- The document discusses crystallizing and analyzing the enzyme FBPase from the ancient bacteria Archaeoglobus fulgidus in its oxidized state. It performed assays to determine the concentration of FBPase and whether it was active when oxidized. Crystals of oxidized FBPase were formed and x-rayed to analyze structural changes caused by oxidation. Assays found the oxidized enzyme had very low activity, indicating oxidation inactivated it by changing the active site structure.

1. Crystallizing Enzyme Proteins
Crystallization of Wild-type FBPase from
Ancient Bacteria A. Fulgidus in Oxidized
State
Jaemin Lee
Honor project
Dr. Kimberly A. Stieglitz
10/28/2012

2. Introduction
Enzymes are proteins that catalyze chemical reactions. Mostly, chemical
reactions in a biological cell require enzymes in order to occur at rates sufficient
for life. If Enzymes lose functions or are overactive, it may cause many human
illnesses. Often researchers develop inhibitors of enzymes to block these
reactions to make potent drugs for specific diseases. Recombinant-technology
can be used to understand how enzymes work and how to inhibit them. Since
enzymes are selective for their substrates and speed up specific reactions from
among many possibilities, the set of enzymes made in a cell determines which
metabolic pathways occur in that cell. Genes coding for specific enzymes and
genes expressed determine the function in the cell.
Numerous hyperthermophilic organisms have an unusual phosphatase
that has dual activity toward inositol monophosphates and fructose 1,6-
bisphosphate. The structures of phosphatases from Archaeoglobus fulgidus
(AF2372) are anaerobic organisms, and resemble the dimeric unit of the
tetrameric human and pig kidney fructose bisphosphatase (FBPase) (Stieglitz,
2002).
This project focused on recombinant FBPase (AF2372) ancient bacterial
enzyme. This ancient enzyme active site has high sequence homology with the
human enzyme, and is a good model for how the human enzyme works, but is
much easier to over-express and much more stable as a recombinant protein.
The project began by comparing BCA and BSA protein assays and by
graphing the data obtained. The purpose of this experiment is to determine the
concentration of FBPase in order to make protein crystals that may be used to
determine the structure after oxidizing FBPase enzyme. This is done to learn
how the shape of the active site changes when the enzyme is oxidized to use the
structure as a model of the inactivated enzyme for future drug development
targeting the active site of the enzyme in the inactivated conformation (oxidized).
Crystallization refers to the formation of solid crystals from a

3. homogeneous solution. It is essentially a solid-liquid separation technique and a
very important one at that. Seven Crystal systems occur in nature and include
cubic, tetragonal, orthorhombic, hexagonal, monoclinic, triclinic, and trigonal. In
order for crystallization to take place a solution must be
"supersaturated". Supersaturation refers to a state in which the liquid (solvent)
contains more dissolved solids (solute) than can ordinarily be accommodated at
that temperature.
Most Proteins and many biological macromolecules differ from "small"
molecules because the environment in which they function is aqueous. Therefore
most biological macromolecules can be prompted to form crystals when the
solution in which they are dissolved becomes supersaturated. The manner in
which this occurs is typical of many other compounds those crystallize from
solution. Membrane proteins are crystallized after being solubilized in detergent
solutions and certain seed proteins like crambin that is not soluble in water, but in
ethanol are crystallized by adding water instead (USCF official Website).

4. <Figure 1. Cys 150 and Cys 186 distance is shown which illustrates close
proximity of these amino acides to the active site>
UCSF Chimera is a highly extensible program for interactive visualization
and analysis of molecules and related data, including density, maps,
supramolecular assemblies, sequence alignments, docking results, trajectories,
and conformational ensembles (USCF official Website).
The figure 1 represents the two cysteines (Cys150 and Cys186) less than
4 A apart, so it may be easier to link each other through S-S bond reaction
(oxidized) which has been shown to inactive proteins (REFERENCE Stieglitz –
protein science redox). Also the substrate fructose 1,6 bisphospate is shown in
the active site which is less than 10 A from the cysteines. The loop that these
cysteine residues is on contains several active site amino acids which are pulled
out of position when the cysteines are oxidized which would dramatically change
the shape of active site. It has already been shown that oxidation of these
cysteines inactivates the enzyme confirming the active site which must be in an
altered shape. However, a structure of the oxidized protein has not yet been
obtained. If the loss of activity of AF2372 on incubation with O2 is owing to
disulfide bond formation between Cys150 and Cys 186, then the C150S variant
should retain activity under oxidative conditions (figure 1).

