Sandbox Reserved 192

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This Sandbox is Reserved from Feb 02, 2011, through Jul 31, 2011 for use by the Biochemistry II class at the Butler University at Indianapolis, IN USA taught by R. Jeremy Johnson. This reservation includes Sandbox Reserved 191 through Sandbox Reserved 200.
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RNase A



Highlighted here is the kidney bean shape of RNase A with the active site located within the cleft..
Highlighted here is the kidney bean shape of RNase A with the active site located within the cleft..

Ribonucleases [1] or RNA depolymerases are enzymes that catalyze RNA degradation. Ribonucleases are highly active in ruminants [2], such as cows, to digest large amounts of RNA produced by microorganisms in the stomach. Ruminants also have high amounts of ribonucleases to process nutrients from cellulose. One such ribonuclease, bovine ribonuclease A or RNase A, is a model enzyme due to its ease of purification and simple structure.


RNase A is made up of a single polypeptide chain of 124 residues. Of the 20 natural amino acids, RNase A possesses 19 of them, excluding tryptophan. This single polypeptide chain is cross-linked internally by four , which contribute to the stability of RNase A. Long four-stranded anti-parallel and three short make up the of RNase A (Raines). The structure of RNase A is often described as kidney shaped, with the active-site residues located within the cleft. residues aid in catalysis. stabilizes the negative charge in the transition state, while acts as a base and acts as an acid in catalysis. The amino acid sequence was discovered to determine the three-dimensional structure of RNase A by Christian Anfinsen in the 1950s. Urea was used to denature RNase A, and mercaptoethanol was used to reduce and cleave the four disulfide bonds in RNase A to yield eight Cys residues. Catalytic activity was lost due to denaturation. When the urea and mercaptoethanol were removed, the denatured ribonuclease refolded spontaneously into its correct tertiary structure with restoration of its catalytic activity. Disulfide bonds were also reformed in the same position. The Anfinsen experiment provided evidence that the amino acid sequence contained all the information required for the protein to fold into its native three-dimensional structure. Anfinsen received the 1972 Nobel Prize in Chemistry for his work with RNase A. Nevertheless, ensuing work showed some proteins require further assistance, such as molecular chaperones, to fold into their native structure (Nelson and Cox).


RNase A has been used as a foundation enzyme for study due to its stability, small size, and because its three-dimensional structure is fully determined by its amino acid sequence, needing no molecular chaperones. The 1972 Nobel Prize in Chemistry was awarded to three researchers for their work with RNase A on the folding of chains in RNase A and the stability of RNase A. The previously mentioned Christian Anfinsen received the 1972 Nobel Prize in Chemistry for his paper "Principles that govern the folding of protein chains." Stanford Moore and William H. Stein received the 1972 Nobel Prize in Chemistry for their paper "The chemical structures of pancreatic ribonuclease and deoxyribonuclease." The 1984 Nobel Prize in Chemistry was awarded to Robert Bruce Merrifield for his paper "Solid-phase synthesis" using RNase A (Raines). RNase A was the first enzyme and third protein for which its amino acid sequence was correctly determined and the third enzyme and fourth protein whose three-dimensional structure was determined by X-ray diffraction analysis [3]. Disulfide bonds in RNase A were determined after developing a method using Fast Atom Bombardment Mass Spectrometry (FABMS) [4]. The methods of NMR spectroscopy [5] and Fourier transform infrared (FTIR) spectroscopy [6] were developed with RNase A in determining protein structure and protein folding pathways. These new methods, developed with RNase A, could be used for further research to determine the protein structure and protein folding pathways of other proteins (Raines).

Medical Implications

A recent study by Patutina et al. (2011) revealed that tumor propagation is associated with an imbalance in nucleic acid metabolism. In the blood plasma of patients, there were increased levels of circulating nucleic acids and decreased nuclease activity. The abnormally high levels of circulating nucleic acids were associated with the increased expression and secretion of tumor-derived miRNA and DNA. With increased expression, the tumor progresses and the patient has a bad prognosis.

RNase A and DNase I inhibit metastasis [7] by catalyzing metastasis pathomorphosis which is apoptosis, necrosis [8] and destruction of oncocytes [9]. This capability retards the primary tumor growth by 30-40%. The tumor bearing mice received doses of RNase A, DNase I or a mixture of the two and the most significant effect observed was in the mice treated with both enzymes simultaneously. Thus the simultaneous administration of RNase A and DNase I led to an anti-metastatic effect and resulted in an almost complete absence in the metastasis of the tumor. Further observations suggest that RNase A and DNase I are toxic at high levels. So for effective treatment, ultra low doses are required to stay below the level of toxicity.

