-induced association at 20 degrees are: deltaG, 21.5 kcal mol-1; deltaH, 14.0 kcal mol-1; deltaS, -26.6 e.u.     

Defining folding and unfolding reactions of apocytochrome b5 using equilibrium and kinetic fluorescence measurements.

The refolding and unfolding kinetics of a soluble domain of apocytochrome b5 extending from residue 1 to 104 have been characterized using stopped flow and equilibrium-based fluorescence methods. The isolated apoprotein unfolds reversibly in the presence of GuHCl. From cooperative unfolding curves, the conformational stability (Delta G(uw)), in the absence of denaturant, is estimated to be 11.6 +/- 1.5 kJ mol-1 at 10 degrees C. The stability of apocytochrome b5 is lower than that of the corresponding form of the holoprotein (Delta G approximately 25 kJ mol-1) and exhibits a transition midpoint at 1.6 M GuHCl. Kinetic studies support the concept of a two-state model with both unfolding and refolding rates showing an exponential dependence on denaturant concentration with no evidence of the formation of transient intermediates in either limb of the chevron plot. Apocytochrome b5 is therefore an example of a protein in which both kinetics and equilibria associated with folding are described by a two-state model. The values of mku and mkf obtained from kinetic analysis are an indication of a transition state (mku/meq of 0.29) that resembles the native form by retaining similar solvent accessibility and many of the noncovalent interactions found in the apoprotein. The changes in heat capacity support a transition state that resembles the apoprotein with a value for Delta Cpf of -3.6 kJ mol-1 K-1 estimated for the refolding reaction. From these measurements, a model of refolding that involves the rapid nucleation of hydrophobic residues around Trp26 is suggested as a major event in the formation of the native apoprotein.     

Dissociation of purified erythrocyte Ca(2+)-ATPase by hydrostatic pressure.

Subunit interactions in the Ca(2+)-ATPase from erythrocyte plasma membranes were investigated through a combination of fluorescence spectroscopy and high-pressure techniques. Application of hydrostatic pressure in the range of 1 bar to 2.4 kbar promoted full dissociation of the ATPase, as revealed by spectral shifts of the intrinsic fluorescence emission and by changes in the fluorescence polarization of dansyl-conjugated ATPase. Pressure dissociation of the ATPase displayed a dependence on protein concentration compatible with dissociation of a dimer. Calculated from pressure-dissociation curves, the standard volume change dV0 for the association of subunits was 43-50 ml/mol and K0, the dissociation constant at atmospheric pressure, was 6-9 x 10(-8) M. Addition of Ca2+ stabilized the dimeric ATPase structure against pressure dissociation, whereas addition of vanadate facilitated dissociation by pressure. These results suggest that intersubunit interactions depend on the equilibrium between the two major conformational states E1 and E2 of the ATPase. Addition of calmodulin in the presence of Ca2+ had no additional effect when compared to that observed in the presence of Ca2+ alone. This finding is interpreted in terms of the mechanism of calmodulin activation of ATPase catalysis.     

Biophysical Characterization of the Dimer and Tetramer Interface Interactions of the Human Cytosolic Malic Enzyme

The cytosolic NADP⁺-dependent malic enzyme (c-NADP-ME) has a dimer-dimer quaternary structure in which the dimer interface associates more tightly than the tetramer interface. In this study, the urea-induced unfolding process of the c-NADP-ME interface mutants was monitored using fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation and enzyme activities. Here, we demonstrate the differential protein stability between dimer and tetramer interface interactions of human c-NADP-ME. Our data clearly demonstrate that the protein stability of c-NADP-ME is affected predominantly by disruptions at the dimer interface rather than at the tetramer interface. First, during thermal stability experiments, the melting temperatures of the wild-type and tetramer interface mutants are 8–10°C higher than those of the dimer interface mutants. Second, during urea denaturation experiments, the thermodynamic parameters of the wild-type and tetramer interface mutants are almost identical. However, for the dimer interface mutants, the first transition of the urea unfolding curves shift towards a lower urea concentration, and the unfolding intermediate exist at a lower urea concentration. Third, for tetrameric WT c-NADP-ME, the enzyme is first dissociated from a tetramer to dimers before the 2 M urea treatment, and the dimers then dissociated into monomers before the 2.5 M urea treatment. With a dimeric tetramer interface mutant (H142A/D568A), the dimer completely dissociated into monomers after a 2.5 M urea treatment, while for a dimeric dimer interface mutant (H51A/D90A), the dimer completely dissociated into monomers after a 1.5 M urea treatment, indicating that the interactions of c-NADP-ME at the dimer interface are truly stronger than at the tetramer interface. Thus, this study provides a reasonable explanation for why malic enzymes need to assemble as a dimer of dimers.     

Interaction of rabbit secretory component with rabbit IgA dimer.

