Ribonuclease disulfide bonds

The ROA spectra of partially unfolded denatured hen lysozyme and bovine ribonuclease A, prepared by reducing all the disulfide bonds and keeping the sample at low pH, together with the ROA spectra of the corresponding native proteins, are displayed in Figure 5. As pointed out in Section II,B, the short time scale of the Raman scattering event means that the ROA spectrum of a disordered system is a superposition of snapshot ROA spectra from all the distinct conformations present at equilibrium. Because of the reduced ROA intensities and large... 

Klink TA, Woycechowsky KJ, Taylor KM, et al. Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A. Eur. J. Biochem. 2000 267 566-572. 

Many extracellular proteins like immunoglobulins, protein hormones, serum albumin, pepsin, trypsin, ribonuclease, and others contain one or more indigenous disulfide bonds. For functional and structural studies of proteins, it is often necessary to cleave these disulfide bridges. Disulfide bonds in proteins are commonly reduced with small, soluble mercaptans, such as DTT, TCEP, 2-mercaptoethanol, thioglycolic acid, cysteine, etc. High concentrations of mercaptans (molar excess of 20- to 1,000-fold) are usually required to drive the reduction to completion. 

By comparing time-resolved and steady-state fluorescence parameters, Ross et alm> have shown that in oxytocin, a lactation and uterine contraction hormone in mammals, the internal disulfide bridge quenches the fluorescence of the single tyrosine by a static mechanism. The quenching complex was attributed to an interaction between one C — tyrosine rotamer and the disulfide bond. Swadesh et al.(()<>> have studied the dithiothreitol quenching of the six tyrosine residues in ribonuclease A. They carefully examined the steady-state criteria that are useful for distinguishing pure static from pure dynamic quenching by consideration of the Smoluchowski equation(70) for the diffusion-controlled bimolecular rate constant k0,... 

The disulfide bonds of ribonuclease. Journ. Biol. Chem. 235,... 

The fact that a denatured protein can spontaneously return to its native conformation was demonstrated for the first time with ribonuclease, a digestive enzyme (see p. 266) consisting of 124 amino acids. In the native form (top right), there are extensive pleated sheet structures and three a helices. The eight cysteine residues of the protein are forming four disulfide bonds. Residues His-12, Lys-41 and His-119 (pink) are particularly important for catalysis. Together with additional amino acids, they form the enzyme s active center. 

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 ... 

Urea is used to denature ribonuclease, and mercaptoethanol (HOCH2CH2SH) to reduce and thus cleave the disulfide bonds to yield eight Cys residues. Renaturation involves reestablishment of the correct disulfide cross-links. 

The intrinsic viscosity of native ribonuclease is very low. Harrington and Schellman (247) reported 3.3 ml/g at neutral pH in 0.1 M KC1. Buzzell and Tanford (265) found values of 3.3-3.5 ml/g over the entire pH range from 1 to 11 and ionic strengths from 0.05 to 0.25 M. This value increases dramatically on denaturation even without oxidation or reduction of the disulfide bonds to 8.5 ml/g (266). In the presence of reducing agents and 6 M guanidine hydrochloride the value is 16.0 ml/g (267). 

Figure 2.41 Denaturing and self-reassembly of ribonuclease. The correct folding occurs only when disulfide bond formation is made reversible by the addition of P-mercaptoethanol (RSH) allowing the system to equilibrate (Reproduced from Section Key Reference by permission of The Royal Society of Chemistry).

Ribonuclease, the enzyme that hydrolyzes ribonucleic acids (Chap. 7), contains four disulfide bonds that help to stabilize its conformation. In the presence of 6 M guanidine hydrochloride, to weaken hydrogen bonds and hydrophobic interactions, and 1 mM mercaptoethanol, to reduce the disulfide bonds, all enzymatic activity is lost, and there is no sign of residual secondary structure. On removing the guanidine hydrochloride by dialysis or gel filtration, enzymatic activity is restored, the native conformation is regained, and correct disulfide bonds are reformed. 

There are four disulfide bonds in ribonuclease. If these are reduced to their component sulfhydryl groups and allowed to reoxidize, how many different combinations of disulfide bonds are possible ... 

Modifying the Active Conformation of Biomolecules. Class B metals such as Hg have been shown to alter the steric conformation of proteins via interactions with sulfur atoms, particularly disulphide bonds. For example, Hg insertion between the two sulfurs involved in a disulfide bond can significantly alter the shape of the protein and reduce or abolish its activity. This has been demonstrated with ribonuclease, for example. 

Susceptibility to oxidation of disulfides built into proteins is strongly dependent on their location in the protein molecule (G3). Since the disulfides have a crucial role in maintaining protein tertiary structure, oxidation of certain —S—S— bridges may expose further disulfides and cause unfolding of the protein molcule. The final disulfide oxidation is a sulfone residue, which is stable and does not tend to reverse to sulfide. Therefore oxidative breakage of disulfides is irreversible. The spatial location of disulfides inside protein molecules influences their susceptibility to oxidation. The ribonuclease molecule has four —S—S— bonds, and at least three correctly located disulfide bonds are necessary to retain the ribonuclease enzyme properties. The compact ribonuclease molecule is relatively resistant to HOC1 oxidation (D18). 

How is the elaborate three-dimensional structure of proteins attained, and how is the three-dimensional structure related to the one-dimensional amino acid sequence information The classic work of Christian Anfinsen in the 1950s on the enzyme ribonuclease revealed the relation between the amino acid sequence of a protein and its conformation. Ribonuclease is a single polypeptide chain consisting of 124 amino acid residues cross-linked by four disulfide bonds (Figure 3.51). Anfmsen s plan was to destroy the three-dimensional structure of the enzyme and to then determine what conditions were required to restore the structure. 

Figure 3.51. Amino Acid Sequence of Bovine Ribonuclease. The four disulfide bonds are shown in color. [After C. H. W. Hirs, S. Moore, and W. H. Stein, J. Biol. Chem. 235 (1960) 633.]... 

The predominant contribution of the folded native structure is amply illustrated by the findings that complete reduction of disulfide bonds and 5-carboxymethylation completely alters the immunochemical reactivity of protein antigens, such as ribonuclease, lysozyme, and other antigens.However, reduction of but two of the four disulfide bonds in ribonuclease had a negligible effect on the ability to precipitate with antibody to native ribonuclease. 

Epstein CJ and Anfinsen CB. The reversible reduction of disulfide bonds in trypsin and ribonuclease coupled to carboxymethyl cellulose. J. Biol. Chem. 1962 237 2175-2179. 

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