comparison of the protein crystal structure between wild-type...
TRANSCRIPT
Comparison of the Protein Crystal Structure between Wild-Type and Per-acetylated
Bovine Carbonic Anhydrase
Christine Cuddemi
Senior Thesis
Emmanuel College
Chemistry and Chemical Biology Department at Harvard University
April 22, 2013
© Christine Cuddemi
Abstract
Understanding how proteins crystalize is a fundamental question in biochemistry,
and has important implications in molecular recognition. Carbonic anhydrase,
specifically bovine carbonic anhydrase (SCA) is a good model system to study
crystallization because SCA is structurally rigid, easily produced in large quantities, and
well-studied. SCA contains 18 lysine amino groups, each of which is on the surface of
the protein. When the lysine amino groups are acetylated with acetic anhydride, the
overall negative charge of the protein increases substantially. We explored three
physical properties of SCA and per-acetylated SCA: protein crystallization
thermostability of the proteins and the binding affinity of each protein to a given ligand.
We successfully labeled the lysine amino groups ((SCA-(NHAc)18), as well as obtained
a crystallized structure. With differential scanning calorimetry we were able to gather
stability data of both proteins. Further studies will look into the binding affinity of
arylsulfonamide ligands to per-acetylated SCA using isothermal titration calorimetry.
Overall, this research will aid pharmaceutical studies with the understanding of protein
ligand interactions and protein stability in regards to the functionality of drugs.
Cuddemi 1
Table of Contents
ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
TABLE OF CONTENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1. INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 -11
PROTEIN CRYSTALLIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 -5
BOVINE CARBOINC ANHYDRASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 -7
THESIS HyPOTHESiS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 -8
METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 -10
FUTURE OF RESEARCH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 -11
SUMMARY OF RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2. MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 -15
2.1 ACETYLATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 -13
2.2 CRYSTALLIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 -15
2.2.1 BCA CRYSTALLIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.2.2 BCA-(Ac)18 CRYSTALLIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
2.3 THERMOSTABILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
3 RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 -28
ACETYLATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 -17
CRySTALLIZATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18 -19
PROTEIN - PROTEIN INTERACTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 -26
THERMOSTABILITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26 -28
4 DiSCUSSiON ................................................................................ 29 -32
SUMMARY OF RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29 -30
FUTURE DIRECTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
5 REFERENCES .............................................................................. 33 -34
6 ApPENDiX ................................................................................... 35 -37
Cuddemi 2
2. List of Figures
[1] BCA CRYSTAL STRUCTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
[2] CAPILLARY ELECTROPHORESIS SCHEMATIC ................................ 9
[3] DIFFERENTIAL SCANNING CALORIMETRY SCHEMATIC ................... 10
[4]lsOTHERMIAL TITRATION CALORIMETRY SCHEMATIC .................... 10
[5] CAPILLARY ELECTROPHEROGRAM .............................................. 1 7
[6] BCA CAPILLARY ELECTROPHEROGRAM COMPARISON ................. 1 7
[7] COMPARISON OF CRYSTAL STRUCTURES .................................... 19
[8] DSC COMPARISON DATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
[9] BTA & TA liGAND STRUCTURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
[10] NMR OF HCA DATA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
2. List of Tables
[1] ACETYLATION VOLUMES ............................................................ 1 2
[2] PROTEIN-PROTEIN INTERACTIONS KEY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 - 22
[3] BCA CRYSTAL INTERACTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
[4] BCA-(Ac)18 CRYSTAL INTERACTIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23 - 25
[5] INTERACTIONS SUMMARY TABLE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25 - 26
Cuddemi 3
1 Introduction
Crystallization is one of the fundamental processes that allow a three
dimensional structure of proteins and nucleic acids to be observed. The first published
observation of a crystallized protein was in the year 1840 by Hunefel [1]. Hemoglobin
from an earthworm was obtained as a flat plate-like crystal when the worm's blood was
pressed between two slides of glass. It was then noted that in a controlled environment
of evaporation, a protein solution can be crystallized, also known as slow dehydration
[1]. Fast forward about eighty years to when the first enzyme, urease, was crystallized
by Sumner in the year 1925. This was around the same time that insulin, a hormone,
was successfully crystallized. With the advantages of protein crystal observations, more
questions began to be asked about the processes of crystallization; how exactly
different molecules crystallized and under what conditions this occurred.
