User:R. Jeremy Johnson/Folding Synthesis: Difference between revisions
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== '''Introduction''' == | == '''Introduction''' == | ||
Ribonuclease A has been the subject of Nobel Prizes on Protein Folding and Solid Phase Peptide Synthesis.<ref name="Raines"> PMID:11848924</ref> The observation of ribonuclease folding helped Christian Anfinsen win the Nobel Prize in 1972 for his work on protein folding <ref>'Anfinsen Nobel Lecture' [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-lecture.html]</ref>. The presence of four disulfide bonds and two ''cis'' proline residues in the structure of RNase A greatly | Ribonuclease A has been the subject of Nobel Prizes on Protein Folding and Solid Phase Peptide Synthesis.<ref name="Raines"> PMID:11848924</ref> The observation of ribonuclease folding helped Christian Anfinsen win the Nobel Prize in 1972 for his work on protein folding <ref>'Anfinsen Nobel Lecture' [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-lecture.html]</ref>. The presence of four disulfide bonds and two ''cis'' proline residues in the structure of RNase A greatly affects the structure and folding kinetics of RNase A <ref>'Anfinsen Nobel Biography' [http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-bio.html]</ref>. When RNase A undergoes reductive denaturation, it spontaneously folds back on itself to form the same structure. The development of solid phase synthesis by Bruce Merrifield was a radical departure from traditional methods of bio-molecular synthesis that greatly increased efficiency. His method made possible the syntheses of much larger and more complex molecules; however, solid phase synthesis was not fully embraced until he demonstrated its full ability with the complete synthesis of Ribonuclease A.[http://nobelprize.org/nobel_prizes/chemistry/laureates/1984/merrifield-lecture.pdf] | ||
== '''Protein Folding''' == | == '''Protein Folding''' == | ||
[[Image:Proteopedia final 2d.png|thumb|280px|left|Residues important to the proper folding of RNase A. Locations of internal residues Pro-114, Pro-117, Cys-58, and Cys-72 are highlighted and labeled.]] | [[Image:Proteopedia final 2d.png|thumb|280px|left|Residues important to the proper folding of RNase A. Locations of internal residues Pro-114, Pro-117, Cys-58, and Cys-72 are highlighted and labeled.]] | ||
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. Although RNase A has 105 possible disulfide bond pairings, only one set of four bonds occurs. This unique observation leads to the "thermodynamic hypothesis", that a protein's native state is determined by the thermodynamic favorability of the whole system; thus the tertiary structure must be predetermined by intramolecular interactions within the amino acid sequence. Since thermodynamic stability of a protein is affected by the environment's temperature, pH, and ionic strength, among other factors, the protein structure can only exist under physiological conditions. Today, the correlation between the amino acid sequence and the tertiary structure of RNase A continues to serve as a model for protein folding. Among the most important attributes of this model are noncovalent interactions, proline conformation, and disulfide bonding <ref name = 'Lehninger'>'Lehninger A., Nelson D.N, & Cox M.M. (2008) Lehninger Principles of Biochemistry. W. H. Freeman, fifth edition.' </ref>. | Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [http://en.wikipedia.org/wiki/Protein_folding]. Several of these interactions have been identified by the use of site directed mutagenesis to wildtype RNase A and subsequent comparison of the crystal structure to the wildtype. Although RNase A has 105 possible disulfide bond pairings, only one set of four bonds occurs. This unique observation leads to the "thermodynamic hypothesis", that a protein's native state is determined by the thermodynamic favorability of the whole system; thus the tertiary structure must be predetermined by intramolecular interactions within the amino acid sequence.<ref>PMID:132421663</ref> Since thermodynamic stability of a protein is affected by the environment's temperature, pH, and ionic strength, among other factors, the protein structure can only exist under physiological conditions. Today, the correlation between the amino acid sequence and the tertiary structure of RNase A continues to serve as a model for protein folding. Among the most important attributes of this model are noncovalent interactions, proline conformation, and disulfide bonding <ref name = 'Lehninger'>'Lehninger A., Nelson D.N, & Cox M.M. (2008) Lehninger Principles of Biochemistry. W. H. Freeman, fifth edition.' </ref>. | ||
==='''Proline Conformation'''=== | ==='''Proline Conformation'''=== | ||
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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 <ref name ='Tyr92'>PMID:9605332</ref>. 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.<ref name="Tyr92" /> | 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 <ref name ='Tyr92'>PMID:9605332</ref>. 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.<ref name="Tyr92" /> | ||
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 | 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 <ref>PMID:16199662</ref>. 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' target='0'/> | <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'/> | ||
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==='''Disulfide Bonds'''=== | ==='''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' 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. | 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' 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 <ref | 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 <ref name="Tyr92" />. | ||
==='''Medical Importance of Protein Folding'''=== | ==='''Medical Importance of Protein Folding'''=== | ||
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=='''References'''== | =='''References'''== | ||
<references /> | <references /> | ||
=='''External Resources'''== | |||
*[http://nobelprize.org/nobel_prizes/chemistry/laureates/1972/anfinsen-lecture.pdf Anfinsen Nobel Prize Lecture] | |||
*[http://nobelprize.org/nobel_prizes/chemistry/laureates/1984/merrifield-lecture.pdf Merrifield Nobel Prize Lecture] | |||
*[http://en.wikipedia.org/wiki/Ribonuclease_A RNase A Wikipedia] | |||
*[http://en.wikipedia.org/wiki/Solid_phase_peptide_synthesis#Solid-phase_synthesis Peptide Synthesis Wikipedia] | |||
*[http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2845543/?tool=pmcentrez Chemical Synthesis of Proteins] | |||
*[http://www.ncbi.nlm.nih.gov/pubmed/3680234 Refined Crystal Structure] | |||
=='''Student Contributors'''== | |||
*[http://www.proteopedia.org/wiki/index.php/User:Liz_Ellis Liz Ellis] | |||
*[http://www.proteopedia.org/wiki/index.php/User:Diana_Trautmann Diana Trautmann] | |||
*[http://www.proteopedia.org/wiki/index.php/User:Michael_Slack Lin Liu and Michael Slack] | |||