The effect of net charge on the solubility, activity, and stability of ribonuclease Sa.

The net charge and isoelectric pH (pI) of a protein depend on the content of ionizable groups and their pK values. Ribonuclease Sa (RNase Sa) is an acidic protein with a pI = 3.5 that contains no Lys residues. By replacing Asp and Glu residues on the surface of RNase Sa with Lys residues, we have created a 3K variant (D1K, D17K, E41K) with a pI = 6.4 and a 5K variant (3K + D25K, E74K) with a pI = 10.2. We show that pI values estimated using pK values based on model compound data can be in error by >1 pH unit, and suggest how the estimation can be improved. For RNase Sa and the 3K and 5K variants, the solubility, activity, and stability have been measured as a function of pH. We find that the pH of minimum solubility varies with the pI of the protein, but that the pH of maximum activity and the pH of maximum stability do not.

Increasing protein stability by altering long-range coulombic interactions.

It is difficult to increase protein stability by adding hydrogen bonds or burying nonpolar surface. The results described here show that reversing the charge on a side chain on the surface of a protein is a useful way of increasing stability. Ribonuclease T1 is an acidic protein with a pI approximately 3.5 and a net charge of approximately -6 at pH 7. The side chain of Asp49 is hyperexposed, not hydrogen bonded, and 8 A from the nearest charged group. The stability of Asp49Ala is 0.5 kcal/mol greater than wild-type at pH 7 and 0.4 kcal/mol less at pH 2.5. The stability of Asp49His is 1.1 kcal/mol greater than wild-type at pH 6, where the histidine 49 side chain (pKa = 7.2) is positively charged. Similar results were obtained with ribonuclease Sa where Asp25Lys is 0.9 kcal/mol and Glu74Lys is 1.1 kcal/mol more stable than the wild-type enzyme. These results suggest that protein stability can be increased by improving the coulombic interactions among charged groups on the protein surface. In addition, the stability of RNase T1 decreases as more hydrophobic aromatic residues are substituted for Ala49, indicating a reverse hydrophobic effect.

Heat capacity change for ribonuclease A folding.

The change in heat capacity deltaCp for the folding of ribonuclease A was determined using differential scanning calorimetry and thermal denaturation curves. The methods gave equivalent results, deltaCp = 1.15+/-0.08 kcal mol(-1) K(-1). Estimates of the conformational stability of ribonuclease A based on these results from thermal unfolding are in good agreement with estimates from urea unfolding analyzed using the linear extrapolation method.

Conformational stability and thermodynamics of folding of ribonucleases Sa, Sa2 and Sa3.

Ribonucleases Sa, Sa2, and Sa3 are three small, extracellular enzymes produced by different strains of Streptomyces aureofaciens with amino acid sequences that are 50% identical. We have studied the unfolding of these enzymes by heat and urea to determine the conformational stability and its dependence on temperature, pH, NaCl, and the disulfide bond. All three of the Sa ribonucleases unfold reversibly by a two-state mechanism with melting temperatures, Tm, at pH 7 of 48.4 degrees C (Sa), 41.1 degrees C (Sa2), and 47.2 degrees C (Sa3). The Tm values are increased in the presence of 0.5 M NaCl by 4.0 deg. C (Sa), 0.1 deg. C (Sa2), and 7.2 deg. C (Sa3). The Tm values are decreased by 20.0 deg. C (Sa), 31.5 deg. C (Sa2), and 27.0 deg. C (Sa3) when the single disulfide bond in the molecules is reduced. We compare these results with similar studies on two other members of the microbial ribonuclease family, RNase T1 and RNase Ba (barnase), and with a member of the mammalian ribonuclease family, RNase A. At pH 7 and 25 degrees C, the conformational stabilities of the ribonucleases are (kcal/mol): 2.9 (Sa2), 5.6 (Sa3), 6.1 (Sa), 6.6 (T1), 8.7 (Ba), and 9.2 (A). Our analysis of the stabilizing forces suggests that the hydrophobic effect contributes from 90 to 110 kcal/mol and that hydrogen bonding contributes from 70 to 105 kcal/mol to the stability of these ribonucleases. Thus, we think that the hydrophobic effect and hydrogen bonding make large but comparable contributions to the conformational stability of these proteins.

Purification of ribonucleases Sa, Sa2, and Sa3 after expression in Escherichia coli.

The genes for three small ribonucleases from different strains of Streptomyces aureofaciens have been cloned and expressed in Escherichia coli. The purification of these ribonucleases from the periplasmic space is described. The yields range from 10 to 50 mg of protein per liter of culture medium. The molar absorption coefficients, isoelectric pH values, and pH of optimum activity are reported.

