User:R. Jeremy Johnson/Folding Synthesis: Difference between revisions

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==='''Proline Conformation'''===

The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding. In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in the ''cis'' conformation than any other amino acid. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. The importance of these conformations are demonstrated based on the structure of RNase A variants with several mutations to the wild type amino acid sequence.

Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/3'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/7'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. When proline was mutated to alanine, <scene name='Sandbox_Reserved_197/P93a/9'>P93A</scene>, a ''cis'' conformation still forms at position 93 which is an energetically unfavorable conformation for an alanine residue [http://www.ncbi.nlm.nih.gov/pubmed/9605332]. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the folded protein structure. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation, suggesting an important kinetic contribution of ''cis'' prolines to protein folding.

Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/3'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/7' target='0'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. When proline was mutated to alanine, <scene name='Sandbox_Reserved_197/P93a/9' target='0'>P93A</scene>, a ''cis'' conformation still forms at position 93 which is an energetically unfavorable conformation for an alanine residue [http://www.ncbi.nlm.nih.gov/pubmed/9605332]. Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the folded protein structure. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation, suggesting an important kinetic contribution of ''cis'' prolines to protein folding.

The <scene name='Sandbox_Reserved_197/Cis-proline114/3'>Asn113-Pro114</scene> peptide bond also resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. Unlike P93A, the insertion of a <scene name='Sandbox_Reserved_197/P114g/3'>P114G</scene> point mutation causes the peptide bond to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop [http://onlinelibrary.wiley.com/doi/10.1110/ps.051610505/full]. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein can lead to formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein.

The <scene name='Sandbox_Reserved_197/Cis-proline114/3' target='0'>Asn113-Pro114</scene> peptide bond also resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. Unlike P93A, the insertion of a <scene name='Sandbox_Reserved_197/P114g/3' target='0'>P114G</scene> point mutation causes the peptide bond to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop [http://onlinelibrary.wiley.com/doi/10.1110/ps.051610505/full]. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein can lead to formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein.

Another important role of proline residues is their involvement in β turns. β turns are 180° turns commonly found in globular proteins to allow for a compact structure by connecting the ends of adjacent antiparallel β sheets [http://en.wikipedia.org/wiki/Beta_sheet]. The turn consists of a sequence of four amino acid residues. The carbonyl of the first amino acid hydrogen bonds with the amino group of the fourth amino acid. Proline is involved in β turns because it is small, flexible, and assumes a ''cis'' conformation, all attributes that allow for formation of a turn. In RNase A both Pro93 and Pro114 are involved in β turns. Proline residues are important to protein folding because their ability to form a favorable ''cis'' conformation allows for thermodynamic favorability of β turn formation. With β turns, amino acids can fold back on themselves allowing the protein to reside in a compact, globular structure.

==='''Disulfide Bonds'''===

Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/6'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/6'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/8'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/7'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet which is the main contributor to the thermodynamic stability of the enzyme.

Another important feature of the folding of RNase A is the presence of four disulfide bonds. These bonds contribute to the thermal stability and the rate of folding of RNase A. The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/6' target='0'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/6' target='0'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/8' target='0'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/7' target='0'>Cys65-Cys72</scene>. Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet which is the main contributor to the thermodynamic stability of the enzyme.

Measurements of protein activity upon removal of disulfide bridges show that the change in enzymatic activity is very small and that not all disulfide bridges are essential for the structure or the reactivity of the protein. However, removal of disulfide bonds does destabilize the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/8'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. In the variant, only 3 disulfide bonds are present, but the overall structure is only changed slightly. The differences occur in residues in close proximity to the location of the missing disulfide bond, <scene name='Sandbox_Reserved_197/Residues_34-45/1'>34-45</scene> and <scene name='Sandbox_Reserved_197/Residues_83-101/1'>83-101</scene>, where there are increased levels of disorder and a destabilized hydrophobic core [http://www.ncbi.nlm.nih.gov/pubmed/9605332].

Measurements of protein activity upon removal of disulfide bridges show that the change in enzymatic activity is very small and that not all disulfide bridges are essential for the structure or the reactivity of the protein. However, removal of disulfide bonds does destabilize the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/8' target='0'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. In the variant, only 3 disulfide bonds are present, but the overall structure is only changed slightly. The differences occur in residues in close proximity to the location of the missing disulfide bond, <scene name='Sandbox_Reserved_197/Residues_34-45/1' target='0'>34-45</scene> and <scene name='Sandbox_Reserved_197/Residues_83-101/1' target='0'>83-101</scene>, where there are increased levels of disorder and a destabilized hydrophobic core [http://www.ncbi.nlm.nih.gov/pubmed/9605332].