Secretory component (SC), a glycoprotein with an apparent molecular weight of approximately 80,000, has been isolated from rabbit milk and found to be heterogenous in size and charge. Functionally intact IgA dimer has been dissociated from milk secretory IgA using a chaotropic agent and further purified to homogeneity. The interaction between SC and IgA dimer is a reversible time- and temperature-dependent process. At 23 degrees C, the association rate constant (2.4 x 10(5) M-1 min-1) and the dissociation rate constant (1.8 x 10(-3) min-1) have been measured independently and the affinity constant based on these rates (1.3 x 10(8) M-1) is similar to that calculated from Scatchard plots (1.9 x 10(8) M-1). One class of binding sites has been estimated from Scatchard plots in spite of the observed heterogeneity of SC. The interaction is tighter at low temperatures because the decrease in dissociation rate is greater than the decrease in association rate. The thermodynamic calculations reveal a delta G of -11.0 kcal . mol-1, a delta H of -8.9 kcal . mol-1 and a delta S of +7.0 cal. mol-1 degree-1. The pH range over which interaction occurs is rather large (5 to 8) with no significant differences in apparent Ka.     

Conformational change coupling the dimerization and activation of KSHV protease

The mechanism of herpesviral protease activation upon dimerization was studied using two independent spectroscopic assays augmented by directed mutagenesis. Spectroscopic changes, attributable to dimer interface conformational plasticity, were observed upon dimerization of Kaposi's sarcoma-associated herpesvirus protease (KSHV Pr). KSHV Pr's dissociation constant of 585 +/- 135 nM at 37 degrees C was measured by a concentration-dependent, 100-fold increase in specific activity to a value of 0.275 +/- 0.023 microM product min(-1) (microM enzyme)(-1). A 4 nm blue-shifted fluorescence emission spectrum and a 25% increase in ellipticity at 222 nm were detected by circular dichroism upon dimer association. This suggested enhanced hydrophobic packing within the dimer interface and/or core, as well as altered secondary structures. To better understand the structure-activity relationship between the monomer and the dimer, KSHV Pr molecules were engineered to remain monomeric via substitution of two separate residues within the dimer interface, L196 and M197. These mutants were proteolytically inactive while exhibiting the spectroscopic signature and thermal stability of wild type, dissociated monomers (T(M) = 75 degrees C). KSHV Pr conformational changes were found to be relevant in vivo, as the autoproteolytic inactivation of KSHV Pr at its dimer disruption site [Pray et al. (1999) J. Mol. Biol. 289, 197-203] was detected in viral particles from KSHV-infected cells. This characterization of structural plasticity suggests that the structure of the KSHV Pr monomer is stable and significantly different from its structure in the dimer. This structural uniqueness should be considered in the development of compounds targeting the dimer interface of KSHV Pr monomers.     

Human platelet factor 4 subunit association/dissociation thermodynamics and kinetics

Platelet factor 4 (PF4) monomers (7800 daltons) form dimers and tetramers in varying molar ratios under certain solution conditions [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469]. The presence of a simplified aromatic region (one Tyr and two His) and resolved monomer, dimer, and tetramer Y60 3,5 ring proton resonances makes study of PF4 aggregate association/dissociation thermodynamics and kinetics possible. PF4 protein subunit association/dissociation equilibrium thermodynamic parameters have been derived by 1H NMR (500MHz) resonance line-fitting analysis of steady-state Y60 3,5 ring proton resonance monomer-dimer-tetramer populations as a function of temperature from 10 to 40 degrees C. Below 10 degrees C and above 40 degrees C, resonance broadening and overlap severely impaired analysis. Enthalpic and entropic contributions to dimer association Gibb's free energy [-5.1 kcal/mol (30 degrees C)] are +2.5 +/- 1 kcal/mol and +26 +/- 7 eu, respectively, and for tetramer association Gibb's free energy [-5.7 kcal/mol (30 degrees C)], they are -7.5 +/- 1 kcal/mol and -7 +/- 3 eu, respectively. These thermodynamic parameters are consistent with low dielectric medium electrostatic/hydrophobic interactions governing dimer formation and hydrogen bonding governing tetramer formation. Association/dissociation kinetic parameters, i.e., steady-state jump rates, have been derived from exchange-induced line-width increases and from 1H NMR (500 MHz) saturation-transfer and spin-lattice (Tl) relaxation experiments. From dissociation jump rates and equilibrium constants, association rate constants were estimated. For dimer and tetramer equilibria at 30 degrees C, unimolecular dissociation rate constants are 35 +/- 10 s-1 for dimer dissociation and 6 +/- 2 s-1 for tetramer dissociation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sedimentation equilibrium measurements of recombinant DNA derived human interferon gamma