Protein growth and crystallization have many applications, especially in the
pharmaceutical industry. The first published observation of crystallized proteins dates
back about 160 years [1]. In relation to pharmaceutical implications, the understanding
of protein crystals can be helpful in the development of novel drugs. Knowledge of the
three-dimensional structure of a target protein can be used to design compounds that
selectively bind to sites of this protein and thereby inhibit or stimulate its activities [1].
Proteins are composed of different amino acid residues, some of which have polar side
chains, specifically the amino groups on ends of molecules may be charged. There are
four amino acids with "charged" side chains at the physiological pH; arginine and lysine
are positively charged; aspartic acid and glutamic acid are negatively charged. When a
protein is in solution, these charged groups interact extensively with water and help to
Cuddemi 4
solvate the protein [1]. The net charge of a protein depends on both the amino acid
sequence and environmental factors (ie. the pH of the solution, the ionic strength of the
solution and the presence of other co-solutes).
Model systems have been used to hundreds of years and many of the common
scientific techniques have been brought about by these systems. Common model
systems used today are; E.coli, C.elegans, Zebrafish and mice. For this research, we
focused on carbonic anhydrase (CA), which is a well-developed model system for
studies in biophysics, bioanalysis, physical-organic chemistry and medicinal chemistry
[2]. Carbonic anhydrase catalyzes the hydration of COz and the dehydration of
bicarbonate: COz + 2HzO;='o HC03 - + H30+ Scientists believe that carbonic
anhydrase is present in all living organisms, because the ability to maintain a (near)
constant cellular pH is important for all living organisms. In eukaryotes, the enzyme
plays a role in various physiological functions; including the inter-conversion between
COz and HC03 - in intermediary metabolism, facilitated diffusion of COz, pH homeostasis
and io n tra nspo rt [3].
There are a number of isozymes of carbonic anhydrase, ranging from human to
bovine carbonic anhydrase. Bovine carbonic anhydrase II (BCA) is well characterized
and a model protein because it can be obtained in large quantities, BCA is derived from
the erythrocytes of Bos taurus, more simply, cow's red blood cells. BCA is a monomeric
protein that is 86% identical in its amino acid sequence to that of human carbonic
anhydrase [4]. Native BCA contains eighteen positively charged lysine residues each of
which are on its surface, as well as a binding cavity surrounded by catalytic Znll ions
[4]. The structure of BCA makes it a convenient protein to study the role of charged
Cuddemi 5
amino acids on protein-protein interactions, protein-ligand interactions and
thermostability. We removed the positively charged lysine residues from the surface of
SCA, with an acetylation reaction, to better understand the relationship between the net
charge of a protein and protein interactions.
SCA is an ideal model protein for studying the crystallization of proteins because
it is structurally rigid and stable under a large number
of pH and salt conditions. SCA does not undergo a
change in the secondary or tertiary structure when the
eighteen lysine residues on the surface are
acetylated; the conditions needed to crystallize SCA
are known and crystal structures of wild-type SCA are
available (Figure 1). The structural rigidity of SCA is
not a common feature among proteins, because gross
changes in the surface of a protein may typically result in
changes of the secondary or tertiary structure. Changes
Figure 1: BCA crystal structure
[3].
in the secondary structure of a protein are less dramatic than tertiary effects and often
result from distortions in the beta sheets or alpha helices of a protein. On the other
hand, changes in the tertiary structure of a protein will cause the protein to appear
sufficiently different.
There are three main reasons that SCA is widely used in physical-organic studies.