==='''Medical Importance of Protein Folding'''===

Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding because proteins can form aberrant aggregates when they do not fold correctly. This abnormality can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/2'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/3'>hydrophobic patches</scene> and, in turn, cause several proteins to stick together and form a plaque. In the future researchers hope to design drugs that combat mistakes in protein folding [http://www.sciencedaily.com/releases/2009/10/091013105324.htm]. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent protein misfolding.

Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding because proteins can form aberrant aggregates when they do not fold correctly. This abnormality can be toxic to human nerve cells. All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/2' target='0'>hydrophobic and hydrophilic residues</scene>. The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/3' target='0'>hydrophobic patches</scene> and, in turn, cause several proteins to stick together and form a plaque. In the future researchers hope to design drugs that combat mistakes in protein folding [http://www.sciencedaily.com/releases/2009/10/091013105324.htm]. The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent protein misfolding.

==='''Summary of Protein Folding'''===

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''Semisynthetic RNase A''

The synthesis of semisynthetic RNasa A clearly exhibits the structure to function relationship that defines proteins. In the RNase A protein, the removal of six C terminal residues, leaving <scene name='Sandbox_Reserved_198/Rnase_1-118/1'>RNase 1-118</scene>, completely halts enzymatic activity<ref>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.</ref>. However, a complex of RNase 1-118 with a synthetic polypeptide comprising the C terminal residues <scene name='Sandbox_Reserved_198/Synthetic_component/3'>111-124</scene> restores enzymatic activity to RNase A. Upon the addition of the synthetic chain, the <scene name='Sandbox_Reserved_198/Interface/7'>semisynthetic enzyme</scene> <scene name='Sandbox_Reserved_198/Interface/8'>(Zoom)</scene> adopts a structure that closely resembles that of <scene name='Sandbox_Reserved_198/Wild_type/1'>Wild Type RNase A</scene><ref>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.</ref>. The restoration of the structure reconstitutes the enzymatic activity of RNase to 98%<ref>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.</ref>.

The synthesis of semisynthetic RNasa A clearly exhibits the structure to function relationship that defines proteins. In the RNase A protein, the removal of six C terminal residues, leaving <scene name='Sandbox_Reserved_198/Rnase_1-118/1' target='1'>RNase 1-118</scene>, completely halts enzymatic activity<ref>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.</ref>. However, a complex of RNase 1-118 with a synthetic polypeptide comprising the C terminal residues <scene name='Sandbox_Reserved_198/Synthetic_component/3' target='1'>111-124</scene> restores enzymatic activity to RNase A. Upon the addition of the synthetic chain, the <scene name='Sandbox_Reserved_198/Interface/7' target='1'>semisynthetic enzyme</scene> <scene name='Sandbox_Reserved_198/Interface/8' target='1'>(Zoom)</scene> adopts a structure that closely resembles that of <scene name='Sandbox_Reserved_198/Wild_type/1' target='1'>Wild Type RNase A</scene><ref>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.</ref>. The restoration of the structure reconstitutes the enzymatic activity of RNase to 98%<ref>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.</ref>.

The semi-synthetic RNase A comprises of residues 1-118 and the synthetic analog of residues 111-124. The RNase 1-118 was prepared by successive digestion of RNase A pepsin and carboxypeptidase A<ref>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</ref>. The synthetic component, RNase 111-124, was prepared by the use of solid-phase peptide synthetic methods, in which the peptide chain was assembled in the stepwise manner while it was attached at one end to a solid support. The peptide chain was extended by repetitive steps of de-protection, neutralization and coupling until the desired sequence was obtained<ref>Lin, M. C. (1970) Journal of Biological Chemistry, 245, 6726-6731</ref>. It was important that the synthesis proceeds rapidly and in high yields to prevent side reactions or by-products.