Recombinant DNA derived human interferon gamma (IFN-gamma) from Escherichia coli was examined by equilibrium ultracentrifugation. Short-column equilibrium experiments at pH 6.9 in 0.1 M ammonium acetate buffer gave a z-average molecular weight of 33,500 +/- 1400 at infinite dilution, corresponding to 1.98 +/- 0.08 times the formula weight. Long- (2.6 mm) column experiments at pH 7.5 in 0.04 M imidazole buffer gave a molecular weight of 33,400 +/- 500. Under the latter conditions IFN-gamma behaves somewhat nonideally, with the departure from ideality accounted for by an effective (Donnan) charge of about 6+. No association of this dimer to form tetramer or higher polymers was observed, with the association constant for formation of tetramer from dimer K24 found to be less than 34 L mol-1. Similarly, no dissociation to monomers was observable, with the dissociation constant to monomer K21 being less than 5 X 10(-8) mol L-1. At pH 3.55 in 0.02 M buffer (acetate plus acetic acid), there was virtually complete dissociation of the dimer to monomer. Extreme nonideality was seen in this low ionic strength system, and the effective charge on the protein was estimated to be about 11+. The reduced molecular weight M(1 -upsilon rho) of the monomer was found to be about 4.09 +/- 0.20 kg mol-1; this corresponds to a molecular weight of 16,410 +/- 820, with the Scatchard definition of components. A small amount of a polymer with a molecular weight of about 0.5 X 10(6) was detected under these conditions.     

The effect of pressure and guanidine hydrochloride on azurins mutated in the hydrophobic core.

The unfolding of the blue-copper protein azurin from Pseudomonas aeruginosa by guanidine hydrochloride, under nonreducing conditions, has been studied by fluorescence techniques and circular dichroism. The denaturation transition may be fitted by a simple two-state model. The total free energy change from the native to the unfolded state was 9.4 +/- 0.4 kcal.mol-1, while a lower value (6.4 +/- 0.4 kcal.mol-1) was obtained for the metal depleted enzyme (apo-azurin) suggesting that the copper atom plays an important stabilization role. Azurin and apo-azurin were practically unaffected by hydrostatic pressure up to 3000 bar. Site-directed mutagenesis has been used to destabilize the hydrophobic core of azurin. In particular either hydrophobic residue Ile7 or Phe110 has been substituted with a serine. The free energy change of unfolding by guanidinium hydrochloride, resulted to be 5.8 +/- 0.3 kcal.mol-1 and 4.8 +/- 0.3 kcal.mol-1 for Ile7Ser and Phe110Ser, respectively, showing that both mutants are much less stable than the wild-type protein. The mutated apoproteins could be reversible denatured even by high pressure, as demonstrated by steady-state fluorescence measurements. The change in volume associated to the pressure-induced unfolding was estimated to be -24 mL.mol-1 for Ile7Ser and -55 mL.mol-1 for Phe110Ser. These results show that the tight packing of the hydrophobic residues that characterize the inner structure of azurin is fundamental for the protein stability. This suggests that the proper assembly of the hydrophobic core is one of the earliest and most crucial event in the folding process, bearing important implication for de novo design of proteins.     

Dissociation of dimers of human hemoglobins A and F into monomers

Dissociation of alpha beta and alpha gamma dimers of human hemoglobins (Hb) A and F into monomers was studied by alpha chain exchange (Shaeffer, J. R., McDonald, M. J., Turci, S. M., Dinda, D. M., and Bunn, H. F. (1984) J. Biol. Chem. 259, 14544-14547). Unlabeled carbonmonoxy-Hb A was incubated with trace amounts of preparatively purified, native, 3H-alpha subunits in 10 mM sodium phosphate, pH 7.0, at 25 degrees C. At appropriate times, free alpha monomers were separated from Hb A tetramers by anion exchange high performance liquid chromatography. Transfer of radioactivity from the alpha chain pool into Hb A was measured, yielding a first order dimer dissociation rate constant, k2 = (3.2 +/- 0.3) X 10(-3) h-1. The Arrhenius plot of k2 was linear between 7 and 37 degrees C, yielding an enthalpy of activation of 23 kcal/alpha beta dimer. As the chloride concentration was raised from 0 to 0.2 M, the dissociation rate increased 3-fold; with higher salt concentrations, however, the rate gradually returned to baseline. This rate was not altered by raising the pH from 6.5 to 7.2, but as pH was further raised to 8.4, kappa 2 increased about 3-fold. Hb F, which has an increased stability at alkaline pH, dissociated into alpha and gamma monomers 3 times more slowly than Hb A. Moreover, the dimer-monomer dissociation of Hb F was characterized by a significantly reduced pH dependence. These results demonstrate that both alpha beta and alpha gamma dimers of Hb A and Hb F dissociate reversibly into monomers under physiologic conditions. The differential pH dependence for dimer dissociation between Hb A and Hb F suggests that specific amino acid replacement at the alpha 1 gamma 1 interface confers increased resistance to alkaline denaturation.     

