Talk:Sandbox Reserved 198

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<Structure load='1SRN' size='650' frame='true' align='right' caption='Semisynthetic Ribonuclease A ' scene='Sandbox_Reserved_198/Semisynthetic_rnase_a/1' />
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<Structure load='1srn' size='500' frame='true' align='right' caption='Semisynthetic RNase A (PDB: 1srn)>
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<scene name='Talk:Sandbox_Reserved_198/Ybff_active_site/1'>TextToBeDisplayed</scene>
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<scene name='Sandbox_Reserved_198/Fully_synthetic/1'>Fully Synthetic</scene>
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<scene name='Sandbox_Reserved_198/Semisynthetic_rnase_a/1'>Semisynthetic RNasa A</scene>
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<scene name='Sandbox_Reserved_198/Synthetic_component/3'>Synthetic Component</scene>
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<scene name='Sandbox_Reserved_198/Rnase_1-118/1'>RNase 1-118</scene>
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<scene name='Talk:Sandbox_Reserved_198/Mzoom/1'>Mzoom</scene>
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<Structure load='1srn' size='250' frame='true' align='right' caption='Semisynthetic RNase A '
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<scene name='Talk:Sandbox_Reserved_198/Maybe/3'>Maybe 3</scene>
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scene='Talk:Sandbox_Reserved_198' name ='1srn'/>
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<scene name='Talk:Sandbox_Reserved_198/Surface/1'>Surface</scene>
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<scene name='Talk:Sandbox_Reserved_198/K90/2'>K90</scene>
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<scene name='Talk:Sandbox_Reserved_198/116/1'>Y116</scene>
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<scene name='Talk:Sandbox_Reserved_198/Y116/3'>LY116</scene>
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<scene name='Talk:Sandbox_Reserved_198/Y116/4'>Tyr116</scene>
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== Introduction ==
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<scene name='Talk:Sandbox_Reserved_198/Tyr_209/2'>116M</scene>
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RNase B is a glycoprotein with N-linked carbohydrates. This sugar chain aids in the folding of the protein as well as well as cell to cell signaling<ref name="first">PMID:20301239</ref>. This plays an important role in tumor formation because it has been found that N-linked glycans recognized by the CD337 receptor on "natural killer cells" are mutated in tumor cells, stopping the death of these cells<ref name="second">http://en.wikipedia.org/wiki/Glycan#Functions_and_importance</ref>. RNase B is structurally the same as RNase A, however it has additional catalytic activity caused by the attachment of polysaccharrides at the <scene name='Sandbox_Reserved_196/Rbb_basic/8'>Asn-34</scene>. This small change allows RNase B to hydrolyze double-stranded RNA at ionic strengths where RNase A has no activity, showing that small changes in the active sites of very similar molecules can lead to totally new roles and activities <ref name="third">PMID:3680242</ref>. <scene name='Sandbox_Reserved_196/Rbb_basic/1'>(Return to original scene)</scene>
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<scene name='Talk:Sandbox_Reserved_198/Lys90/5'>1</scene>
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== Structure and Biology of RNase B ==
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<scene name='Talk:Sandbox_Reserved_198/Lys90/3'>2</scene>
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[[Image:RNase B.jpg | thumb|left|RNase B]]
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RNase A and RNase B have identical primary structures; however, RNase B is bound to mannose carbohydrates. This glycosylation increases the kinetic stability of RNase B by 3 kj/mol compared to RNase A <ref name="fourth">PMID:10600722</ref>. Gycosylated, RNase B however, is not significantly different by NMR in protein <Structure load='1rbj' size='250' frame='true' align='right' caption='Ribonuclease B with a strand of DNA in active site' scene='Sandbox_Reserved_196/Secondary_structure/8' name ='1rbj' />conformation to RNase A <ref name="fifth">PMID:1322837</ref>.
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Slight differences indclude changes in crystal packing. RNase B crystals have two slightly asymmetrical units. The crystals are dimeric with two separate molecules, I and II, which are linked by a salt bridge at Asp-121 and Arg-85. This linkage determines the orientation of the two molecules in relation to one another. Not only does a salt bridge link this dimer-type molecule, but other ions also interact via cross-linkage to stabilize the structure <ref name="third" />.
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<scene name='Talk:Sandbox_Reserved_198/Lys90/4'>3</scene>
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The crystallization of RNase B provided the structure of the active site in which double stranded RNA is hydrolyzed. The active site, a triangle formation of <scene name='Sandbox_Reserved_196/Secondary_structure/11' target='1rbj'>Lys-41, His-12, and His-119</scene> was shown to be the most intense active site and is found in both molecules I and II of RNase B. In molecule II, the most drastic difference is the proximity of the active site to Lys-66, because ions can ligand to <scene name='Sandbox_Reserved_196/Secondary_structure/10' target='1rbj'>Lys-66, Arg-39 and Lys-1</scene>. Even though both active sights are close to identical, the two separate molecules are packed very differently from one another. These active sights have been seen to deviate less from their “true” positions than those molecules in RNase A. Shown in the image, the region of <scene name='Sandbox_Reserved_196/Rbb_basic/9' target='1rbb'>residues 15-23</scene> (in top applet) appear to have more flexibility, and upon looking at the structure could provide the opening for the active site. This catalytic site, with all the structures shown, has still not been an aid in providing the mechanism by which RNase performs its duty of hydrolyzing double stranded RNA <ref name="third" />. <scene name='Sandbox_Reserved_196/Secondary_structure/8' target='1rbj'>(Return to original scene)</scene>
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<scene name='Talk:Sandbox_Reserved_198/T/2'>H</scene>
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<scene name='Talk:Sandbox_Reserved_198/T/3'>H1</scene>
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==Semisynthetic Ribonuclease A==
 +
----
-
== References ==
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2-D Semisynthetic RNase A
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<references />
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== Additional Resources ==
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==Introduction==
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<ref group="xtra">http://www.pdb.org/pdb/explore/explore.do?structureId=1RBJ</ref><ref group="xtra">http://www.pdb.org/pdb/explore/explore.do?structureId=1RBB</ref><ref group="xtra">[[Ribonuclease]]</ref><ref group="xtra">[[User:R. Jeremy Johnson]]</ref>
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<references group="xtra"/>
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The synthesis of a fully active semi-synthetic RNase A supports the
 +
hypothesis that the amino acid sequence of a protein solely dictates
 +
the formation of an active enzyme and demonstrates that an enzyme with
 +
the catalytic activity and specificity of a naturally produced enzyme can
 +
be made in laboratory. Semisynthetic RNase A illustrates that functional
 +
enzymes can be produced from merely the individual constituent amino acid
 +
residues. Polypeptide synthesis can provide new routes to the study of
 +
enzymes through the selective modification of natural proteins to
 +
assay individual roles of amino acids in folding and catalysis.
 +
 