Another member in the ribonuclease family and structural homologue to bovine RNase A is frog onconase [10] or ONC. ONC is found in oocytes [11] and early embryos of northern leopard frogs. The frog ribonuclease variant shows both cytostatic (cell growth suppression) and cytotoxic (prevents cell divisions) characteristics when it interacts with tumor cells. According to Gahl et al. (2008), no side effects have been determined for ONC. Leland et al. (2001) looked to determine the interactions that control the folding of ONC in order to develop effective mimics of ONC. In order to determine the interactions that controlled folding, the regeneration of RNase A was studied. Although RNase A and ONC were structurally very similar, there were significant differences in their folding pathways. While ONC forms a stable disulfide intermediate, RNase A does not. ONC was also found to be missing a disulfide bond that RNase A possesses. In the case of both enzymes, entropy is lost in the formation of the disulfide bonds, but folding may be driven by enthalpically favorable interactions of the side chains. Further experiments are being done to identify intramolecular interactions that account for the increased rate and formation of the structured intermediate in ONC (Gahl).

RNase A

Further Research with the Hydrophobic Core

The phenylalanine-46 (Phe46) residue located within the of RNase A was experimentally replaced with other hydrophobic residues: leucine, valine and alanine. The goal was to conclude how the change would affect the conformational stability. It was concluded that the replacement of Phe46, which is key to the formation of the hydrophobic core, causes the destabilization of the RNase A by preventing the core from being tightly packed. The protein folds with its hydrophobic amino acids facing inward and its hydrophilic amino acids facing outward to reduce the amount of water that interacts with the least number of hydrophobic residues (Kadonosono).

Evolutionary Significance

RNase variants have undergone duplication six times since amphibians and mammals diverged, giving rise to RNase A and other homologues. RNase A was believed to have become more specified within bovids[12] 35 million years ago (Opitz et al. 1997). RNase A homologues have been found in frogs and humans by comparing the amino acid sequences of these particular enzymes with RNase A and seeing what residues were conserved. , shown here for the homologues of RNase A, can either support or refute theories of protein structure and function. There have been over 40 different RNase homologues that have been sequenced. Conservation of amino acids Lys41 and His12 and His119 maintain the catalytic function within RNase A homologues. However, these RNase A homologues differ in cytotoxicity and also have slight differences in sequences which may lead to different functions. One homologue, angiogenin, promotes neovascularization [13]. Unusual homologues include other RNase homologues in the human body such as in urine and red blood cells. (Raines)

Literary Citations

Ferreri, Carla; Chryssostomos Chatgillaloglu, Armida Torreggiani, Anna Marla Salzano, Giovanni Rensone, and Andrea Scaloni. “The Reductive Desulfurization of Met and Cys Residues in Bovine RNase A Is Assoicated with trans Lipid Formation in a Mimetic Model of Biological Membranes.” Journal of Proteom. 7 (2008): 2007-2015[14]

Gahl, R. F. et al. “Dissimilarity in the oxidative folding of onconase and ribonuclease A, two structural homologues.”Proetin Engineering, Design & Selection. 21 (2008) 223-231[15]

H. P. Avey; M. O. Boles; C. H. Carlisle; S. A. Evans; S. J. Morris; R. A. .Palmer; B. A. Woolhouse.”Structure of Ribonuclease.” Nature. 213 (1967) 557-562[16]

H. W. Wyckoff, Karl D. Hardman; N. M. Allewell; Tadash Inagam; L. N. Johnson. “The Structure of Ribonuclease-S at 3.5 A Resolution.” Department of Molecular Biophysics, Yale University. 242 (1967): 3984-3988 [17]

Kadonosono, Tetsuya; Eri Chatani, Rikimaru Hayashi, Hideaki Moriyama, and Tatzuo Ueki. “Minimization of Cavity Size Ensures Protein Stability and Folding: Structures of Phe-46-Replaced Bovine Pancreatic RNase A.” Biochemistry. 42 (2003): 10651-10658 [18]

Nelson, L. D., M. Cox. "Lehninger Principles of Biochemistry" New York, NY. 2008 (Fifth Edition)

Opitz, J. G. et al. “Origin of the catalytic activity of bovine seminal ribonuclease against double-stranded RNA.” Biochemistry 1998. 37 (4023-4033)

Patutina, Olga; Nadezda Mironova, Elena Ryabchikova, Nelly Popova, Valery Nikolin, Vasily Kaledin, Valentin Valssov, Marina Zenkova. “ Inhibition of Metastasis Development by Daily Administration of Ultralow Doses of RNase A and DNase I” Biochimie. 93 (2011) 689-696 [19]

Raines, Ronald T. “Ribonuclease A.” Chemistry Review. Madison Wisconsin. 98 (1998): 1045-1065 [20]

Wlodawer, Alexander; L. Anders Svensson, Lennart Sjolin, Gary L. Gilliland. “Structure of Phosphate-Free Ribonuclease A Refined at 1.26 A.” American Chemical Society. 27 (1988) 2705-2717 [21]

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