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

[[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.

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

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.  

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.  

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].  

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.