5. Results
<Determine The Activity Of Concentrated Enzyme Is
Whether Oxidized Or Not By Using Spectrophotometer >
➔ Continue to explain on discussion
<Figure 2. Greenish blue represents enzyme is active. Enzyme
activity assay confirms that the enzyme has very low activity. 1A and
1B were blanks without enzyme but had FBP substrate in the tubes.
2A and 2B were oxidized for 6 hours. 3A and 3B were used same
protein as 2A and 2B, but observed before oxidation.>

6. Discussion
<Determine The Activity Of Concentrated Enzyme Is
Whether Oxidized Or Not Using Spectrophotometer >
Blank is control which doesn`t include enzyme and used 5 mm of FBP
and 20 mm of MgCl2. Spectrophotometer at 660 nm, you can find values using
formula y = mx + b. X value represents Phosphate. If the graph is a direct
proportion graph, phosphate is not oxidized. However, if you find a graph, such
as y = b, then the graph proves you that phosphate is oxidized. My result was Y
= b. In other words, the phosphate group was oxidized (Fiugre 2).
At the preparatory stage for crystallographic experiments, the substrate
specificity as well as the metal ion affinity of AF2372 was evaluated kinetically (at
85 °C). It is possible that the presence of two phosphate groups in FBP facilitate
metal ion interact within the active site. Similar to the phosphatase from M.
jannaschii (25), Ca2+
was an inhibitory metal ion for both Ins-1-P and FBP
hydrolysis catalyzed by AF2372 (Figure 2).

7. <Figure 3. BCA assay - Higher concentration of proteins raises purple
color and lower concentration of proteins raises green color>
BCA is a biochemical assay for determining the total concentration of
protein in a solution, similar to Bradford protein assay. The total protein
concentration is exhibited by color change of a sample solution from green to
purple in proportion of protein concentration, which can then be measured using
colorimetric techniques (Figure 3).
BCA proteins with copper were in a water bath for 30 minutes at 37`C to
find standard absorbance using spectrophotometer to determine concentration of
FBPase by comparing with standard curve (BCA) and FBPase curve. It is very
important to keep at 37`C not to denature protein. Standard curve can determine
protein concentration of oxidized FBPase. The samples were incubated in a
water bath for 30 minutes to interact between BSA, and then 2 of BCA slowly
bound copper to make BCA copper complex (figure 2).

8. <figure 4. Reaction schematic for the BCA protein Assay>
The BCA assay was read at 560 nm. The reaction is similar to the Lowry
reaction, except that bicinchoninic acid (BCA) is used in place of the Folin-
Ciocalteu reagent Cu2+ that is reduced to Cu1+ by protein in alkaline solution
(Figure 4). Two molecules of BCA chelate to a cuprous ion resulting in an intense
purple color with an absorbance maximum at 560 nm. The color continues to
develop slowly over time. To solve this problem, I could incubate for 30 minutes
at 37`C; the color was sufficiently stable for reliable measurements (figure 3).
After AF2372 was crystallized by vapor using 5 ul of hanging, Dr.
Kimberly and I took an X-ray to find how oxidized AF2372 was changed their
structures. After AF2372 was oxidized, AF2372 maybe not function as before,
but oxidized AF2372 may be given new functions. Thus, it is very important to
find how the denatured AF2372 works accurately on the human bodies. In
General, scientists do animal experiments to find any side effects because most
enzymes still work on human bodies and in many cases without side effects.

