RNaseA Nobel Prizes: Difference between revisions

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.  

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.<ref name="Raines" /> 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.  

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 name="Martin" /> 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 name="Martin" />. The restoration of the structure reconstitutes the enzymatic activity of RNase to 98%<ref name="Martin" />.

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>PMID: 6615822 </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>PMID: 4921569</ref>. It was important that the synthesis proceeds rapidly and in high yields to prevent side reactions or by-products.

* [[RNase A NMR]]

* [[RNaseS RNaseB|RNase S and RNase B]]

==See Also==