Line 43:

''Fully Synthetic RNase A''

The <scene name='Sandbox_Reserved_198/Fully_synthetic/4'>Fully Synthetic RNase A</scene> demonstrates similar structural and functional characteristics (such as catalytic activity) as those of the <scene name='Sandbox_Reserved_198/Wild_type/1'>Wild Type</scene> RNase A<ref>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.</ref>. The crystal data, X-ray data collection and refinement statistics show that the fully synthetic protein shares identical molecular structures with the wild type RNase A, and that the active sites of both emzymes contain no walter molecules and have no substrate ligand<ref>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.</ref>. The crystal structure similarities of the <scene name='Sandbox_Reserved_198/Wild_type_as/1'>Wild Type Active Site</scene>, <scene name='Sandbox_Reserved_198/Fully_synthetic_active_site/1'>Fully Synthetic Active Site</scene>, and <scene name='Sandbox_Reserved_198/Active_site/1'>Semisynthetic Active Site</scene> are further evidence that amino acid sequence dictates folded structure formation.

The <scene name='Sandbox_Reserved_198/Fully_synthetic/4' target='1'>Fully Synthetic RNase A</scene> demonstrates similar structural and functional characteristics (such as catalytic activity) as those of the <scene name='Sandbox_Reserved_198/Wild_type/1' target='1'>Wild Type</scene> RNase A<ref>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.</ref>. The crystal data, X-ray data collection and refinement statistics show that the fully synthetic protein shares identical molecular structures with the wild type RNase A, and that the active sites of both emzymes contain no walter molecules and have no substrate ligand<ref>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.</ref>. The crystal structure similarities of the <scene name='Sandbox_Reserved_198/Wild_type_as/1'>Wild Type Active Site</scene>, <scene name='Sandbox_Reserved_198/Fully_synthetic_active_site/1' target='1'>Fully Synthetic Active Site</scene>, and <scene name='Sandbox_Reserved_198/Active_site/1' target='1'>Semisynthetic Active Site</scene> are further evidence that amino acid sequence dictates folded structure formation.

The peptide ligation chemistry in addition to solid-phase peptide synthesis is used to synthesize relatively longer peptide molecules with typical length of 125 residues<ref>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.</ref>. The ligation methods overcome the length limitation of solid-phase synthesis, because the chemical ligation involves the joining of mutually reactive peptide segments created by solid-phase synthesis. The peptide bond in ligation is formed between an unprotected peptide and a peptide-thioester<ref>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.</ref>. The shorter peptide segments are more rapidly prepared and are less susceptible to solubility issues in longer peptide chains.

The <scene name='Sandbox_Reserved_198/Fully_synthetic/1'>Fully Synthetic RNase A</scene> (124 residues) is prepared by two consecutive sets of one-pot ligations and related chemical transformations of six peptide segments (residues <scene name='Sandbox_Reserved_198/1-25/1'>1-25</scene>, <scene name='Sandbox_Reserved_198/26-39/1'>26-39</scene>, <scene name='Sandbox_Reserved_198/40-64/1'>40-64</scene>, <scene name='Sandbox_Reserved_198/65-83/1'>65-83</scene>, <scene name='Sandbox_Reserved_198/84-94/1'>84-94</scene>, <scene name='Sandbox_Reserved_198/95-124/1'>95-124</scene>, as highlighted in red)<ref>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.</ref>,which can prevent undesired byproduct formation. The six unprotected peptide segments were synthesized by highly optimized, stepwise solid-phase synthesis. This synthetic pathway is simple, has high overall yields, and it eliminate the need for the isolation of intermediate products.

The <scene name='Sandbox_Reserved_198/Fully_synthetic/1' target='1'>Fully Synthetic RNase A</scene> (124 residues) is prepared by two consecutive sets of one-pot ligations and related chemical transformations of six peptide segments (residues <scene name='Sandbox_Reserved_198/1-25/1' target='1'>1-25</scene>, <scene name='Sandbox_Reserved_198/26-39/1' target='1'>26-39</scene>, <scene name='Sandbox_Reserved_198/40-64/1' target='1'>40-64</scene>, <scene name='Sandbox_Reserved_198/65-83/1' target='1'>65-83</scene>, <scene name='Sandbox_Reserved_198/84-94/1' target='1'>84-94</scene>, <scene name='Sandbox_Reserved_198/95-124/1' target='1'>95-124</scene>, as highlighted in red)<ref>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.</ref>,which can prevent undesired byproduct formation. The six unprotected peptide segments were synthesized by highly optimized, stepwise solid-phase synthesis. This synthetic pathway is simple, has high overall yields, and it eliminate the need for the isolation of intermediate products.

