Harold Edelhoch

The Thermodynamic Basis of the Stability of Proteins, Nucleic Acids, and Membranes

Publisher Summary The contributions of the polar and nonpolar components of the proteins and nucleic acids can be regarded as constituting the bricks and mortar, respectively, of the two structures. This chapter describes the changes in enthalpy and entropy of reactions as diverse as denaturation, ligand binding, and self-association display a characteristic dependence on temperature, which is not evident in the free energy changes. The chapter focuses on comparing the energetic data on protein reactions with similar data on model systems to evaluate the contributions of the various groups to the thermodynamic parameters of protein reactions. The folding of the proteins is different from that of the nucleic acids, because the energetics of the interactions with water of the nonpolar side chains and bases are different, and owing to the planarity and partial polar properties of the purine and pyrimidine bases, they cluster by forming stacks. The thermodynamic parameters responsible for polypeptide folding are similar to those found for other protein reactions—that is, ligand binding, or subunit association, because they are controlled by the interactions of the same groups with water. The stability of the folded conformations is controlled largely by the interactions of the nonpolar side chains of the amino acids and the purine and pyrimidine bases with water. Although, nucleic acids do not show the low-temperature dissociation found with numerous protein aggregates, however, they show only positive values for these two functions.

The rates of dissociation and reassociation of the subunits of human chorionic gonadotropin

Abstract A large change in quantum yield of the fluorescent probe 1,8-anilinonaphthalene sulfonate is produced when it combines with the glycoprotein hormone, human chorionic gonadotropin. A method of analyzing for the hormone in the presence of its subunits has been developed based on the finding that the subunits have no effect on 1,8-anilinonaphthalene sulfonate fluorescence. Quantitative rates of dissociation and recombination can be obtained with very small concentrations of hormone since fluorescence measurements are fast and sensitive. The effects of temperature, pH, and urea concentration on the rate of human chorionic gonadotropin dissociation have been measured. The rates of recombination of subunits have been studied as a function of temperature, pH, and KCl concentration. Human chorionic gonadotropin is stable in water to pH 12 and pH 4.5 at 37 °C.

Rates of dissociation and recombination of the subunits of bovine thyrotropin.

Abstract The rates of dissociation and recombination of the subunits of bovine thyrotropin have been measured under a variety of conditions using the fluorescence probe 1,8-anilinonaphthalenesulfonate. The method is based on the fact that the native hormone strongly enhances the fluorescence of 1,8-anilinonaphthalenesulfonate whereas the subunits have very little effect. The hormone can be easily dissociated into subunits, either in dilute acid (pH m ) urea solutions at pH 8.O. The rate of dissociation is first order with time and increases strongly with increasing temperature. The hormone is very stable in alkali, showing little tendency to dissociate below pH 12. After dissociation in acid, the subunits can be recombined between pH 7 and 9 at a rate which increases with increasing temperature and subunit concentration. The recombination is intermediate between first and second order suggesting a two-step mechanism: association of the subunits followed by a first-order refolding process in which the subunits acquire the tertiary structure characterisitc of the native hormone. Difference absorption measurements indicate that the dissociation is accompanied by the exposure of a substantial fraction of the 16 tyrosine residues to the more polar aqueous environment, suggesting major conformational changes in one or both subunits.

Thyroxine-binding prealbumin. Conformation in urea and guanidine

Abstract The effects of urea and guanidine on the stability of thyroxine-binding prealbumin have been evaluated by absorption and fluorescence measurements on the native protein and by fluorescence and polarization measurements on the dansyl-conjugated protein. Thyroxine-binding prealbumin neither dissociates into subunits nor unfolds in concentrations of urea as large as 8 m or in guanidine solutions less than 6 m . On the basis of the stability toward these two denaturants, thyroxine-binding prealbumin appears to be one of the most stable globular proteins yet examined. It is also clear that 6 m guanidine cannot be considered as a universal denaturant in dissociating and unfolding native proteins.

