1. Mutation studies of the b
subunit of E.coli ATP
synthase
Senior research: CHEM 4300
Lucy Ingaiza
8/6/2015
The process of ATP synthase translocation through the electric and chemical motor involves the
interaction between the stator stalk and the different subunits between the two sub complexes
F0F1. This paper examines how different mutations in E.coli change the function of ATP
synthase and similarly how the deletions made in the b subunit of the stator stalk affect its
stability to function.

2. Introduction:
ATP synthase is an important primary source of ATP for the majority of organisms on
earth including humans. By catalyzing the synthesis of ATP in oxidative phosphorylation from
ADP and Pi, ATP synthase powers the cellular processes in the human body and other
organisms. The enzyme is found in the inner membrane of mitochondria, the thylakoid
membrane of chloroplasts, in the plasma membrane of bacteria and the central enzyme of energy
metabolism in most organisms. ATP synthesis is composed of two rotary motors, each powered
by a different fuel. The motor at the top per diagram below, termed F0 is the electric motor that
consists of three different subunits an α subunit, two b subunits and a set of c subunits that varies
in number from species to species. It is embedded in a membrane and is powered by the flow of
hydrogen ions across the membrane. As the protons flow through the motor, they turn a circular
rotor. This rotor is connected to the second motor, termed F1. The F1 motor is a chemical motor,
powered by ATP and consists of five different subunits which are three α, three β, and a single
copy of γ, δ and ε. The two motors are connected together by a stator shown on the right in the
diagram, so that when F0 turns, F1 turns too. The ring of the stator contains three catalytic
nucleotide binding sites one of which contains the static portion of the proton- translocating
machinery. The three are held together by the stator stalk. The stator stalk experiences elastic
strain during rotation. The experiment to be performed involves the stator stalk. In E.coli, the
structure of the stator stalk consists of two copies of subunit b and one subunit of δ that are
genetically identical. Subunit b has 156 subunits. The δ subunit is essential for a functional ATP
synthase. In photosynthesis and mycobacterium there are two types’ b and b’. Previously in the
lab, we generated mycobacterium like E.coli stator stalk with b’ and b. Later plasmid PCG-26
was generates which expresses b’, b and δ where b’ ranges from 1-140 and b 30-156 subunits.

3. Originally, b is 156 subunits, starting with PCG26 different strains were generated using site
directed mutagenesis. In this experiment, the mutants were already generated prior to the
procedures. The study involved ATPase assay and western blots that lead to methods that
warranted the use of limited glucose growth assay and the preparation of membrane proteins
which allowed for further studies regarding the function of the ATP synthase.

4. Methods:
1. Limited Growth assay
The function of this assay was to examine the ability to support oxidative
phosphorylation and the function of ATP synthase.
Medium preparation: We started by preparing minimal media with 3mm of glucose.
The procedure called for 10.45g of Potassium phosphate dibasic, 5.52g sodium dihydrogen
phosphate, 1.98g ammonium sulfate, .15g casamine acids and 0.022g of uracil all per one liter.
After a period of autoclaving the addition of other compounds per liter were needed to the
different strains we were experimenting with. It is critical for them to be added after autoclaving
and cooling to hand-tolerable temperature. After addition further sterilization by Millipore
filtration was performed this in return ensured safety and eliminated the risk of contamination
which would have intern affected the ability for the growth of the strains. After everything was
mixed, ampicillin was added and the flasks were then inoculated with the respectful strains and
left overnight to grow. The flasks were labeled with PU11.8 a negative control which contained
no ATP synthase and PBWU13.4 that had the wild type ATP synthase. A record of growth was
then collected at different intervals following the overnight growth to examine whether there was
a consistent growth or not. The growth was then measured using a spectrophotometer at a
wavelength of 590nm and results were collected within a two day period. Following the recorded
measurements the results were as follows.

5. July 10th
set A
STRAINS 10AM 11:20AM 2PM 3:15 PM 4:15PM CORRECTED
VALUE 0F
% CONC.
PuC11.8 .387 .376 .358 .360 .355 0.000
PB013 .381 .374 .370 .371 .366 0.011
PB09 .405 .400 .389 387 .403 0.037
PB012 .405 .407 .402 .404 .406 0.051
PB08 .412 .406 .400 .401 .388 0.033
PB011 .447 .448 .447 .446 .445 0.090
PB07 .400 .402 .412 .416 .421 0.066
PB010 .562 .568 .561 .540 .536 0.181
PCG26 .742 .730 .721 .712 .709 0.354
PBU13.4 .792 .799 .777 .768 .756 0.401
Set B
STRAINS 10:10AM 11:30AM 2:15PM 3:25PM 4:25PM CORRECTED
VALUE OF
% CONC.
PuC11.8 .315 .312 .272 .314 .289 0.000
PB013 .359 .367 .349 .351 .333 0.044
PB09 .356 .355 .332 .323 .323 0.034
PB012 .387 .372 .356 .355 .351 0.062
PB08 .395 .365 .350 .348 .331 0.042
PB011 .384 .387 .391 .394 .412 0.123
PB07 .415 .421 .441 .459 .462 0.173
PBO10 .535 .534 .493 .498 .509 0.220
PCG26 .607 .599 .589 .580 .578 0.289
PBU13.4 .673 .688 .675 .687 .678 0.389
The following two sets were prepared on Friday some were collected on Friday and then again
collected on Monday. The result wasn’t as consistent due the spread out time.
July 17th
–July 20th
SET A
STRAINS 2:20pm 3:30pm 4:23pm 5:17pm 10:30am 11:30am CORRECTED
VALUE OF
% CONC.
PuC11.8 .216 .189 .190 .290 .128 .104 0.000
PB013 .250 .216 .229 .240 .314 .305 0.039
PB09 .313 .294 .304 .320 .330 .320 0.114
PB012 .289 .259 .273 .295 .350 .340 0.083
PB08 .412 .382 .389 .403 .395 .385 0.199
PB011 .498 .468 .484 .490 .547 .526 0.294
PB07 .407 .396 .430 .480 .548 .527 0.240
PBO10 .287 .256 .265 .284 .306 .288 0.075
PCG26 .502 .474 .487 .498 .553 .540 0.297
PBU13.4 .543 .517 .539 .549 .616 .590 0.349