=='''References'''==

@@ Line 8: / Line 8: @@
 ==='''Proline Conformation'''===
 The presence of ''cis'' [http://en.wikipedia.org/wiki/Cis_configuration]proline residues plays a large role in protein folding.  In nature, most amino acids reside in a ''trans'' conformation, but due to their cyclic structure, prolines are more stable in the ''cis'' conformation than any other amino acid. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation.  The importance of these conformations are demonstrated based on the structure of RNase A variants with several mutations to the wild type amino acid sequence.
-Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/3'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/7'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. When proline was mutated to alanine, <scene name='Sandbox_Reserved_197/P93a/9'>P93A</scene>, a ''cis'' conformation still forms at position 93 which is an energetically unfavorable conformation for an alanine residue [http://www.ncbi.nlm.nih.gov/pubmed/9605332].   Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the folded protein structure. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation, suggesting an important kinetic contribution of ''cis'' prolines to protein folding.
+Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/3'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/7' target='0'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. When proline was mutated to alanine, <scene name='Sandbox_Reserved_197/P93a/9' target='0'>P93A</scene>, a ''cis'' conformation still forms at position 93 which is an energetically unfavorable conformation for an alanine residue [http://www.ncbi.nlm.nih.gov/pubmed/9605332].   Upon unfolding, Tyr92-Ala93 undergoes isomerization to form its more favorable ''trans'' conformation demonstrating that the ''cis'' conformation is favored by other interactions within the folded protein structure. Although the overall structure of RNase A is not affected by this mutation, the rate of folding greatly decreases upon insertion of the P93A mutation, suggesting an important kinetic contribution of ''cis'' prolines to protein folding.
-The <scene name='Sandbox_Reserved_197/Cis-proline114/3'>Asn113-Pro114</scene> peptide bond also resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. Unlike P93A, the insertion of a <scene name='Sandbox_Reserved_197/P114g/3'>P114G</scene> point mutation causes the peptide bond to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop [http://onlinelibrary.wiley.com/doi/10.1110/ps.051610505/full]. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein can lead to formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein.
+The <scene name='Sandbox_Reserved_197/Cis-proline114/3' target='0'>Asn113-Pro114</scene> peptide bond also resides in a ''cis'' conformation in its folded structure, but exists in the ''trans'' conformation in its unfolded state; therefore, steric restraints imposed by the rest of the protein must be responsible for this ''cis'' conformation. Unlike P93A, the insertion of a <scene name='Sandbox_Reserved_197/P114g/3' target='0'>P114G</scene> point mutation causes the peptide bond to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop [http://onlinelibrary.wiley.com/doi/10.1110/ps.051610505/full]. The kinetic rate and overall native conformation are not significantly effected by this mutation; however, locally, a rearrangement of the hydrogen-bonding network occurs. Results of this mutation confirm that steric hinderance of the protein can lead to formation of the ''cis'' conformation by a proline and is further energetically stabilized by hydrogen bonding, Van der Waals, and electrostatic interactions within the protein.
-<Structure load='7RSA' size='380' frame='true' align='right' caption='RNase A: Important prolines, disulfide bonds, and hydrophobic packing involved in its proper folding' scene='Sandbox_Reserved_197/Rnase_a_wild_type/7' />
+<Structure load='7RSA' size='380' frame='true' align='right' caption='RNase A: Important prolines, disulfide bonds, and hydrophobic packing involved in its proper folding' scene='Sandbox_Reserved_197/Rnase_a_wild_type/7' target='0'/>
 Another important role of proline residues is their involvement in β turns. β turns are 180° turns commonly found in globular proteins to allow for a compact structure by connecting the ends of adjacent antiparallel β sheets [http://en.wikipedia.org/wiki/Beta_sheet]. The turn consists of a sequence of four amino acid residues. The carbonyl of the first amino acid hydrogen bonds with the amino group of the fourth amino acid. Proline is involved in β turns because it is small, flexible, and assumes a ''cis'' conformation, all attributes that allow for formation of a turn. In RNase A both Pro93 and Pro114 are involved in β turns. Proline residues are important to protein folding because their ability to form a favorable ''cis'' conformation allows for thermodynamic favorability of β turn formation. With β turns, amino acids can fold back on themselves allowing the protein to reside in a compact, globular structure.