SCA is a relatively easy protein to handle and purify because of its stability under
standard experimental conditions [2]. Secondly, changes in the overall net charge of
SCA protein can be monitored with a capillary electrophoresis machine using charge
Cuddemi 6
ladders for reference. A charge ladder is a family of derivatives for a protein that differ in
integral units of charge [5]. The final reason for its model stature is that it is readily
available and sufficiently cheap to acquire (as crystallography and calorimetry
experiments require milligram levels of protein for each experiment) [2]. Model systems
are important in studies of protein structure because the more that is known about a
particular protein the easier it is to make accurate predictions about relationships
between structure and function. Once the model system (BCA) is understood, it is
simpler to test predictions made on less characterized proteins. Using BCA as a model
protein system for our research will aid in further research of the functionality of drugs
and their targets.
Crystal structures of wild-type BCA (BCA) are available in the Protein Database. We
recently solved the structure of per-acetylated BCA (BCA-(Ac)18) in which each amino
group on the side chain lysine was acetylated. A comparison of the crystal structures of
BCA and BCA-(Ac)18 show that the secondary or tertiary structure of the protein is
conserved after acetylation. Given these findings, we hypothesized that the overall
charge of BCA (1) decreased its ability to crystalize, (2) decreased its stability in
solution, and (3) increased the binding affinity of a ligand to BCA. We were interested to
see if a change in the net charge of BCA affected its ability to crystallize, and in
particular if removing the positively-charged residues would decrease the stability of the
protein crystal because the number of ion-ion interactions that could form between two
proteins in the crystal may be decreased. We crystallized the acetylated BCA, obtained
their X-ray crystal structure of the protein with the help of the staff at National
Synchrotron Light Source at Brookhaven National Laboratory, and compared the
Cuddemi 7
regions of contact between molecules of SCA and SCA-(Ac)18. As previously
mentioned, the change in the protein's charge does not affect the secondary or tertiary
structure, which is important for a comparison of protein-protein interactions as only a
single amino acid residue is altered upon acetylation. The second question asked if a
change in surface chemistry by increasing the net negative charge of SCA reduced its
thermostability in solution (i.e. the temperature at which the protein could be
successfully unfolded in solution). We measured the overall stability of SCA and SCA
(Ac)18 with differential scanning calorimetry (DSC), which monitors the heat generated
from the protein (as it unfolds) as a function of temperature. We found that SCA-(Ac)18 is
less stable in solution than wild-type SCA. The third question asked if a change in the
surface chemistry by increasing the net negative charge of SCA maintain the binding on
an arylsulfonamide ligand (i.e. thiazolesulfonamide and benzothiazolesulfonamide) to
the protein. If the structure of a protein does not change when the net charge of the
protein is increased, then does the affinity of the interaction between a ligand a protein
remain unchanged?
The approach of our research was to successfully acetylate the eighteen lysine
residues on the surface of SCA, to determine at which temperature the newly acetylated
protein would denature and what the binding affinity of a ligand to the acetylated SCA
was. There were three methods used in this research to complete the above objectives.
(1) Capillary electrophoresis (CE), which is commonly used to separate ionic species by
their charge and frictional forces [5]. A CE machine was used to determine how many of
the eighteen lysine residues were acetylated on the SCA molecules (because each
acetylation increases the overall net negative charge of the protein by about 0.9).
Cuddemi 8
Separations in CE are similar to the separations that occur in gel electrophoresis, where
electrically charged particles move in a conductive buffer medium under the influence of
an electric field. As shown in Figure 2, the setup
Deled"" 1190.600 rom)
""'., (10-100,.ml.d._
--
-� 2O-100cm,""gIh)
<'/---
of the CE machine is relatively simple. The main
components are a sample vial, a glass capillary, ""
ANODE v tl"",wm (02-W) CATHODE
�V
Eledrode -
-
electrodes, a high-voltage power supply, and a
detector combined with a computer for data r- I-, EIec:trrMyle cootairw
(5-150mM)
output. The separated chemical compounds '-- '--
I: n HighvcMrnge SOIXoe appear as peaks monitored from a UV absorptio
(o.30 kV)
measurement with different migration times in Figure 2: CE schematic.
an electropherogram CE has been established http://www.doping.chuv.chlenilad-schema-ec-eng.jpg
as an independent and reliable analytical technique in separating protein samples in
which the physical or chemical properties are altered [6].
