Ribonuclease, conformation

In a more recent study using circular dichroism, Pflumm and Beychok (313) have fitted the observed curve for RNase-A (see Figs. 11c and d) weighted mixtures of the characteristic bands for helix from poly-L-glutamic acid and / structure from poly-L-lysine. The data are compatible with 11.5% helix and 33% / conformation. Ribonuclease-S and RNase-A have almost identical CD spectra from 198 to 300 nm. The spectrum of S-protein is markedly different from the other two. 

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. 

The secondary and tertiary structures of myoglobin and ribonuclease A illustrate the importance of packing in tertiary structures. Secondary structures pack closely to one another and also intercalate with (insert between) extended polypeptide chains. If the sum of the van der Waals volumes of a protein s constituent amino acids is divided by the volume occupied by the protein, packing densities of 0.72 to 0.77 are typically obtained. This means that, even with close packing, approximately 25% of the total volume of a protein is not occupied by protein atoms. Nearly all of this space is in the form of very small cavities. Cavities the size of water molecules or larger do occasionally occur, but they make up only a small fraction of the total protein volume. It is likely that such cavities provide flexibility for proteins and facilitate conformation changes and a wide range of protein dynamics (discussed later). 

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],... 

Lu, X. M., Benedek, K., and Karger, B. L., Conformational effects in the high-performance liquid chromatography of proteins. Further studies of the re-versed-phase chromatographic behavior of ribonuclease, ]. Chromatogr., 359, 19, 1986. 

S. J. Wearne and T. E. Creighton, Effect of protein conformation on rate of deamidation Ribonuclease A, Proteins Struct. Funct. Genet, 5, 8 (1989). 

Osterhout JJ Jr, Baldwin RL, York EJ, Stewart JM, Dyson HJ, Wright PE (1989) 11 NMR studies of the solution conformations of an analogue of the C-peptide of ribonuclease A. Biochemistry 28 7059-7064. 

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. 

Crestfield, A. M., Stein, W. H., and Moore, S. (1963). Properties and conformation of the histidine residues at the active site of ribonuclease./. Biol. Chem. 238, 2421-2428. DeLano, W. L. (2002). The PyMol Molecular Graphics System. DeLano Scientific, San Carlos, CA. 

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]. 

In recent years attention has focused on the role of intrinsic binding energy and entropic factors as major contributors to enzyme catalytic efficiency (Page and Jencks, 197l Jencks, 1975,1981). The ribonuclease mechanism conforms to expectations based on these ideas. In particular, distortion occurs to raise the ground state of the substrate in the S complex, and the bound substrate interacts with the enzyme in a manner such that the enzyme becomes complementary to the transition state of the reaction during the catalytic cycle. 

No large conformational changes occur in the enzyme during catalysis, but many small movements take place. The structural basis for the catalytic power of ribonuclease thus resides in several different features tight, specihc binding of a strained conformation of the substrate, general acid-base catalysis by His-12 and His-119, and preferential stabilization of the transition state by ionic interactions with Lys-41. 

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. 

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