 ==='''Disulfide Bonds'''===
-Another important feature of the folding of RNase A is the presence of four disulfide bonds.  These bonds contribute to the thermal stability and the rate of folding of RNase A.  The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/6'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/6'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/8'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/7'>Cys65-Cys72</scene>.  Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet which is the main contributor to the thermodynamic stability of the enzyme.
+Another important feature of the folding of RNase A is the presence of four disulfide bonds.  These bonds contribute to the thermal stability and the rate of folding of RNase A.  The residues involved in these linkages include <scene name='Sandbox_Reserved_197/Cys26-cys84/6' target='0'>Cys26-Cys84</scene>, <scene name='Sandbox_Reserved_197/Cys58-cys110/6' target='0'>Cys58-Cys110</scene>, <scene name='Sandbox_Reserved_197/40-95_disulfide_native_form/8' target='0'>Cys40-Cys95</scene>, and <scene name='Sandbox_Reserved_197/Cys65-cys72/7' target='0'>Cys65-Cys72</scene>.  Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet which is the main contributor to the thermodynamic stability of the enzyme.
-Measurements of protein activity upon removal of disulfide bridges show that the change in enzymatic activity is very small and that not all disulfide bridges are essential for the structure or the reactivity of the protein. However, removal of disulfide bonds does destabilize the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate.  An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/8'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here.  In the variant, only 3 disulfide bonds are present, but the overall structure is only changed slightly. The differences occur in residues in close proximity to the location of the missing disulfide bond, <scene name='Sandbox_Reserved_197/Residues_34-45/1'>34-45</scene> and <scene name='Sandbox_Reserved_197/Residues_83-101/1'>83-101</scene>, where there are increased levels of disorder and a destabilized hydrophobic core [http://www.ncbi.nlm.nih.gov/pubmed/9605332].
+Measurements of protein activity upon removal of disulfide bridges show that the change in enzymatic activity is very small and that not all disulfide bridges are essential for the structure or the reactivity of the protein. However, removal of disulfide bonds does destabilize the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate.  An analog of this, <scene name='Sandbox_Reserved_197/C40-95a_variant/8' target='0'>C[40,95]A</scene>, shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here.  In the variant, only 3 disulfide bonds are present, but the overall structure is only changed slightly. The differences occur in residues in close proximity to the location of the missing disulfide bond, <scene name='Sandbox_Reserved_197/Residues_34-45/1' target='0'>34-45</scene> and <scene name='Sandbox_Reserved_197/Residues_83-101/1' target='0'>83-101</scene>, where there are increased levels of disorder and a destabilized hydrophobic core [http://www.ncbi.nlm.nih.gov/pubmed/9605332].
 ==='''Medical Importance of Protein Folding'''===
-Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding because proteins can form aberrant aggregates when they do not fold correctly.  This abnormality can be toxic to human nerve cells.  All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/2'>hydrophobic and hydrophilic residues</scene>.  The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/3'>hydrophobic patches</scene> and, in turn, cause several proteins to stick together and form a plaque.  In the future researchers hope to design drugs that combat mistakes in protein folding [http://www.sciencedaily.com/releases/2009/10/091013105324.htm].  The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent protein misfolding.