 +
 
 +
 
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=='''Function'''==
 +
 
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The structure to function relationship is clearly exhibited by semisynthetic
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RNase A. In the RNase A protein, the removal of six C terminal residues,
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leaving <scene name='Sandbox_Reserved_198/Rnase_1-118/1'>RNase 1-118</scene>,
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completely halts enzymatic activity (Martin, 1987). However, a complex of
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RNase 1-118 with a synthetic polypeptide comprising the <scene name='Talk:Sandbox_Reserved_198/Synthetic_component_114-124/1'>C terminal residues 114-124</scene>
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restores enzymatic activity to RNase A. Upon the addition of the synthetic
 +
chain, the semisynthetic enzyme adopts a structure that closely resembles
 +
that of natural RNase (Martin, 1987). The restoration of the structure
 +
reconstitutes the enzymatic activity of RNase to 98% (Martin, 1987).
 +
 
 +
 
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=='''Synthetic Method'''==
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The RNase 1-118 was prepared by successive digestion of RNase A pepsin and
 +
carboxypeptidase A (Doscher, 1983). The synthetic component, RNase 111-124,
 +
was prepared by the use of solid-phase peptide synthetic mothods, in which
 +
the peptide chain was assembled in the stepwise mannar while it was attached
 +
at one end to a solid support. The peptide chain was extented by repetitive
 +
steps of deprotection, neutralization and coupling until the desired sequence
 +
was obtained (Lin, 1970). It was important that the synthesis proceeds rapidly
 +
and in high yields to prevent side reactions or by-products.
 +
 
 +
'''Related Web-links'''
 +
 
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1. Introduction to Ribonuclease A by Raines:
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http://www.uta.edu/faculty/sawasthi/Enzymology-4351-5324/Class%20Syllabus%20Enzymology/ribonucleaseA.pdf
 +
 
 +
2. Solid Phase Synthesis by Merrifield (Nobel Prize Winner):
 +
http://nobelprize.org/nobel_prizes/chemistry/laureates/1984/merrifield-lecture.pdf
 +
 
 +
3. Chemical Synthesis of Proteins:
 +
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845543/?tool=pmcentrez
 +
 