Structural characterization of lactate dehydrogenase dissociation under high pressure studied by synchrotron high-pressure small-angle X-ray scattering.

The effect of high pressure on lactate dehydrogenase (LDH) was studied using small-angle X-ray scattering (SAXS). The SAXS results are interpreted in terms of the dissociation and association of LDH within a compression and decompression cycle and its temperature dependence. LDH consists of four identical subunits. At 120 MPa and 25 degrees C, 50% of the LDH dissociates into subunits, while at 10 degrees C, this occurs at 78 MPa. The hysteresis in the dissociation and association under pressure was confirmed in terms of the radius of gyration and was seen to be more conspicuous at low temperature. Forward scattering, I(0)/C, which is proportional to molecular weight, showed that LDH dissociated into dimer (not monomer) subunits under pressure. The application of high pressure to dissociated dimers induced irreversible aggregation. This result is in sharp contrast with the result of fluorescence spectroscopy suggesting a dissociated monomer [King, L., and Weber, G. (1986) Biochemistry 25, 3637-3640]. As for structural change after reassociation, there was little structural difference between native and drifted LDH. The difference was smaller than the structure change by ligand binding. At 200 MPa, the presence of five scattering peaks in the medium-angle region indicates that the dissociated dimer does not have a molten globule-like structure but a core structure. We propose a model of the dissociated dimer, based on the SAXS profile, in which the volume is reduced without disrupting the core structure.     

Folding of a de novo designed native-like four-helix bundle protein

The folding of a model native-like dimeric four-helix bundle protein, (alpha(2))(2), was investigated using guanidine hydrochloride, hydrostatic pressure, and low temperature. Unfolding by guanidine hydrochloride followed by circular dichroism and intrinsic fluorescence spectroscopy revealed a highly cooperative transition between the native-like and unfolded states, with free energy of unfolding determined from CD data, DeltaG(unf) = 14.3 +/- 0.8 kcal/mol. However, CD and intrinsic fluorescence data were not superimposable, indicating the presence of an intermediate state during the folding transition. To stabilize the folding intermediate, we used hydrostatic pressure and low temperature. In both cases, dissociation of the dimeric native-like (alpha(2))(2) into folded monomers (alpha(2)) was observed. van't Hoff analysis of the low temperature experiments, assuming a two-state dimer 171-monomer transition, yielded a free energy of dissociation of (alpha(2))(2) of DeltaG(diss) = 11.4 +/- 0.4 kcal/mol, in good agreement with the free energy determined from pressure dissociation experiments (DeltaG(diss) = 10.5 +/- 0.1 kcal/mol). Binding of the hydrophobic fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) to the pressure- and cold-dissociated states of (alpha(2))(2) indicated the existence of molten-globule monomers. In conclusion, we demonstrate that the folding pathway of (alpha(2))(2) can be described by a three-state transition including a monomeric molten globule-like state.     

Spectroscopic investigations of the single tryptophan residue and of riboflavin and 7-oxolumazine bound to lumazine apoprotein from Photobacterium leiognathi

Spectroscopic techniques have been applied to investigate the conformation, local structure, and dynamic properties of the apoprotein of the lumazine protein from Photobacterium leiognathi and the holoprotein reconstituted with either the natural ligand 6,7-dimethyl-8-ribityllumazine or the closely related analogues riboflavin and 6-methyl-7-oxo-8-ribityllumazine (7-oxolumazine). The analogues are bound similarly to the natural prosthetic group. They exhibit similar shifts on binding in their absorption and fluorescence spectra, single-exponential fluorescence decays, and no independent motion from the protein as evident from a long-lived anisotropy decay (single-exponential phi = 10 ns, 20 degrees C) and high initial anisotropy. Steady-state anisotropy measurements result in similar KD's (40 nM, 20 degrees C, 50 mM inorganic phosphate) for all ligands. Circular dichroism in the far-UV region (190-250 nm) indicates no change in secondary structure on binding to the apoprotein. In the spectral region of 250-310 nm relatively large changes occur, indicating changes in the environment of the tyrosine and tryptophan residues. The single tryptophan residue shows a three-exponential decay of its fluorescence in both the apoprotein and the holoprotein. Radiationless energy transfer also occurs from the tryptophan to the bound ligand, especially evident with 7-oxolumazine. We have designed a new method for evaluation of the rate constant of energy transfer by measuring the (picosecond) rise time of the acceptor fluorescence. The anisotropy decay of the tryptophan residue shows two correlation times, a short one (phi approximately equal to 0.4 ns) representing rapid but restriced oscillation of this residue and a longer one (phi 2 = 5-7 ns, 20 degrees C) representing the motion of a larger segment of the protein.