We altered the surface of bovine carbonic anhydrase by acetylating the lysine
amino groups and produced a protein with an equivalent likelihood of crystallizing as
wild-type SCA. We modified the surface of SCA by introducing the proteins to a solution
of acetic anhydride (to acetylate the Iysines) or an N-hydroxysuccinimide (NHS)-
activated formic acid (to formylate the Iysines). This chemical modification increases the
overall negative charge of SCA (Z = -3.4 at pH = 8.0) by I'lZ = -15.6 to yield a protein
with an overall negative charge of I'lZ = -19.
(2) Differential scanning calorimetry (DSC) is used to measure thermostability of
the acetylated SCA protein crystals. DSC is the most direct experimental technique that
measures the heat capacity of a solution of a molecule as a function of temperature [7].
Cuddemi 9
DSC gives the immediate access to the
thermodynamic stability of molecules in
solution, and there are many examples in the
sample polymer sample reference
p
\ I pan
;" I
literature of using DSC to monitor the unfolding \� u I C�I I I
I of proteins and other polymers in solution [7].
Our research used this method on the basis of
I I • �" / ,
heaters I computer to morutor temperature
monitoring the thermal unfolding of BCA and and re gulate he at flow
Figure 3: DSC schematic. BCA-(Ac)1s. The schematic of the DSC machine is http://pslc.ws/macrog/images/dscOl.gif
shown in Figure 3.
(3) Isothermal titration calorimetry (ITC) is used
to study the binding affinity of ligands to proteins. ITC is
important in drug discovery studies as this method
determines the affinity of a ligand for a protein, and
provides information about the binding constant
(L'.GObind), the enthalpy of binding (L'.Hobind), and the
entropy of binding (-TL'.Sobind) [7]. Figure 4
shows the basic setup of an ITC machine.
Pr<>l�h in
�amplucell
Figure 4: ITC schematic. http://www.nature.cominprotijoumal/v6/n2iimages/n prot.20 10. 187-F2.jp g
For the future of this research we hope to accomplish three real-life applications;
the first involving protein-protein interactions. There is no way to currently predict what
set of reaction conditions will cause a protein to crystallize. Crystal structures offer a
large amount of insight into the active site of a protein -- the areas of a protein that are
exposed to solvent -- the way complicated molecules interact in the solid state.
Understanding the interactions between proteins will help scientists in numerous fields
Cuddemi 10
of chemistry and biology. The second application entails protein-ligand interactions. The
majorities of drugs bind to a protein (active site, exterior, etc) and inhibit a structure or a
function. We are incapable of predicting how tightly a molecule will bind to a protein
(even if we know what the protein and molecule look like). Understanding these
interactions will allow us to better rationalize what and how a drug should look. The third
application involves protein stability in solution when changes are introduced, which is
important for protein-based therapeutics. Masking the charge of a molecule allows it to
more easily cross the membrane of a cell, or it also could allow the protein to last longer
in the body without denaturing or succumbing to enzymatic breakdown. Helping
medicinal chemistry and pharmaceuticals research is the target for the future
implications of our research.
We were interested in determining if the overall charge of SCA affects its (1)
ability to crystalize, (2) its stability in solution, and (3) the binding affinity of a ligand to
SCA. We successfully labeled the eighteen lysine amino groups ((SCA-(NHAc)18) on a
SCA protein with acetic anhydride. We were then able to obtain a crystallized structure
of SCA-(Ac)18 to compare to a wild-type SCA protein crystal. With differential scanning
calorimetry we were able to gather stability data of both wild-type SCA and per
acetylated SCA proteins, to answer the second question of our research. Further
studies will look into the binding affinity of arylsulfonamide ligands to per-acetylated
SCA using isothermal titration calorimetry. Overall, this research will aid pharmaceutical
studies with the understanding of protein-ligand interactions and protein stability in
regards to the functionality of drugs.
