Ribonuclease protein structural studies

Reported applications of DMA include the cross-linking of bovine pancreatic ribonuclease A (Hartman and Wold, 1967), treatment of erythrocyte membranes to reduce the effects of sickle cell anemia (Waterman et al., 1975), conjugation and analysis of the outer membrane proteins of Neisseria gonorrhoeae (Newhall et al., 1980), protein structural studies of bovine a-crystalline (Siezen et al., 1980), cross-linking of hemoglobin S (Pennathur-Das et al., 1982), and forming S-carbomethoxy-valeramidine during hydrolysis of DMA (Mentzer et al., 1982). 

The great specificity of tyrosinase, the simplicity of monitoring its action spectrally, and in some cases, the discrimination shown among the several tyrosyl groups of a protein [it oxidizes only one of the six tyrosyl groups of ribonuclease (Yasuiiobu and Dandliker, 1957)] appear to recommend its serious consideration as a tool in protein structure studies. 

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. 

Ribonuclease H is a small, single domain protein that eleaves RNA from RNA-DNA hybrids. RNase H from E. coli T = 66 °C) has been structurally studied by NMR spectroscopy.Hydrogen exchange NMR experiments have been used to examine the structural distribution of stability in RNase H from T. thermophiliis = 86 °C) and to compare its stability with that of the homologous RNase H from the mesophilic E. The general distribution... 

The tertiary structure of a protein refers to the arrangement of amino acid side chains in the protein. Generally, the information for protein structure is contained within the amino acidjequence of the protein itself This important principle of biochemistry was first determined by the biochemist Christian Anfinsen in studies of the enzyme ribonu-clease. Ribonuclease catalyzes a simple hydrolysis of ribonucleic acid. The native enzyme has 124 amino acids 8 of these are cysteines, forming 4 disulfide bonds. When ribonuclease was treated with mercaptoethanol to destroy the disulfide bonds and urea to disrupt its secondary and tertiary structure, all enzymatic activity was... 

Tilton Jr. RF, Dewan JC, Petsko GA Effect of temperature on protein structure and dynamics x-ray crystahographic studies of the protein ribonuclease-A at nine different temperatures from 98 to 320K. Biochemistry 1992,31 2469-2481. 

However, there are a number of other miscellaneous biological roles played by this complex. The [Co(NH3)6]3+ ion has been shown to inhibit the hammerhead ribozyme by displacing a Mn2+ ion from the active site.576 However, [Co(NH3)6]3+ does not inhibit ribonuclease H (RNase),577 topoisomerase I,578 or hairpin ribozyme,579 which require activation by Mg2+ ions. The conclusions from these studies were that an outer sphere complex formation between the enzyme and Mgaq2+ is occuring rather than specific coordination of the divalent ion to the protein. These results are in contrast to DNase I inhibition by the same hexaammine complex. Inhibition of glucose-induced insulin secretion from pancreatic cells by [Co(NH3)6]3+ has been found.580 Intracellular injection of [Co(NH3)6]3+ into a neurone has been found to cause characteristic changes to the structure of its mitochondria, and this offers a simple technique to label neuronal profiles for examination of their ultrastructures.581... 

This technique has been described as a general method of studying protein-protein interactions as well as a method for investigating the three-dimensional structure of individual proteins (Muller et al., 2001 Back et al., 2003 Dihazi and Sinz, 2003 Sinz, 2003 Sinz, 2006). It also has been used for the study of the interactions of cytochrome C and ribonuclease A (Pearson et al., 2002), to investigate the interaction of calmodulin with a specific peptide binder (Kalkhof et al., 2005a Schmidt et al., 2005), and for probing laminin self-interaction (Kalkhof et al., 2005b). 

A relative of the kinases is adenylate cyclase, whose role in forming the allosteric effector 3, 5 -cyclic AMP (cAMP) was considered in Chapter 11. This enzyme catalyzes a displacement on Pa of ATP by the 3 -hydroxyl group of its ribose ring (see Eq. 11-8, step a). The structure of the active site is known.905 Studies with ATPaS suggest an in-line mechanism resembling that of ribonuclease (step a, Eq. 12-25). However, it is Mg2+ dependent, does not utilize the two-histidine mechanism of ribonuclease A, and involves an aspartate carboxylate as catalytic base.906 All isoforms of adenylate cyclase are activated by the a subunits of some G proteins (Chapter 11). The structures907 of Gsa and of its complex with adenylate kinase905 have been determined. The Gsa activator appears to serve as an allosteric effector. 

The enzyme consists of a single polypeptide chain of Mr 13 680 and 124 amino acid residues.187,188 The bond between Ala-20 and Ser-21 may be cleaved by subtilisin. Interestingly, the peptide remains attached to the rest of the protein by noncovalent bonds. The modified protein, called ribonuclease S, and the native protein, now termed ribonuclease A, have identical catalytic activities. Because of its small size, its availability, and its ruggedness, ribonuclease is very amenable to physical and chemical study. It was the first enzyme to be sequenced.187 The crystal structures of both forms of the enzyme were solved at 2.0-A resolution several years ago.189,190 Subsequently, crystal structures of many complexes of the enzyme with substrate and transition analogues and products have been solved at very high resolution.191 Further, because the catalytic activity depends on the ionizations of two histidine residues, the enzyme has been extensively studied by NMR (the imidazole rings of histidines are easily studied by this method—see Chapter 5). 

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