We have determined the coexistence curves (plots of phase-separation temperature T versus protein concentration C) for aqueous solutions of purified calf lens proteins. The proteins studied, calf y'Ila-, yIlb-, and yIVacrystallin, have very similar amino acid sequences and threedimensional structures. Both ascending and descending limbs of the coexistence curves were measured. We find that the coexistence curves for each of these proteins and for yIcrystallin can be fit, near the critical point, to the function W(Cc -C)/CJI = A[(Tc -T)/TCJ, where fi = 0.325, Cc is the critical protein concentration in mg/ml, T, is the critical temperature for phase separation in K, and A is a parameter that characterizes the width of the coexistence curve. We find that A andCc are approximately the same for all four coexistence curves (A = 2.6 +-0.1, Cc = 289 ± 20 mg/ml), but that Tc is not the same. For yIH-and yIIIb-crystallin, Tc7-5°C, whereas for yIIa-and yIVa-crystallin, Tc 38C. By comparing the published protein sequences for calf, rat, and human y-crystallins, we postulate that a few key amino acid residues account for the division of y-crystallins into low-Tc and high-Tc groups.The y-crystallins constitute a family of highly homologous mammalian lens proteins (1-4). Concentrated aqueous solutions of y-crystallins (5-8) exhibit the phenomena of binaryliquid-phase separation (9-11), also known as coacervation (12). These solutions separate into two coexisting liquid phases of unequal protein concentration at temperatures less than the critical temperature for phase separation Tc. From previous studies (5,7,8), it is known that location of the coexistence curve depends sensitively on the amino acid sequence of the crystallin molecule. Two distinct groups of y-crystallins have been identified in rat (7) and human (8) lenses: high-Tc crystallins and low-Tc crystallins. In these rat and human studies, the precise values of Tc for each crystallin, though inferred from the data, were not determined explicitly. For the high-Tc crystallins, only the ascending limb of the coexistence curves was measured. For the low-Tc crystallins, only an upper bound for the Tc values was established.In this paper, we report on measurements of coexistence curves for three purified calf y-crystallins-yIIIa, yIIIb, and yIVa-[in Table 2 we indicate the current nomenclature for mammalian y-crystallins (2)] and for the native calf y-crystallin mixture yIV. We have determined both the ascending and descending limbs of each coexistence curve. This information enables us to characterize the coexistence curves in detail and to determine explicitly the values of the critical concentration Cc and the critical temperature Tc for each protein. Such detailed analysis of y-crystallin phase separation, which requires gram quantities of purified protein, has been performed only for calf yII (6).We find that the purified calf y-crystallins, in accord with the purified rat and human y-crystallins, fall into two distinct groups: high-Tc (Tc > 350C) proteins, ...

We report measurement of the solid-liquid phase boundary, or liquidus line, for aqueous solutions of three pure calf y6-crystallin proteins: y$I, yHIa, and yIHIb. We also studied the liquidus line for solutions of native yIV-crystallin calf lens protein, which consists of 85% yIVa/15% yIVb. In all four proteins the liquidus phase boundaries lie higher in temperature than the previously determined liquid-lquid coexistence curves. Thus, over the range of concentration and temperature for which liquid-liquid phase separation occurs, the coexistence of a protein crystal phase with a protein liquid solution phase is thermodynamically stable relative to the metastable separated liquid phases. The location ofthe liquidus lines clearly divides these four crystallin proteins into two groups: those in which liquidus lines flatten at temperatures >70rC: yMa and yIV, and those in which liquidus lines flatten at temperatures <500C: yll and -yIlb. We have analyzed the form of the liquidus lines by using specific choices for the structures of the Gibbs free energy in solution and solid phases. By applying the thermodynamic conditions for equilibrium between the two phases to the resulting chemical potentials, we can estimate the temperature-dependent free energy change upon binding of protein and water into the solid phase.Maintenance ofthe lens proteins in a single homogenous fluid phase is an essential condition for transparency of the eye lens (1, 2). Consequently, we previously investigated the location of the coexistence curve (3-5) for liquid-liquid phase separation for four pure calf y-crystallin protein solutions. In those studies, preliminary findings at a few points in the phase diagram suggested that the coexistence curve for solid-liquid phase equilibrium might be higher in temperature than the liquid-liquid coexistence curve (4, 5). We, therefore, undertook the present systematic investigation of the location of the solid-liquid coexistence curve for three pure lens crystallin proteins 'yII, yIIIa, and yIIIb, as well as for native 'yIV protein, which is a mixture of yIVa and yIVb in relative proportion of 85% to 15%, respectively, by number. We report here the measurement for each protein of the ascending limb of the solid-liquid coexistence curve. This limb is called the liquidus line; it is defined as the locus of points in the concentration (c) and temperature (T) plane that corresponds to equilibrium between protein crystals and an aqueous liquid solution of the same protein having concentration c. This locus can be designated by TL(c), or alternatively cL(T). At fixed temperature T, the concentration CL is the solubility of the protein in aqueous solution. The descending limb ofthe solid-liquid coexistence curve is called the solidus line c5(T), and it is the locus of points showing the protein concentration in the solid phase for each temperature T. For cL(T) < c < c,(T), the equilibrium state of the solution consists of a mixture of protein crystals of protein concentration c5(T) and aqueous liquid ...