Cuddemi 11
2 Materials and Methods
2.1 Acetylation of BCA
Ten different acetylation reactions were run before testing the extent of
acetylation with capillary electrophoresis. We chose those reaction conditions to
determine the appropriate amounts of reagents needed to acetylate all eighteen lysine
residues on BCA. These ten test tubes were prepared with varying volumes of acetic
anhydride (Sigma-Aldrich) and 100mM NaOH (VWR International). The following
Table 1 depicts the amount of each reagent added into each of the ten tubes:
Tube # Amount of Amount of Amount of BCA (mL) AA (mL) NaOH (!.IL)
BCA = bovine carbonic
anhydrase
AA = acetic anhydride
NaOH = sodium hydroxide
(100mM)
1A 0.5 0.5 1B 0.5 0.5
1C 0.5 0.5 10 0.5 0.5 1E 0.5 0.5
2A 0.5 2.0 2B 0.5 2.0
2C 0.5 2.0 20 0.5 2.0
2E 0.5 2.0
Table 1: Volumes of reagents added to the reaction tubes for the acetylation of BCA.
0 43
86
172 344
0
43
86
172 344
The ten reactions were prepared into 100mM HEPBS (pH 9.0), incubated at 4°C
for two hours, and the reaction was then quenched with 0.5mL of 1 OOmM NaOH and
added to each tube to stop the reaction. The products of the reaction were exchanged
into 1 X TrisGly buffer in anion exchange spin columns following a standard protocol
prepared by the manufacturer (Thermo Scientific).
Cuddemi 12
A fraction of each reaction (1 OOI-lL) was added into sample vials, loaded into the
capillary electrophoresis machine (CE), and separated with a standard program
07feb2013_BCA. This program included: a ten minute wash cycle in which the column
was rinsed with 1 M HCl, 1 M NaOH and 1 X TrisGly buffer; a sample injection of 5nL of
solution onto the column; a ten minute separation at 1 O.OV; and a ten minutes wash
cycle with deionized water. The CE instrument contained freshly prepared vials of H20,
MeOH, NaOH, HCl, and 1X TrisGlybuffer.
We used the results from the above procedure to design a second set of
experiments, in which the amount of acetic anhydride was decreased, in the attempt to
acetylate the entire eighteen lysine residues on the BCA protein. Nine reaction tubes
were prepared before running the solutions on the CE machine for results.
The acetylation reactions were carried out in 1.5mL centrifuge tubes with the
following amounts of reagents: 200l-iL of a stock solution of BCA (1 mg/mL solution of
BCA in HEPBS, pH = 9.0); 40l-lL of neat acetic anhydride; 60l-lL of 1 M NaOH, which was
added to each tube after a five minute incubation at 4°C. The reaction mixture was
incubated for thirty minutes at 4°C, and then another 40l-lL of acetic anhydride was
added, five minutes in the cold room and 60l-lL of NaOH.
As mentioned previously, the products of the reaction were exchanged into 1X
TrisGly buffer in anion exchange spin columns following a standard protocol prepared
by the manufacturer (Thermo Scientific). The amount of 1 00 1-1 L of each reaction solution
was added into a separate sample vial and loaded into the capillary electrophoresis
instrument and separated with the method discussed above (14march2013_BCA).
Cuddemi 13
2.2 Crystallization
After the successful completion of acetylation to form BCA-(Ac)18, we crystallized
both wild-type BCA (unmodified) and per-acetylated BCA-(Ac)18. The following
procedures were carried out to crystalize the proteins.
2.2.1 BCA CRYSTALLIZATION
Wild-type BCA was crystallized in small tubes (2 mL) with round bottom, with a
protocol published by Saito, Saito and Ikai. A concentrated solution of BCA (200 IJM) in
50 mM Tris-HCI (with 2.4 M ammonium sulfate at pH 7.5) was placed in the small tube
(-100 IJL) and stored at 40C until crystallized.