+Protein folding has several medical implications. Diseases such as ALS, Alzheimer's Disease, and Parkinson's Disease can all be traced back to protein folding because proteins can form aberrant aggregates when they do not fold correctly.  This abnormality can be toxic to human nerve cells.  All proteins contain <scene name='Sandbox_Reserved_197/Hydrophobic-hydrophilic/2' target='0'>hydrophobic and hydrophilic residues</scene>.  The hydrophilic residues lie on the outer part of the protein and the hydrophobic residues bury themselves within the interior of the protein due to the hydrophobic effect [http://en.wikipedia.org/wiki/Hydrophobic_effect]. Mistakes made during protein folding may cause a protein to expose <scene name='Sandbox_Reserved_197/Hydrophobic/3' target='0'>hydrophobic patches</scene> and, in turn, cause several proteins to stick together and form a plaque.  In the future researchers hope to design drugs that combat mistakes in protein folding [http://www.sciencedaily.com/releases/2009/10/091013105324.htm].  The use of ribonuclease A in protein folding research has been an instrumental feature in designing experiments to determine these "misfolding" snapshots and in developing therapies to prevent protein misfolding.
 ==='''Summary of Protein Folding'''===
@@ Line 35: / Line 35: @@
 ''Semisynthetic RNase A''
-The synthesis of semisynthetic RNasa A clearly exhibits the structure to function relationship that defines proteins. In the RNase A protein, the removal of six C terminal residues, leaving <scene name='Sandbox_Reserved_198/Rnase_1-118/1'>RNase 1-118</scene>, completely halts enzymatic activity<ref>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.</ref>. However, a complex of RNase 1-118 with a synthetic polypeptide comprising the C terminal residues <scene name='Sandbox_Reserved_198/Synthetic_component/3'>111-124</scene> restores enzymatic activity to RNase A. Upon the addition of the synthetic chain, the <scene name='Sandbox_Reserved_198/Interface/7'>semisynthetic enzyme</scene> <scene name='Sandbox_Reserved_198/Interface/8'>(Zoom)</scene> adopts a structure that closely resembles that of <scene name='Sandbox_Reserved_198/Wild_type/1'>Wild Type RNase A</scene><ref>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.</ref>. The restoration of the structure reconstitutes the enzymatic activity of RNase to 98%<ref>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.</ref>.
+The synthesis of semisynthetic RNasa A clearly exhibits the structure to function relationship that defines proteins. In the RNase A protein, the removal of six C terminal residues, leaving <scene name='Sandbox_Reserved_198/Rnase_1-118/1' target='1'>RNase 1-118</scene>, completely halts enzymatic activity<ref>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.</ref>. However, a complex of RNase 1-118 with a synthetic polypeptide comprising the C terminal residues <scene name='Sandbox_Reserved_198/Synthetic_component/3' target='1'>111-124</scene> restores enzymatic activity to RNase A. Upon the addition of the synthetic chain, the <scene name='Sandbox_Reserved_198/Interface/7' target='1'>semisynthetic enzyme</scene> <scene name='Sandbox_Reserved_198/Interface/8' target='1'>(Zoom)</scene> adopts a structure that closely resembles that of <scene name='Sandbox_Reserved_198/Wild_type/1' target='1'>Wild Type RNase A</scene><ref>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.</ref>. The restoration of the structure reconstitutes the enzymatic activity of RNase to 98%<ref>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.</ref>.
-<Structure load='1SRN' size='380' frame='true' align='right' caption='Semisynthetic Ribonuclease A: Residues 114-124 are highlighted in the surface representations of the Wild Type, Fully Synthetic, and Semisynthetic enzymes to emphasize similarity in structure. Also, the surface representation of semisynthetic RNase A illustrates the interface between the synthetic analog and the natural enzyme  ' scene='Sandbox_Reserved_198/Fully_synthetic/4' />
+<Structure load='1SRN' size='380' frame='true' align='right' caption='Semisynthetic Ribonuclease A: Residues 114-124 are highlighted in the surface representations of the Wild Type, Fully Synthetic, and Semisynthetic enzymes to emphasize similarity in structure. Also, the surface representation of semisynthetic RNase A illustrates the interface between the synthetic analog and the natural enzyme  ' scene='Sandbox_Reserved_198/Fully_synthetic/4' target='1'/>
 The semi-synthetic RNase A comprises of residues 1-118 and the synthetic analog of residues 111-124. The RNase 1-118 was prepared by successive digestion of RNase A pepsin and carboxypeptidase A<ref>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</ref>. The synthetic component, RNase 111-124, was prepared by the use of solid-phase peptide synthetic methods, in which the peptide chain was assembled in the stepwise manner while it was attached at one end to a solid support. The peptide chain was extended by repetitive steps of de-protection, neutralization and coupling until the desired sequence was obtained<ref>Lin, M. C. (1970) Journal of Biological Chemistry, 245, 6726-6731</ref>. It was important that the synthesis proceeds rapidly and in high yields to prevent side reactions or by-products.
@@ Line 43: / Line 43: @@
 ''Fully Synthetic RNase A''