The binding of different inhibitors to glucoamylase G2 from Aspergillus niger and its temperature and pH dependencies have been studied by titration calorimetry. The enzyme binds the inhibitors 1-deoxynojirimycin and the pseudo-tetrasaccharide acarbose with association constants of 3 x 10(4) and 9 x 10(11) M-1, respectively, at 27 degrees C. The binding free energy for both ligands is remarkably temperature-invariant in the interval from 9 to 54 degrees C as the result of large compensating changes in enthalpy and entropy. Acarbose and 1-deoxynojirimycin bound with slightly different free energy-pH profiles, with optima at 5.5 and 5.5-7.0, respectively. Variations in delta H degrees and T delta S degrees as a function of pH were substantially larger than variations in delta G degrees in a partly compensatory manner. Two titratable groups at or near subsite 1 of the catalytic site were found to change their pKa slightly upon binding. The hydrogenated forms of acarbose, D-gluco- and L-ido-dihydroacarbose, bind with greatly reduced association constants of 3 x 10(7) and 2 x 10(5) M-1, respectively, and the pseudo-disaccharide methyl acarviosinide, lacking the two glucose units at the reducing end compared to acarbose, has a binding constant of 8 x 10(6) M-1; these values all result from losses in both enthalpy and entropy compared to acarbose. Three thio analogues of the substrate maltose, methyl alpha- and beta-4-thiomaltoside and methyl alpha-4,5'-dithiomaltoside, bind with affinities from 3 x 10(3) to 6 x 10(4) M-1.(ABSTRACT TRUNCATED AT 250 WORDS)

We have investigated the binding of mutant forms of glucoamylase from Aspergillus niger to the inhibitors 1-deoxynojirimycin and acarbose. The mutants studied comprise a group of single amino acid replacements in conserved regions near the active site of the enzyme. For each mutant we have determined both the affinities for the two inhibitors and the thermodynamic state functions for binding using titration microcalorimetry. We find that acarbose binds to all the mutants with a wide range of binding constants (10(4) < Ka < 10(13) M-1). In contrast, 1-deoxynojirimycin shows either binding at near wild-type affinity (Ka approximately equal to 10(4) M-1) or no detectable binding. The changes in the affinities of the mutant enzymes are rationalized in terms of the known three-dimensional structure of the wild-type enzyme with subsites 1, 2, and 3 being important for acarbose binding while only subsite 1 is critical for 1-deoxynojirimycin binding. In most of the mutants studied the magnitudes of the enthalpies and the entropies of binding of the mutant enzymes differed from those of the wild-type enzyme with the mutant enzymes having a relatively large portion of their binding energy composed of enthalpy and a relatively small proportion composed of entropy. The pattern of changes in the enthalpy and entropy is hypothesized to be due to changes in the structural complementarity of the binding pocket and the inhibitor.

The kinetics and energetics of the binding between barley alpha-amylase/subtilisin inhibitor (BASI) or BASI mutants and barley alpha-amylase 2 (AMY2) were determined using surface plasmon resonance and isothermal titration calorimetry (ITC). Binding kinetics were in accordance with a 1:1 binding model. At pH 5.5, [Ca(2+)] = 5 mM, and 25 degrees C, the k(on) and k(off) values were 8.3 x 10(+4) M(-1) s(-1) and 26.0 x 10(-4) s(-1), respectively, corresponding to a K(D) of 31 nM. K(D) was dependent on pH, and while k(off) decreased 16-fold upon increasing pH from 5.5 to 8.0, k(on) was barely affected. The crystal structure of AMY2-BASI shows a fully hydrated Ca(2+) at the protein interface, and at pH 6.5 increase of [Ca(2+)] in the 2 microM to 5 mM range raised the affinity 30-fold mainly due to reduced k(off). The K(D) was weakly temperature-dependent in the interval from 5 to 35 degrees C as k(on) and k(off) were only increasing 4- and 12-fold, respectively. A small salt dependence of k(on) and k(off) suggested a minor role for global electrostatic forces in the binding and dissociation steps. Substitution of a positively charged side chain in the mutant K140L within the AMY2 inhibitory site of BASI accordingly did not change k(on), whereas k(off) increased 13-fold. ITC showed that the formation of the AMY2-BASI complex is characterized by a large exothermic heat (Delta H = -69 +/- 7 kJ mol(-1)), a K(D) of 25 nM (27 degrees C, pH 5.5), and an unfavorable change in entropy (-T Delta S = 26 +/- 7 kJ mol(-1)). Calculations based on the thermodynamic data indicated minimal structural changes during complex formation.