2.2.2 BCA-(Ac)18 CRYSTALLIZATION
We screened a number of values of pH, and a number of concentrations of
ammonium sulfate, to determine the conditions needed to crystalize [BCA-(Ac)18]; thin
plates of [BCA-(Ac)18r19 formed at pH = 7.0 and [(NH4hS04] = 1.6 M over a two-month
period. We prepared solutions of protein by combining 2 IJL of protein (200 IJM) with 2
IJL of the precipitant solution; a droplet of solution was placed on a glass slide, and was
sealed over 1 mL of precipitant solution in a well of a 24-well plate. Amorphous
precipitates of BCA-(Ac)18 formed, in less than one day, in droplets containing
concentrations of ammonium sulfate greater than 1.6 M.
Cuddemi 14
2.2.3 COLLECTION AND SOLVING X-RAY
We captured a crystal of wild-type BCA or BCA-(Ac)1B (thin plates approximately 200
!-1m x 200 !-1m x 25 !-1m in size) in a nylon loop, transferred the crystal to a reservoir
containing a solution of 17.5 % glycerol / 82.5% cryoprotectant, and soaked it briefly
before rapidly freezing the crystal in liquid nitrogen. We collected the X-ray diffraction
data at 77 K on an RAXIS IV instrument equipped with a rotating copper anode.
To solve the X-ray crystal structure, we indexed, scaled, and integrated the X-ray
diffraction data with the HLK2000 software package. The structure of [BCA-(Ac)1Br
19 was refined with the CCP4i suite of programs; molecules of water were added
automatically to regions of density greater or equal to 1 cr.
2.3 Thermostability
We measured the thermostability of the BCA and BCA-(Ac)1B with a differential
scanning calorimeter (nano-DSC, TA Instruments). A 25 uM solution of protein in 10
mM sodium phosphate buffer (pH = 7.4) was placed in the calorimeter, degassed, and
then scanned at a rate of 1 DC / minute. We fit the DSC data with DSC-fit software
provided by TA instruments.
Cuddemi 15
3 Results
3.1 Acetylation
To acetylate all eighteen lysine groups that are present on a structure of SCA, we
used acetic anhydride and analyzed the results on a CE machine. A CE machine
separates molecules by their charge and by the absorption. The machine itself, referring
back to Figure 2, contains a wire that is negative on one end and positive on the other.
As the SCA protein becomes more negative, it will move towards to positive end (right
side of the figure). Therefore, we can interpret that the data as a protein containing
more acetylated lysine groups. The capillary electropherogram, shown in Figure 5,
depicts the separation of charge and absorption of SCA; wild-type and per-acetylated.
In Figure 5, the lines are representative of SCA with different numbers of lysine
residues labeled with acetyl groups. Each peak in the electropherogram is a protein of a
different charge. Since the overall charge of SCA is typically positive (charged lysine
groups), the top line shows SCA that was only contained one acetylated lysine group.
The bottom line shows a SCA protein that contained all eighteen acetylated lysine
groups. The left-most line is that of N-N dimethylformamide (OM F), which serves as a
neutral marker in the results. This allows us to compare each run to one another.
In Figure 6, the same information is shown, expect in simpler terms. The top line
is a SCA protein with all eighteen labeled lysine residues, while the bottom line is wild
type SCA (zero acetylated lysine residues). The middle line is a charge ladder, serving
as a marker to understand the previous Figure 6. Each peak represents a protein with a
different number of labeled lysine residues. The SCA charge ladder helps to emphasize
the movement of protein within the CE machine.
Cuddemi 16
�
N e!'ative ( ) I I Positive (+)
E c
'<t
0.16
0.14
0.12
0.10
0."
0.06
0.04
"
W-214nm W-214nm W-214nm W-214nm W-214nm w- 214nm W-214nm
1.9.001.dlt 1.9.003 .dll 1.S.002.d;1t 1.S.oo3.dll 1.9.004.dat 1.9.00�.dM 1.9.006.dat 1.9.001.dlt U.OO8.cbt
I DMF I 0.18
I 1 Ivsine I ./\ , 0.14
LA 0.12
�A
� 0.10
'"'--' �
"'" � ,..
