3 Alanine scanning is a technique to determine the import pr

3. Alanine scanning is a technique to determine the import protein. This technique uses site directed mutagenesis to createa single amino acid to alanine. h amino acid to the function of the series of mutants, each of which changes a tion to the original (wild type) protein, this indicates that the If the mutant protein maintains c mutated amino acid is not essential. However, if the function decreases or is lost, there are several possible effects. For example, certain mutations of the primary structure arc so disruptive the protein is targeted for degradation by the unfolded protein response (UPR). Because the protein is degraded immediately after production, it is not present long enough in the cell to show any arable 0 function. Using what you\'ve learned about proteins, conciscly summarize 4 additional ways that alanine mutation onal ways that alanine mutatio uld result in a loss of protein activity In each case, be sure to explicitely relate how disruption wonld resalt co in a Joss of protein activity MacBook Pro 4) Fn F1O FV

Solution

1.

Location at the active site: If the residue under consideration form a part of the active site of the enzyme and is involved in catalysis, it can result in a loss of activity. Eg. Serine, histidine and aspartate in serine proteases forms the catalytic triad and are involved in the catalysis. Mutating any of the residues to alanine during scanning mutagenesis results in loss of activity of the enzyme.

2.

Away from the active site: Although certains residues lie at a distant site from the active site of an enzyme, it has structural role in catalysis but do not form a part of the active site of an enzyme. Alanine muatation of those residues can result in a conformational change of the enzyme which has a global effect at the active site, resulting in loss of activity.

3.

Surface of proteins: If the residue to be mutated is located a the surface of a protein which is involved in protein-protein or protein-DNA interactions, it can result in decreased affinity of the subject protein towards the partner protein or DNA. Eg. Mutating lysine or arginine residues on the surface of proteins which are involved in DNA binding can negatively effect its binding property.

4.

Disulfides and knotted proteins: Cyclotides are cyclic proteins which has conserved cysteine residues that are involved in the formation of disulfide bridges to form knots. Alanine mutation of any of these residues can result in the loss of structural integrity (cannot form the knot) and thus loss of its insecticidal properties.