-The <scene name='Sandbox_Reserved_198/Fully_synthetic/4'>Fully Synthetic RNase A</scene> demonstrates similar structural and functional characteristics (such as catalytic activity) as those of the <scene name='Sandbox_Reserved_198/Wild_type/1'>Wild Type</scene> RNase A<ref>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.</ref>. The crystal data, X-ray data collection and refinement statistics show that the fully synthetic protein shares identical molecular structures with the wild type RNase A, and that the active sites of both emzymes contain no walter molecules and have no substrate ligand<ref>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.</ref>. The crystal structure similarities of the <scene name='Sandbox_Reserved_198/Wild_type_as/1'>Wild Type Active Site</scene>, <scene name='Sandbox_Reserved_198/Fully_synthetic_active_site/1'>Fully Synthetic Active Site</scene>, and <scene name='Sandbox_Reserved_198/Active_site/1'>Semisynthetic Active Site</scene> are further evidence that amino acid sequence dictates folded structure formation.
+The <scene name='Sandbox_Reserved_198/Fully_synthetic/4' target='1'>Fully Synthetic RNase A</scene> demonstrates similar structural and functional characteristics (such as catalytic activity) as those of the <scene name='Sandbox_Reserved_198/Wild_type/1' target='1'>Wild Type</scene> RNase A<ref>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.</ref>. The crystal data, X-ray data collection and refinement statistics show that the fully synthetic protein shares identical molecular structures with the wild type RNase A, and that the active sites of both emzymes contain no walter molecules and have no substrate ligand<ref>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.</ref>. The crystal structure similarities of the <scene name='Sandbox_Reserved_198/Wild_type_as/1'>Wild Type Active Site</scene>, <scene name='Sandbox_Reserved_198/Fully_synthetic_active_site/1' target='1'>Fully Synthetic Active Site</scene>, and <scene name='Sandbox_Reserved_198/Active_site/1' target='1'>Semisynthetic Active Site</scene> are further evidence that amino acid sequence dictates folded structure formation.
 The peptide ligation chemistry in addition to solid-phase peptide synthesis is used to synthesize relatively longer peptide molecules with typical length of 125 residues<ref>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.</ref>. The ligation methods overcome the length limitation of solid-phase synthesis, because the chemical ligation involves the joining of mutually reactive peptide segments created by solid-phase synthesis. The peptide bond in ligation is formed between an unprotected peptide and a peptide-thioester<ref>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.</ref>. The shorter peptide segments are more rapidly prepared and are less susceptible to solubility issues in longer peptide chains.
-The <scene name='Sandbox_Reserved_198/Fully_synthetic/1'>Fully Synthetic RNase A</scene> (124 residues) is prepared by two consecutive sets of one-pot ligations and related chemical transformations of six peptide segments (residues <scene name='Sandbox_Reserved_198/1-25/1'>1-25</scene>, <scene name='Sandbox_Reserved_198/26-39/1'>26-39</scene>, <scene name='Sandbox_Reserved_198/40-64/1'>40-64</scene>, <scene name='Sandbox_Reserved_198/65-83/1'>65-83</scene>, <scene name='Sandbox_Reserved_198/84-94/1'>84-94</scene>, <scene name='Sandbox_Reserved_198/95-124/1'>95-124</scene>, as highlighted in red)<ref>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.</ref>,which can prevent undesired byproduct formation. The six unprotected peptide segments were synthesized by highly optimized, stepwise solid-phase synthesis. This synthetic pathway is simple, has high overall yields, and it eliminate the need for the isolation of intermediate products.
+The <scene name='Sandbox_Reserved_198/Fully_synthetic/1' target='1'>Fully Synthetic RNase A</scene> (124 residues) is prepared by two consecutive sets of one-pot ligations and related chemical transformations of six peptide segments (residues <scene name='Sandbox_Reserved_198/1-25/1' target='1'>1-25</scene>, <scene name='Sandbox_Reserved_198/26-39/1' target='1'>26-39</scene>, <scene name='Sandbox_Reserved_198/40-64/1' target='1'>40-64</scene>, <scene name='Sandbox_Reserved_198/65-83/1' target='1'>65-83</scene>, <scene name='Sandbox_Reserved_198/84-94/1' target='1'>84-94</scene>, <scene name='Sandbox_Reserved_198/95-124/1' target='1'>95-124</scene>, as highlighted in red)<ref>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.</ref>,which can prevent undesired byproduct formation. The six unprotected peptide segments were synthesized by highly optimized, stepwise solid-phase synthesis. This synthetic pathway is simple, has high overall yields, and it eliminate the need for the isolation of intermediate products.
 =='''References'''==