Ribonuclease denatured conformations

For many solubilized enzymes the greatest catalytic activity and/or changes in conformation are found at R < 12, namely, when the competition for the water in the system between surfactant head groups and biopolymers is strong. This emphasizes the importance of the hydration water surrounding the biopolymer on its reactivity and conformation [13], It has been reported that enzymes incorporated in the aqueous polar core of the reversed micelles are protected against denaturation and that the distribution of some proteins, such as chymotrypsine, ribonuclease, and cytochrome c, is well described by a Poisson distribution. The protein state and reactivity were found markedly different from those observed in bulk aqueous solution [178,179],... 

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... 

AAA nucleotidases share the common property of altering the conformation or association state of proteins, so it is not surprising that the RC has been shown to prevent aggregation of several denatured proteins including citrate synthase and ribonuclease A [59-61]. The chaperone activity of the RC may explain why the RC plays a role in transcription apparently in the absence of an attached 20S proteasome [62]. 

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. 

In ribonuclease A 13C spin-lattice relaxation of the carbonyl and a and / carbon atoms is slower in the denaturated protein than in the native sample [177]. Apparently, the skeleton of this macromolecule becomes more flexible on denaturation, probably owing to conformational changes. However, the s carbons of lysine in the native protein exhibit relatively large T, values which change only insignificantly on denaturation [177]. This behavior is attributed to a considerable segmental mobility of the lysine side chain (Table 3.17 [177]). 

Several rapid and sensitive methods have been developed to detect mutations in cDNA and genomic DNA. These include ribonuclease (RNase) protection analysis, denaturing gradient gel electrophoresis, and single-strand conformation polymorphism analysis. These methods can be used in conjunction with the PCR technique to detect small deletions or insertions and single base substitutions. 

These points arc illustrated by the elegant studies of Nelson and Hummel (1962) on the kinetics of denaturation of ribonuclease by urea. As is shown in Fig. 16, the difference spectrum generated with ribonuclease using 7 M urea versus aqueous reference solvent is markedly time-dependent. Difference spectra taken shortly after mixing protein and urea solvent reflect the refractive index red-shift of urea on the tyrosyl and phenylalanyl groups of the protein in its native conformation. As the urea denaturation... 

Haas E, McWherter C, Scheraga H. Conformational unfolding in the N-terminal region of ribonuclease A detected by nonradiative energy transfer Distribution of interresidue distances in the native, denatured, and reduced-denatured states. Biopolymers 1988 27 1-21. 

Most polypeptide chains devoid of cross-links assume a random-coil conformation in 8 M urea or 6 M guanidinium chloride, as evidenced by physical properties such as viscosity and optical activity. When ribonuclease was treated with P-mercaptoethanol in 8 M urea, the product was a fully reduced, randomly coiled polypeptide chain devoid of enzymatic activity. In other words, ribonuclease was denatured by this treatment (Figure 3.53). 

When active pancreatic ribonuclease A is treated with 8M urea or P-mercaptoethanol, it is converted to an inactive, denatured molecule. When urea or mercaptoethanol is removed, it attains its native (active) conformation. 

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. 

Protein folding the folding of a random coil polypeptide into its native structure, i.e. its 3-dimensional, biologically functional structure, also known as the native conformation. Loss of this native structure is known as denaturation, and the re-establishment of native structure is known as renaturation. (For the reductive denaturation and oxidative renaturation of pancreatic ribonuclease, see Protein). 

Christian Anfinsen (1916-1995), American biochemist, obtained, the Nobel Prize in 1972 for his work on ribonuclease, especially concerning the connection between the amino acid sequence and the biologically active conformation He made an important contribution showing that after denaturation (a large change of conformation) some proteins fold back spontaneously to their native conformation. 

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