 +
4. Refined Crystal Structure: http://www.ncbi.nlm.nih.gov/pubmed/3680234
 +
 
 +
'''References'''
 +
 
 +
Martin, Philip D., Marilynn S. Doscher, and Brian F. P. Edwards. "The Redefined
 +
Crystal Structure of a Fully Active Semisynthetic Ribonuclease at 1.8-A Resolution."
 +
The Journal of Biological Chemistry
 +
262.33 (1987): 15930-5938.
 +
 
 +
Marilynn S. Doscher, Philip D. Martin and Brian F.P. Edwards, "Characerization
 +
of the Histidine Proton Nuclear Magnetic Resonance of a Semisynthetic Ribonuclease."
 +
Biochemistry, 1983,22,4125-4131.
 +
 
 +
Lin, M. C. (1970) Journal of Biological Chemistry, 245, 6726-6731.
 +
 
 +
David J. Boerema, Valentina. A. T., Stephen B. H. Kent, "Total Synthesis by
 +
Modern chemical Ligation Methods and High Resolution (1.1-A) X-ray structure
 +
of Ribonuclease A. Biopolymers. 2008;90(3):278-86.
 +
[[Link title]]

Current revision

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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Semisynthetic Ribonuclease A

Drag the structure with the mouse to rotate

Contents

Semisynthetic Ribonuclease A


2-D Semisynthetic RNase A

Introduction

The synthesis of a fully active semi-synthetic RNase A supports the hypothesis that the amino acid sequence of a protein solely dictates the formation of an active enzyme and demonstrates that an enzyme with the catalytic activity and specificity of a naturally produced enzyme can be made in laboratory. Semisynthetic RNase A illustrates that functional enzymes can be produced from merely the individual constituent amino acid residues. Polypeptide synthesis can provide new routes to the study of enzymes through the selective modification of natural proteins to assay individual roles of amino acids in folding and catalysis.


Function

The structure to function relationship is clearly exhibited by semisynthetic RNase A. In the RNase A protein, the removal of six C terminal residues, leaving , completely halts enzymatic activity (Martin, 1987). However, a complex of RNase 1-118 with a synthetic polypeptide comprising the restores enzymatic activity to RNase A. Upon the addition of the synthetic chain, the semisynthetic enzyme adopts a structure that closely resembles that of natural RNase (Martin, 1987). The restoration of the structure reconstitutes the enzymatic activity of RNase to 98% (Martin, 1987).


Synthetic Method

The RNase 1-118 was prepared by successive digestion of RNase A pepsin and carboxypeptidase A (Doscher, 1983). The synthetic component, RNase 111-124, was prepared by the use of solid-phase peptide synthetic mothods, in which the peptide chain was assembled in the stepwise mannar while it was attached at one end to a solid support. The peptide chain was extented by repetitive steps of deprotection, neutralization and coupling until the desired sequence was obtained (Lin, 1970). It was important that the synthesis proceeds rapidly and in high yields to prevent side reactions or by-products.

Related Web-links

1. Introduction to Ribonuclease A by Raines: http://www.uta.edu/faculty/sawasthi/Enzymology-4351-5324/Class%20Syllabus%20Enzymology/ribonucleaseA.pdf

2. Solid Phase Synthesis by Merrifield (Nobel Prize Winner): http://nobelprize.org/nobel_prizes/chemistry/laureates/1984/merrifield-lecture.pdf

3. Chemical Synthesis of Proteins: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845543/?tool=pmcentrez

4. Refined Crystal Structure: http://www.ncbi.nlm.nih.gov/pubmed/3680234

References

Martin, Philip D., Marilynn S. Doscher, and Brian F. P. Edwards. "The Redefined Crystal Structure of a Fully Active Semisynthetic Ribonuclease at 1.8-A Resolution." The Journal of Biological Chemistry 262.33 (1987): 15930-5938.

Marilynn S. Doscher, Philip D. Martin and Brian F.P. Edwards, "Characerization of the Histidine Proton Nuclear Magnetic Resonance of a Semisynthetic Ribonuclease." Biochemistry, 1983,22,4125-4131.

Lin, M. C. (1970) Journal of Biological Chemistry, 245, 6726-6731.

David J. Boerema, Valentina. A. T., Stephen B. H. Kent, "Total Synthesis by Modern chemical Ligation Methods and High Resolution (1.1-A) X-ray structure of Ribonuclease A. Biopolymers. 2008;90(3):278-86. Link title

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