Sandbox Reserved 197
From Proteopedia
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==='''Proline Conformation'''=== | ==='''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 a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. Importance of these conformations are demonstrated with several mutations to the wilde type. | 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 a ''cis'' conformation. RNase A contains four proline residues, two reside in the ''cis'' conformation and two in the ''trans'' conformation. Importance of these conformations are demonstrated with several mutations to the wilde type. | ||
| - | Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/2'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/6'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a mutation from proline to alanine, <scene name='Sandbox_Reserved_197/P93a/ | + | Located in an outer <scene name='Sandbox_Reserved_197/Tyr92-pro93_loop/2'>loop</scene> of RNase A, the <scene name='Sandbox_Reserved_197/Tyr92-pro93/6'>Tyr92-Pro93</scene> peptide group of RNase A in its native state is found in the ''cis'' conformation. Despite a mutation from proline to alanine, <scene name='Sandbox_Reserved_197/P93a/8'>P93A</scene>, a ''cis'' conformation still forms which is very unlikely for an alanine residue. 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 protein. 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. |
<scene name='Sandbox_Reserved_197/Cis-proline114/1'>Asn113-Pro114</scene> 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. This is further demonstrated with the insertion of a <scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene> point mutation which causes the chain to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop where it is located. 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 causes 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. | <scene name='Sandbox_Reserved_197/Cis-proline114/1'>Asn113-Pro114</scene> 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. This is further demonstrated with the insertion of a <scene name='Sandbox_Reserved_197/P114g/1'>P114G</scene> point mutation which causes the chain to adopt a ''trans'' conformation and causes a 9.3 Å movement of the loop where it is located. 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 causes 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. | ||
Revision as of 18:18, 12 April 2011
| 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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Contents |
Introduction
Ribonuclease A is an enzyme found in the pancreas that is involved in catalyzing RNA degradation. The kidney-shaped structure of RNase A has been determined through crystallography [1] and FABMS [2]. RNase A is composed of four anti-parallel β-sheets and three α-helixes. The lies within the cleft and houses three residues important for catalysis: His12, His119, and Lys41. Presence of four disulfide bonds and four cis proline residues greatly effect the structure and folding kinetics of RNase A [3]. Perhaps most widely known as the protein that helped Christian Anfinsen win the Nobel Prize, RNase A has been shown to spontaneously fold back into its native conformation following degradation to its primary structure. This experiment has ignited the interest in protein folding and its characterisitcs that is observed today and the idea behind protein folding that "sequence determines structure."
Protein Folding
Interatomic interactions, delegated by the amino acid sequence, are responsible for formation of a protein's 3D structure [4]. 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.
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Proline Conformation
The presence of cis [5]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 a cis conformation. RNase A contains four proline residues, two reside in the cis conformation and two in the trans conformation. Importance of these conformations are demonstrated with several mutations to the wilde type. Located in an outer of RNase A, the peptide group of RNase A in its native state is found in the cis conformation. Despite a mutation from proline to alanine, , a cis conformation still forms which is very unlikely for an alanine residue. 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 protein. 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.
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. This is further demonstrated with the insertion of a point mutation which causes the chain to adopt a trans conformation and causes a 9.3 Å movement of the loop where it is located. 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 causes 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.
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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 , , , and . Cys26-Cys84 and Cys58-Cys110 stabilize an interaction between an α-helix and a β-sheet. This connection is the main contributor to the thermodynamic stability of the enzyme. Measurements of protein activity upon removal of disulfide bridges show that the active center is very small and not all disulfide bridges are essential for reactivity of the protein. However, removal of disulfide bonds destabilizes the hydrophobic core and decreases the rate of folding. RNase A actually has a rate-determining three-disulfide intermediate. An analog of this, , shows RNase A, missing the disulfide bond, Cys40-Cys95, that would normally occur here. As you can see in the variant and the 2D structure, there are only 3 disulfide bonds present, shown in red.
Summary
Protein folding is not due to one interaction, but a network of interactions within the protien. Presence of Cu+ upon folding shows that the Cu+ does not dictate folding, but rather binds to a pre-existing structure, therefore protein folding is not due to external forces. When a proline or disulfide bond is removed, the structural changes are usually confined to the site of mutation and minor structural changes occur within close proximity to the mutation. Mutation of a cis proline is often accompanied by an insertion or deletion in order to provide more flexibility for the structure. Although the effects of mutations seem to be localized, mutating proteins greatly effects the stability of the molecule and the rate of folding.
Medical Importance
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 abnormaility can be toxic to human nerve cells. All proteins contain . 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 [6]. Mistakes made during protein folding may cause a protein to expose of the interior that can cause several proteins to stick to one another forming plaque. In the future researchers hope to design drugs that combat mistakes in protein folding. 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 this problem in the future.
References
Pearson, M.A., Karplus, P.A., Dodge, R.W., Laity, J.H, and Scheraga, H.A. (1998) Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A. Protein Science. 7:1225-1258.
Raines, R.T. (1998) Ribonuclease A. Chem. Rev. 98:1045-1065.
Schultz, D.A., Friedman, A.M., White, M.A., and Fox, R.O. (2005). The crystal structure of the cis-proline to glycine variant (P114G) of ribonuclease A. Protein Sci. 14:2862-2870.
Sela, M. (1957) Reductive cleavage of disulfide bridges in ribonuclease. Science 125(3250):691-692.