6. SET B
STRAINS 2:20pm 3:30pm 4:23pm 5:17pm 10:30am 11:30am CORRECTED
VALUE OF
% CONC.
PuC11.8 .272 .267 .267 .263 .142 .137 0.000
PB013 .438 .440 .458 .457 .425 .431 0.194
PB09 .340 .336 .339 .335 .342 .365 0.072
PB012 .341 .343 .350 .355 .333 .328 0.092
PB08 .388 .390 .389 .383 .307 .295 0.120
PB011 .425 .432 .418 .409 .327 .309 0.146
PB07 .421 .443 .470 .473 .529 .518 0.210
PBO10 .296 .292 .298 .290 .363 .341 0.027
PCG26 .558 .553 .547 .544 .582 .580 0.281
PBU13.4 .678 .672 .672 .678 .738 .731 0.415
Summary: The calculations based on the average
STRAINS 7/10 SET 1 7/10 SET 2 7/17-20 SET
1
7/17-20 SET
2
OVER ALL
PuC11.8 0 0 0 0 0
PB013 2.74 11.31 11.17 46.7 17.98
PB09 9.22 8.74 32.6 17.34 16.97
PB012 12.71 15.93 23.78 22.16 18.64
PB08 8.22 10.79 57.02 28.90 26.23
PB011 22.44 31.61 84 35.18 29.74
PB07 16.45 44.47 68.7 50.60 33.80
PBO10 45.1 56.55 21.48 6.5 32.40
PCG26 88.2 74.29 85 67.7 78.79
PBU13.4 100 100 100 100 100
Conclusion:
Based on the limited glucose growth assay, the different deletion mutants show less growth than
the parent PCG26 that had the maximum growth. The interpretations of the results mean that the
ability to support oxidative phosphorylation is affected by the deletion of the subunit via site
directed mutagenesis to some extent.
2. Preparation of Membrane protein
Membrane proteins are found anchored in the phospholipid bilayer. They play important
function in the interaction and communication of information from cell to cell. The following
outlines the process within preparation of the membrane protein.
E.coli Membrane prep.
I. Harvesting cells

7. a). Spin suspension 9500rpm x 15 min in JA10 bottles in pre-cooled JA10 rotor in J21B
centrifuge.
b). Resuspend combined pallets in STEM at 2o C. Spin 9500rpmx15 min.
c). Resuspend weighed pellet in 2ml STEM/g wet cells. Freeze ultra-cold (-70oc). Store
overnight. No longer
II French Press and Tes 50 wash
a). Start with less that 450ml (150g) washed cells in STEM. To thawed cells, add a spatula- sized
pile of N=DNase (sigma). Pass through chilled French press at 20K psi twice. Each 40ml
fraction should take 3-6min.
b). Spin in JA20 rotor 18K rpm x 20min. Keep supernatant, discard pallets.
c). Spin supernatant at maximum speed in 60 Ti or 55.2 Ti rotor for 2hrs. Fill tubes with stem if
they are not full.
d).Resuspend pellets in TES 50. (Paint brush or syringe). Spin maximum speed 2hr, 60 Ti or
55.2 Ti. Discard supernatant. Freeze pallets or go to next step.
III. TES 5 plus PAB wash
a). Resuspend pellets in TES plus PAB. Spin maximum speed 60 Ti or 55.2 Ti, 75-90 mins.
b). Repeat step a.
c). Freeze for storage at -70oC.
At the end of the procedure, tiny reddish brown pellets were obtained.
Conclusion
Unfortunately, I was unable to continue further on the results due to time constraints. Using
prepared membrane protein, ATPase assay and western blot will be performed to quantify the
expression of the protein which will allow for further studies regarding the function of the ATP
synthase. All together with the limited glucose assay, the results will give more insight to the
function of ATP synthase.

8. Reference page
Weber J. “ATP synthase: subunit-subunit interaction in the stator stalk.” Texas Tech University. Web
www.sciencedirect.com
Ebru Dulekgurgen “Proteins Lowry Protocal”. Istanbul Technical University. Web. 4 August 2015
http://web.itu.edu.tr/~dulekgurgen/Proteins.pdf