The noncovalent subunit structure of human thyroglobulin

Abstract The subunit composition of human thyroglobulin has been determined by analytical centrifugation in aqueous solution and by equilibrium centrifugation in 5 m guanidine. In poorly iodinated (goiter) thyroglobulin all of the 19S molecules dissociate into half-sized subunits (12S), whereas in normal thyroglobulin dissociation is incomplete. It is suggested that the nondissociable 19S form is formed by the intermolecular oxidation of sulfhydryl groups by iodine, which is present in much larger amounts in the normal than in the low-iodine protein. The 12S subunits separated into two closely sedimenting boundaries by ultracentrifugation, suggesting the presence of two different subunit species.

The self-association of apoA-II, an apoprotein of the human high density lipoprotein complex.

Abstract The molecular properties of human apoA-II have been investigated as a function of pH, temperature, and concentration by employing sedimentation equilibrium, fluorescence, fluorescent polarization, circular dichroism, and difference absorption. The results reveal that in the concentration range investigated apoA-II self-associates to a dimer with concomitant changes in secondary and tertiary structures. This reversible association was found to be dependent on temperature between 5 and 50 °C, showing a maximum in association near 25 °C.

The migration behavior of proparathyroid hormone, parathyroid hormone, and their peptide fragments during gel filtration☆

Abstract The migration behavior of bovine proparathyroid and parathyroid hormones, as well as several hormonal peptide fragments, has been analyzed by gel filtration on thin-layer plates and by column chromatography. The two hormones migrated appreciably faster than was expected for their molecular weights. Their migration rates decreased with increasing pH and approached values more characteristic of their molecular weights at pH 11.0. Migration rates were the same over a concentration range of 2 × 10 −6 –0.9 × 10 −3 m . These results indicate that parathyroid hormone does not exist in solution as an aggregate at the concentrations used in these experiments. These studies suggest that the two hormones are asymmetrical, unfolded monomers in acid solution which become more folded or globular at alkaline pH values.

Structural transitions of lysozyme in urea solution.

Abstract The molecular state of lysozyme in high concentrations of urea is dependent upon the pH and temperature. At extremes of pH, or at elevated temperatures, a time-dependent transition occurs from the state prevailing at neutral pH and 25° to a new configuration characterized by greatly enhanced ultraviolet fluorescence, increased levorotation, and altered absorption spectrum. The degree of reversibility from alkaline solution depends upon the pH of exposure.

IODOPROTEINS IN THE THYROID

The thyroid gland contains a protein first studied so long ago that it has been noted only recently that little is really known of its structure. This protein is thyroglobulin, which comprises most of the iodine of the thyroid. Recently other iodoproteins in the thyroid present in smaller amounts have been partially characterized. We shall present some new data on the old protein and review our work on the nonthyroglobulin iodoproteins.

Biosynthesis of the 27 S thyroid iodoprotein.

Abstract Biosynthesis of the 27 S thyroid iodoprotein was studied in rats in vivo , and evidence was obtained that 19 S, the major thyroid iodoprotein, is the immediate precursor of 27 S. In untreated rats, labeled 19 S is found within minutes after the administration of [ 3 H]leucine, whereas the appearance of 27 S is preceded by a lag-phase of at least 1 hr. Inhibition of protein synthesis by cycloheximide administration resulted in a several fold increase in the proportion of [ 3 H]leucine-labeled 27 S relative to 19 S. In contrast, inhibition of iodination with antithyroid agents prevented the formation of 27 S, but not of 19 S synthesis. Since formation of 27 S is dependent on iodination, but not on de novo protein synthesis, 27 S must be derived from the preexisting pool of 19 S in the follicular colloid. It is suggested that 27 S is formed by chemical alterations produced as a by-product of iodination of the tyrosyl residues, probably involving oxidative side-reactions. It appears that the quaternary structure of the 27 S iodoprotein is not encoded into its structural gene, but is a result of post-transcriptional events.