9. Procedures
• How to use micropipette;for students
1. To know unit conversion
Ex) 1mL = 1000 uL
1. Understand micropipette
➔ There are many types of micropipettes. Many labs usually
use 5/20, 25/200 and 250/1000.
If you have to measure 750 uL, then pick 250/1000
micropipette and set 0 7 5 0
1. Now you are ready for pipetting, but you have to be careful
when you press the pipette. You must press slowly and
slightly. If you press too much, it won`t be 750 uL.
2. Move your solution to another place where you desire, then you
can press the micropipette till the end.
● Deternimation of FBPase enzymatic activity
1. Adding the 10, 25, 50 and 100 ul of 5 mM FBP (stock) into each cleaned dry
test tubes for blank.
2. You add the 100 ul of 100 mL magnesium chloride (MgCl2) into each test
tube that you already prepared.
3. You have to add 390, 375, 350 and 300 ul of tris-base pH8 (buffer) in regular
sequence 10, 25, 50 and 100 ul of FBP test tubes to make 500 ul of final
volume.
4. Adding the 10, 25, 50 and 100 ul of 5 mM FBP (stock) into each new test
tube.
5. You add the 23 ul of enzyme and 100 ul of 100 mL magnesium chloride

10. (MgCl2) into each test tube that you already prepared.
6. You have to add 367, 352, 327 and 277 ul of tris-base pH8 (buffer) in regular
sequence 10, 25, 50 and 100 ul of FBP test tubes to make 500 ul of final
volume.
7. You should incubate all the 10 test tubes for 3 minutes at 55`C
8. You need to add 1.5 mL of die (malachite green 30mL + ammonium
molybdate 10mL T.S)
9. You can find absorbance of each test tube using spectrophotometer (blank is
without enzyme).
10. You should draw the graph of consequential protein concentration. Then you
can find which tubes have more oxidized proteins.
• Lowry method
1. Make a set of standards using BSA or some other protein source in
the range of 5-500 ug/mL. In the Lowry procedure, you will use 0.5
mL of each sample.
2. Add 2.5 mL of Lowry reagent I into 0.5 mL of your unknown samples
(and dilutions of your samples) and to 0.5 mL of the standards. Mix
well and let stand for 10 min. the unknowns and the standards
should be measured in the same experiment.
3. For each sample, add 0.25 mL of Lowry reagent 2 and mix well.
4. Measure the absorbance at 750 nm ( or with the spec 20, at 650 nm)
against a blank consisting of 0.5 mL sample buffer processed as
the samples.
5. Determine the protein concentrations from standard curve
• BCA method
1. Make a set of standards using BSA or some other protein source in

11. the range of 5-2000 ug/mL. In the BCA procedure, you will use 0.1
mL of each sample.
2. Add 3 mL of BCA working reagent into 0.1 mL of your unknown
samples and 0.1 mL of the standards into labeled test tubes.
3. Incubate tubes at 50 `C for 30 min. the unknowns and the standards
should be measured in the same experiment.
4. Cool down to room temperature and read the absorbance at 560 nm
against a blank of sample buffer processed as above.
5. Determine protein concentration from a standard curve.

12. Reference
Kimberly A. Stieglitz, Crystal structure of a Dual Activity IMPase/FBPase
(AF2372) from Archaeoglobus fulgidus, 01//31/2002
Instructions piperce BCA protein Assay Kit, www.thermo.com/pierce
Kimberly A. Stieglitz, Unexpected similarity in regulation between an archaeal
inositol monophosphatase/fructose bisphosphatase and chloroplast fructose
bisphosphatase, 10/14/2002
Alexander J. Ninfa, Ph.D, Fundamental Laboratory approaches for biochemistry
and biotechnology second edition
UCSF Official Website, http://www.cgl.ucsf.edu/chimera/
Wikipedia,Protein Ctystallization