Lecture Notes

Lecture 13 Site Directed Mutagenesis
SDM- powerful  In protein biology, protein interaction is studied and are important in
technique for finding specific residues in binding sites of ligand
studying protein  Protein interaction with other molecules (proteins, ligands, etc.) are
function often due to specific residues at the interaction site
 In SDM, mutate 1 or 2 residues in the binding site to see if it retains its
biological pathway
 Mutate several residues in the binding site to see which one is the
most vital
 to prove the role of a specific residue at this interaction site, they are
often mutated to alanine.
 Alanine scanning mutagenesis is a common technique employed
 Why use alanine instead of glycine?
- Alanine like glycine has a simple side chain group but the methyl
group is less likely to disrupt the alpha helix or beta strand in the
protein
- Glycine not chosen as it permits a large range of dihedral angles,
enabling secondary structures not likely possiblewith the wildtype
residue
- The only change to be made in the active site is the Side chain and
not the structure of the protein




Exploring protein  Bacterial microcompartments (BMCs) are protein cages, made of
cages- using SDM 1000s of copies of 7 different proteins, interacting to form a closed
to elucidate cage.
(explain) the role  One of the proteins found in this cage is pduA
of residues in  Overexpression of pduA in vivo (within organism) leads to formation
aprotein of nanotubes

,  Nanotubes are tubular molecules composed of large number of
carbon atoms.
Analysis of BMC crystal structure reveals:
- Lysine (K26) at interference between adjacent copies of pduA in
nanotubes may be key to their interaction
- Yellow shows 2 antiparallel lysine side chain interaction
- Based on this structure, a hypothesis can be made
- HYPOTHESIS:
pduA K23A mutation will prevent nanotube formation
- What happen if mutate lysine to alanine? Currently attempting to
do




Introduction to  SDM is a technique that allows us to selectively introduce
SDM - Insertion mutation
- Deletion mutation
- Substitution mutation
Within the nucleotide sequence of a DNA plasmid and not the AA

 One of the first is kunkel mutagenesis in 1985
 Many variations of SDM exist today.
 Amongst most common is cassette mutagenesis
Getting ready for  Before carry out SDM need to amplify the plasmid to be able to work
SDM-amplifying on it
our plasmid  To do this use a strain of E.coli called DH5alpha, which is the same as
the strain of E.coli in the gut but modified to make suitable to amplify
DNA
 There are 3 vital genes modified in this genome:
1. RecA- (recombinant enzyme)
- which recombines DNA inside the cytoplasm because we are
inserting the insert into plasmid and do not want to lose insert.
Therefore, by knocking out this gene =higher stability (less likely
to lose insert from plasmid)
2. endA1- ( endonuclease)
- breaks down extra copies of plasmid in the bacterial cytoplasm
but because trying to purify the plasmid we need to maintain a
high copy of plasmid.
- If knock out this gene we will maintain a high number of the
plasmid copies
3. Dam+/dcm+ (DNA methyl transferase)
- Vital for technique and bacterium as plays important role in the
replication of the plasmid
- Methylates DNA
- Enables efficient DNA replication of plasmid from replication
origin