"'"
0"
I Hllvsines I on,
13 14 " " 17 " " " 21 n " 24 " " 27 " 29 :lO 31
MnUles
Figure 5: Capillary electropherogram, the final product of complete lysine acetylation of a SCA
protein. All eighteen lysine residues were acetylated (overall charged changed). DMF (far left
peaks) are markers. Each peak represents the number of Iysines acetylated. Conditions for
acetylation were; 40l-lM SCA, 20mM acetic anhydride, NaOH, pH = 8.
[BCA-(NH, +),,]-H
DMF
[BCA-(NHCOCH,),l"
i • i • • Iii i • I •• i. i": (NHCOCH1)n o 5 10 15 1.8
Figure 6: Electropherograms of wild-type [SCA-(NH3 +)18r
3.4
(bottom), a charge ladder of wild-type SCA (middle) in
which each peak in the ladder represents the number of
acetylated lysine groups, and [SCA-(NHCOCH3)18r19
(top). Conditions for electrophoresis were: unmodified
capillary, 25 mM Tris-192 mM Gly buffer, pH = 8.4, with
electrophoretic mobilities estimated relative to a neutral
marker, N,N-dimethy/formamide.
-2 0 2 4 6 8 10 12 14 16 18 20 22 24
mobility (em' kV-' min-')
Cuddemi 17
3.2 Crystallization
After acetylating wild-type SCA, understanding how the crystal structure changed
was a crucial part in our research. We know that the change in charge of a protein does
not affect the secondary or tertiary structure, therefore we were able to obtain a crystal
structure of SCA, as well as SCA-(Ac)18. Figure 7 shows the difference in the crystal
structure of SCA and SCA-Ac. The crystal structures were obtained from x-ray
diffraction. The pictures in Figure 7 were gathered from a computer representation of
SCA and SCA-(Ac)18 for the purposes of illustrating the crystal structures.
The results allowed us to understand the different protein-protein interactions that
are happening inside of the SCA and SCA-(Ac)18 crystal structures. The different
colored folded structures represent different SCA or SCA-(Ac)18 proteins in the crystal,
indicating that there are five core proteins present in protein-protein interactions inside
the crystals. The core SCA protein is able to interact with each protein to form bonds to
produce the crystal. In Figure 7, each arrow indicates a unique lysine residue
interaction taking place. We were able to conclude that there are eight lysine
interactions that happen in the wild-type SCA protein crystal. An interesting result was
that the per-acetylated SCA-(Ac)18 protein crystal involved twenty-nine lysine
interactions. We determined this by zooming in on each lysine residue in the core SCA
protein and measuring the length of the chemical bonds between the Iysines. If the
length of the bond measure greater than or equal to 3.oA (angstroms), the bond was
considered a protein-protein interaction.
Cuddemi 18
[BCA-(NH +) ]-34 3 18 [BCA-(NHCOCH3)
Figur. 7: Comparison of packing in crystals of wild-type SCA
[SCA-(NH3+)18] and per-acetylated SCA [SCA-(NHCOCH3)18].
The labEJed regions (A - G) represent unique contacts between
the neighboring molerules in each crystal.
3.3 Protein-protein Interactions
To aid in the comparison of the crystal structures of SCA and SCA(Ac)18, we
measured all of the protein-protein contacts within the crystal lattices with the MOE
program suite (http://www.chemcomp.com). We chose those dimensions (2x2x2 unit
cells) so that the crystal lattices would contain all possible inter- and intra-unit-cell
protein-protein contacts. We wrote software using the OESpicoli toolkit provided by
OpenEye Scientific Software (http://www.eyesopen.com) to calculate the solvent-
19
accessible surface area (SASA) of a crystal lattice, and to measure how much surface
area is contributed by each residue of each protein monomer in the lattice. The
software developed was specific for our research project. For each protein monomer in
the crystal lattice, our program also calrolated the per-residue SASA contributions for
Cuddemi 19
that monomer independently of the crystal lattice (by removing all other protein
monomers and recalculating the SASA). By computing the difference between the
SASA of a residue in the crystal lattice with the SASA of the same residue outside of the
crystal lattice, we determined how much of the SASA of that residue is buried by
protein-protein contacts in the crystal lattice. Our program produced a model of a single
unit cell, in which each residue was labeled with the amount of SASA buried by crystal
contacts [8].
