synthase complexes with two histidine-tagged b subunits bind with a higher affinity than

the complexes containing a b subunit heterodimer, which has only one histidine epitope

tag. For the purposes of Western blot analysis, a total of 5 mg of membrane protein was

brought up to 1 mL with final concentrations of 0.2% tegamineoxide WS-35, 0. 15 M

NaC1, and 1 mM imidazole. The purification procedure was accomplished using the trial

scale miniprep method essentially described by the manufacturer. The 1 mL of clarified

membrane protein was divided up equally into 5 microcentrifuge tubes, containing 100

CIL of packed Ni-resin that had been washed one time with 500 CIL equilibration/wash

buffer (50 mM NaH2PO4-H20, 0.3 M NaC1, 10 mM imidazole, 0.2% tegamineoxide WS-

35, pH8.0), and allowed to mix on a nutator for 5 minutes. The samples were centrifuged

for 30 seconds at 5,000Xg and the supernatant was discarded. The resin was washed 8

times with 1 mL wash buffer by gently mixing for 1 minute, centrifuging for 30 seconds

at 5,000Xg and discarding the supernatant. The histidine-tagged proteins were eluded by

mixing 50 CIL elution buffer (50 mM NaH2PO4-H20, 0.3 M NaC1, 250 mM imidazole,

0.2% tegamineoxide WS-35, pH8.0) in each microcentrifuge tube for 1 minute,

centrifugation, and then the supernatant was extracted. The elution step was repeated one

more time to recover more of the target protein. Finally, the target protein was pooled

together and then concentrated from 500 CIL to about 50 CIL by centrifugation with a

Millipore Amicon Bioseparations Microcon YM-10 (14,000Xg for 30 minutes). To

control for possible enzyme disruption during the solubilization and purification

procedures, the tagged wild-type length b subunits were either mock treated or treated

with the homobifunctional crosslinker, bis(3 -sulfo-N-hydroxysuccinimide ester) (B S3)

after the addition of detergent. Membrane preparations were chemically crosslinked by