Ribonuclease denaturation

Ribonuclease denatured by 8 M urea and a mercaptan (RSH a reagent that reduces disulfides to sulfhydryl groups) can be renaturated by removing the urea and RSH and air oxidizing the reduced disulfides. 

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],... 

Recently, SPE was determined to be useful in a protein refolding process. Goto, Hatton, and coworkers demonstrated that SPE was the most efficient method for solubilizing denatured ribonuclease into AOT-based w/o-MEs [59]. The w/o-ME encapsulated... 

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... 

Fig. 11. Amide F thermal denaturation spectra for ribonuclease A as followed by FTIR (left) and VCD (right), which show the IR peak shifting from the dominant /3-sheet frequency (skewed with a maximum at 1635 cm-1) to the random coil frequency ( 1645-1650 cm-1) and the VCD shape changing from the W-pattern characteristic of an a + p structure to a broadened negative couplet typical of a more disordered coil form. The process clearly indicates loss of one form and gain of another while encompassing recognition of an intermediate form. (This is seen here most easily as the decay and growth back of the 1630 cm-1 VCD feature, but is more obvious after factor analysis of the data set, Fig. 15).

Fig. 12. Thermal denaturation for ribonuclease Tj as followed by VCD, from 20° to 65°C. The matrix descriptors determined for the native state and the unfolded high-temperature data are indicated. The values indicate a loss of the helix segment but maintenance of sheet segments. Also listed are the spectrally determined fractional contributions (FC) to the secondary structure. When combined with the segment analysis, this implies that the residual sheet segments must be very short. Reprinted with permission from Pancoska, P., et al. (1996). Biochemistry 35(40), 13094-13106, the American Chemical Society.

Qi et al. (1998) have demonstrated that ribonuclease A exhibits behavior like that of cytochrome c. The burst phase observed on dilution of Gdm HCl-denatured RNase A is mimicked exactly by reduced RNase A. The latter, when carboxamidomethylated to prevent oxidation, has a CD at 222 nm that is nearly independent of temperature and indicative of extensive unfolding at zero denaturant. 

The cells are lysed in a buffer containing strong chaotropic reagents such as guanidine thiocyanate and 2-mercaptoethanol, which completely denatures any ribonuclease present. The supernatant is then placed on a cushion of CsCl (5.7 mol l-1) and centrifuged at 100000 g for 18 h. The RNA passes through the CsCl and is pelleted, while the DNA and protein remain in the aqueous solution. The RNA pellet is dissolved in buffer and concentrated by precipitation in cold ethanol. 

N. Barboy and J. Feitelson, Fluorescence lifetime study of the denaturation of ribonuclease... 

Poklar, N., N. Petrovcic, M. Oblak, and G. Vesnaver. 1999. Thermodynamic stability of ribonuclease A in alkylurea solutions and preferential solvation changes accompanying its thermal denaturation a calorimetric and spectroscopic study. Protein Sci 8 832-840. 

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 first step in all RNA isolation protocols involves lysing the cell in a chemical environment that denatures ribonucleases. The RNA is then fractionated from other cellular macromolecules by either homogenizing the tissue (dissected brain tissue) or simply vortexing the sample (very small tissues and laser-microdissected sample) without further homogenization. The cell type from which the RNA is to be isolated, the sample size, and the eventual use of the RNA will determine which procedure described here is appropriate. 

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. 

The model polypeptide acetyltetraglycine ethyl ester (ATGEE) behaves in this manner. Citrates, sulfates, phosphates, acetates, and chlorides salt out ATGEE whereas phenol, perchlorates, tosylates, trichloroacetates, thiocyanates, iodides, and bromides salt in (8). The heat denaturation of ribonuclease also fits this cTass. 

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