Conformational stability and mechanism of folding of ribonuclease T1.

Urea and thermal unfolding curves for ribonuclease T1 (RNase T1) were determined by measuring several different physical properties. In all cases, steep, single-step unfolding curves were observed. When these results were analyzed by assuming a two-state folding mechanism, the plots of fraction unfolded protein versus denaturant were coincident. The dependence of the free energy of unfolding, delta G (in kcal/mol), on urea concentration is given by delta G = 5.6 - 1.21 (urea). The parameters characterizing the thermodynamics of unfolding are: midpoint of the thermal unfolding curve, Tm = 48.1 degrees C, enthalpy change at Tm, delta Hm = 97 kcal/mol, and heat capacity change, delta Cp = 1650 cal/mol deg. A single kinetic phase was observed for both the folding and unfolding of RNase T1 in the transition and post-transition regions. However, two slow kinetic phases were observed during folding in the pre-transition region. These two slow phases account for about 90% of the observed amplitude, indicating that a faster kinetic phase is also present. The slow phases probably result from cis-trans isomerization at the 2 proline residues that have a cis configuration in folded RNase T1. These results suggest that RNase T1 folds by a highly cooperative mechanism with no structural intermediates once the proline residues have assumed their correct isomeric configuration. At 25 degrees C, the folded conformation is more stable than the unfolded conformations by 5.6 kcal/mol at pH 7 and by 8.9 kcal/mol at pH 5, which is the pH of maximum stability. At pH 7, the thermodynamic data indicate that the maximum conformational stability of 8.3 kcal/mol will occur at -6 degrees C.

Conformational stability and activity of ribonuclease T1 with zero, one, and two intact disulfide bonds.

Ribonuclease T1 has two disulfide bonds linking cysteine residues 2-10 and 6-103. We have prepared a derivative of ribonuclease T1 in which one disulfide bond is broken and the cysteine residues carboxymethylated, (2-10)-RCM-T1, and three derivatives in which both disulfides are broken and the cysteine residues reduced, R-T1, carboxamidomethylated, RCAM-T1, or carboxymethylated, RCM-T1. The RNA hydrolyzing activity of these proteins has been measured, and urea and thermal denaturation studies have been used to determine conformational stability. The activity, melting temperature, and conformational stability of the proteins are: ribonuclease T1 (100%, 59.3 degrees C, 10.2 kcal/mol), (2-10)-RCM-T1 (86%, 53.3 degrees C, 6.8 kcal/mol), R-T1 (53%, 27.2 degrees C, 3.0 kcal/mol), RCAM-T1 (43%, 21.2 degrees C, 1.5 kcal/mol), and RCM-T1 (35%, 16.6 degrees C, 0.9 kcal/mol). Thus, the conformational stability is decreased by 3.4 kcal/mol when one disulfide bond is broken and by 7.2-9.3 kcal/mol when both disulfide bonds are broken. It is quite remarkable that RNase T1 can fold and function with both disulfide bonds broken and the cysteine residues carboxymethylated. The large decrease in the stability is due mainly to an increase in the conformational entropy of the unfolded protein which results when the constraints of the disulfide bonds on the flexibility are removed. We propose a new equation for predicting the effect of a cross-link on the conformational entropy of a protein: delta Sconf = -2.1 - (3/2)R 1n n, where n is the number of residues between the side chains which are cross-linked. This equation gives much better agreement with experimental results than other forms of this equation which have been used previously.

Ribonuclease T1 is stabilized by cation and anion binding.

The stability of the folded conformation of ribonuclease T1 is increased by 0.8, 1.8, and 3.3 kcal/mol in the presence of 0.1 M NaCl, MgCl2, and Na2HPO4, respectively. This remarkable increase in the conformational stability results primarily from the preferential binding to the native protein of one Mg2+ or two Na+ ions at cation-binding sites and by the binding of one HPO4(2-) ion at an anion-binding site. Only modest binding constants, 6.2 (Na+), 155 (Mg2+), and 282 M-1 (HPO4(2-)), are required to account for the enhanced stability. One important goal of the modification of proteins through genetic engineering is to increase their stability. Our results suggest that the creation of specific cation- and anion-binding sites on the surface of a protein through amino acid substitutions might be a generally useful way of achieving this goal. The design of these sites will be aided by the recent availability of detailed structural information on cation- and anion-binding sites.

Purification of ribonuclease T1.

An improved method for purifying ribonuclease T1 from Aspergillus oryzae is described. The method uses gradient elution from DEAE-cellulose and sulfopropyl-Sephadex columns followed by gel filtration on Sephadex G-50 to give almost 100 mg (50% yield) of ribonuclease T1 from 100 g of starting material in less than 5 days.