8 and a subunit mutations. The 6 subunit has two indigenous cysteines, Scys64

and 6cvsl40, which have been shown to be highly reactive with maleimide reagents (293).

We planned to chemically label both sites specifically, so plasmids were constructed

which expressed only one of the two cysteines by mutating one, either Scys64-ser Of

6cvsl40-ser, to a serine. Also, another cysteine located within the a subunit of F1 has been

shown to be highly reactive to maleimide reagents. This cysteine was mutated to a serine

without loss of enzyme function (198). Plasmid pAES9 (upyGsabc, Cmr) was used to

construct the cysteine mutant 6 and a subunits. Due to the difficulty of performing

Quikchange on a 10.9 kb plasmid, it was necessary temporarily move the genes encoding

the 6 and a subunits into plasmid pBluescript (pB S) by digesting pAES9 (upyisabc,

Cmr) with the restriction enzymes BamHI and EcoRI, flanking the genes of interest, and

then lighting the 3.8 kb cassette into pBS, pTAM2. Sense and antisense mutagenic

oligonucleotides were created for each of the desired 8 and a subunit mutations (Figure

4-2B and C). First, each of the native cysteines found in the 6 subunit were substituted

with series in two different plasmids by site-directed mutagenesis of codon 64 or 140 of

the unc (6) gene to express 6cvs640ser Of 6evsl400ser. Then the native cysteine found in the

a subunit was changed to a serine by mutagenesis of codon 90 of the unc (u) gene to

expreSS aevs90,ser. The Clal sequence was silently knocked out and the EcoRI site was

generated along with Scysl400ser (Figure 4-2B) and the BBBBBBBBBBBBBBBBBamH sequence was added along

with the aevs90,ser mutation (Figure 4-2C) for initial screening purposes and then the

nucleotide sequence was subsequently confirmed by automated sequencing in the ICBR

core facility. The genes encoding the 6 and a subunits with the generated cysteine