Disulfide bonds RNase

This enzyme (RNase A) is a single chain protein of 124 amino acid residues, cross-linked by four intrachain disulfide bonds. Limited proteolysis of the enzyme cuts a single peptide bond between residues 20 and 21 (Richards and Vithayathil, 1959). The derived protein, RNase S, retains enzymic activity although the N-terminal peptide of 20 amino acids (S-peptide) is no longer covalently attached to the balance of the molecule (S-protein). Removal of S-peptide from... 

Many secretory proteins—e. g., pancreatic ribonuclease (RNAse see p. 74)—contain several disulfide bonds that are only formed oxidatively from SH groups after translation. The eight cysteine residues of the RNAse can in principle form 105 different pairings, but only the combination of the four disulfide bonds shown on p. 75 provides active enzyme. Incorrect pairings can block further folding or lead to unstable or insoluble conformations. The enzyme protein disulfide iso-merase [1] accelerates the equilibration between paired and unpaired cysteine residues, so that incorrect pairs can be quickly split before the protein finds its final conformation. 

Ribonuclease A (RNase A) was selected as the target enzyme for solid-phase synthesis because its sequence was known (Scheme S), 22 25 and an X-ray structure had been deduced. 24 Importantly, it had been shown that this 124-residue protein could be reduced and unfolded and then reoxidized to re-form the four disulfide bonds with recovery of full enzymatic activity. 25 ... 

One approach to the understanding of the relationship between the amino acid sequence of a protein and its three-dimensional structure consists of preparing fragments which reconstitute a functional nativelike structure by noncovalent association. Richards first demonstrated that the two fragments of bovine pancreatic ribonuelease, RNase-S-peptide (residues 1-20) and RNase-S-protein (residues 21-124), the latter with four intact disulfide bonds, bind noncovalently to form the original functional structure, RNase-S (73, 74)- The elucidation of the three-dimensional structure of RNase-S by X-ray crystallographic study confirmed these observations (75). The RNase-S-protein-RNase-S-peptide system also provided a way by which chemically synthesized fragments could be used to test the role of individual residues in the formation of the functional structure of the protein (76-79). 

The amino acid sequence of RNase Tx has been elucidated by K. Takahashi. It consists of a single polypeptide chain of 104 amino acid residues cross-linked by two disulfide bonds, essential for the maintenance of the enzymically active structure, as shown in Fig. 2 (51). 

In in vitro experiments prolyl isomerase accelerates the oxidative folding of reduced RNase T1 (i.e., folding coupled with formation of the disulfide bonds) and the catalysis of disulfide bond formation by PDl is markedly improved when PPI is present simultaneously (Schonbrunner and Schmid, 1992). The oxidative folding of RNase T1 in the presence of a mixture of reduced and oxidized glutathione is a slow process and it can be followed by the increase in tryptophan fluorescence (Fig. 7). Folding is strictly linked to disulfide bond formation under the conditions... 

Fig. 7. Oxidative refolding of reduced RNase Tl. Reoxidation conditions were 0.1 M Tris-HCl, pH 7.8, 0.2 Af guanidinium chloride, 4 mM reduced glutathione, 0.4 mM oxidized glutathione, 0.2 mM EDTA, and 2.5 nM RNase Tl at 25°C. The kinetics of oxidative refolding were followed by the increase in tryptophan fluorescence intensity at 320 nm ( ), by an unfolding assay (Kiefhaber el ai, 1990b) that measures the formation of native protein molecules (A), and by the increase in the intensity of the band for native RNase Tl in native polyacrylamide gel electrophoresis ( ). Fluorescence emission in the presence of 10 mM reduced dithioerythritol to block disulfide bond formation (O). The small decrease in signal after several hours is caused by slight aggregation of the reduced and unfolded protein. (From Schonbrunner and Schmid (1992).

The specific activity and thermodynamic stability of the (C2A, ClOA) mutant confirm that the Cys-2 to Cys-10 disulfide bond imparts thermodynamic stability but has litde effect on catalytic activity. Hence this mutant was selected as the starting point for constructing a circularly permuted form of RNase-Tl so that as short a linker as possible could be used to bridge the original N- and C-termini. The activity and stability of the circularly permuted variant indicate that it adopts an overall tertiary fold very similar to that of the native protein. Therefore, transposing the first 34 residues to the C-terminus has little effect on the overall folding to the final tertiary structure. The real effect, however, may be more evident in the kinetics of the specific folding pathway. 

Studies by Anfinsen of the reversible denaturation of the pancreatic enzyme ribonuclease prompted the hypothesis that secondary and tertiary structures are derived inclusively from the primary structure of a protein (Figures 4-11 and 4-12). RNase A, which consists of a single polypeptide chain of 124 amino acid residues, has four disulfide bonds. Treatment of the enzyme with 8 M urea, which disrupts noncovalent bonds, and j8-mercaptoethanol, which reduces disulfide linkages to cysteinyl residues, yields a random coil conformation. 

Remove prehybridization solution without permitting the specimens to dry and add probe in hybridization solution (radiolabeled probes 10—600 ng/ml nonradioactive probes up to 10 times more). Hybridization solution contains 50% formamide, 0.5 M NaCl, 10 mM Tris-HCI (pH 7.6), 2 mM EDTA, 1 x Denhardt s solution, 20 mM DTT, 300 pg/ml tRNA, 300 (xg/ml poly(A) or/and poly(C) (optional), 10% PEG 6000 RNase-free. S label may form nonspecific disulfide bonds in the cell if reductants, such as DTT or 2-mercaptoethanol, are omitted. 

Incubate the RNase-2-pyridyl disulfide derivative with 2 mM DTT (final concentration) for 1 h at room temperature to reduce the 2-pyridyl disulfide bond (see Note 13). 

The disulfide bonds of the RNase are not affected by this concentration of DTT (2.0 mM) (14). If the RNase is stable to dialysis and concentration, the 2-pyridyl disulfide bond can be reduced under acidic conditions. At pH 4.5, the reduction of the protein-2-pyridyl disulfide is very specific. At this pH, the 2-thio-pyridine is a good leaving group (8). To perform the reduction under acidic conditions, follow steps 1-3 in Subheading 3.5. 

Follow the reaction between the RNase and antibody by observing the appearance of thionitrobenzoate (TNB) (12). TNB is released from the antibody as disulfide bonds between the RNase and antibody are formed. This can be observed spectrophotometrically at 412 nm using the molar extinction coefficient of TNB of 13,600. By comparing the number of mols of TNB released with the number of mols of antibody, the number of mols of RNase conjugated per mol of antibody can be determined. 

The classic experiments conducted by Anfinsen and co-workers proved that all the information for the three-dimensional structure of a protein is encoded by its amino acid sequence (Anfinsen, 1973) Bovine pancreatic RNase A, a 124-residue protein that contains four disulfide bonds in its native state, was used as a model protein (Sela et al., 1959 White, 1961). RNase A denatures readily and its disulfide bonds are reduced by incubation in urea and 2-mercaptoethanol. After removal of urea and 2-mercaptoethanol by dialysis, a very slow but nearly complete recovery of catalytic activity is observed. Thus, Anfinsen concluded that it is possible to refold denatured proteins into their active state in the test tube. Based on this observation, he further noted that the information for the assumption of the native secondary and tertiary structure is contained in the amino acid sequence itself (Anfinsen et al., 1961). 

---