Part A: Protein-Protein Interactions Key
Classification Example
charge-charge
charge-dipole
ydrogen bond
Cuddemi 20
dipole-dipole
igher-order
alkyl group - alkyl
group
alkyl group - carbonyl
oxygen atom
alkyl group - oxygen
atom or nitrogen atom
alkyl group - aromatic
carbon
Cuddemi 21
alkyl group - carbonyl
carbon
Table 2: computerized depictions of the types of interactions shown in the corresponding tables. These reactions exist in the crystal structures of BCA and BCA(Ac)1s. This table serves to act as a key to understanding the reactions presented.
Part B: Interactions Present In A BCA Crystal
Wild-type BCA
charge-charge
alkyl C-carbonyl 0
Table 3: computerized images of the lysine interactions between wild-type BCA proteins within the crystal structure. The type of reactions are labeled according to the key in Figure_.
Cuddemi 22
Pari C: Interactions Present In A BCA-(Ac)18 Crystal
BCA-(Ac)18
GlU·212
char�e-dipole
carbonyl-carbonyl
GLN-1.35
A
carbonyl-carbonyl carbonyl N-O
Cuddemi 23
carbonyl N-O high order
alkyl-alkyl C
alkyl-alkyl C
THR·Bl
alkyl-alkyl C
alkyl-alkyl C
(1 )- AtY-44 3� ...... ,,,-...
Cuddemi 24
alkyl C-carbonyl 0
Table 4: computerized images of the lysine interactions between per-acetylated BCA-(Ac)1s proteins within the crystal structure. The types of reactions are labeled
according to the key in Figure_.
Part D: Summary Table of the Interactions
Interaction Types BCA BCA-(Ac)18
charge-charge 2 --
ch arge-d i pole 1 3
hydrogen bond -- 3
dipole-dipole -- --
higher-order -- 2
alkyl group - alkyl group 1 6
alkyl group - carbonyl
oxygen atom 1 6
Cuddemi25
alkyl group - oxygen atom or
nitrogen atom 1 5
alkyl group - aromatic
carbon -- 2
alkyl group - carbonyl
carbon -- 1
TOTAL INTERACTIONS 6 28
Table 5: summary of the interactions between proteins within the crystals of
SCA and SCA-(Ac)18. As shown, there are about 6-8 interactions present in wild-type SCA and 28-29 interactions present in SCA-(Ac)18.
As seen in the comparison Table 5 above, the interactions present in the crystals
of SCA and SCA-(Ac)18 immensely differ. In wild-type SCA, there are about six to eight
lysine protein-protein interactions within the crystal. An interesting finding was the ability
of acetylated SCA-(Ac)18 to interact significantly more within its crystal. Noted in the
table above, there are anywhere from twenty-seven to twenty-nine lysine protein-protein
interactions in the acetylated SCA-(Ac)18 crystal.
3.4 Thermostability
The comparison of melting points between wild-type SCA (SCA) and per-
acetylated SCA was completed by using differential scanning calorimetry (DSC). DSC
measures the amount of heat (kJ) that it takes to denature a protein crystal in solution.
Figure 8 depicts the temperature at which SCA and SCA-Ac crystal structures melted,
either with or without a ligand present. Each point on the graph denotes the kJ of heat
that was required to denature the protein crystals. The y-axis is the measurement of the
heat that was applied and the x-axis represents the protein that was being tested.
Cuddemi 26
Examining the first column on the len, SCA Vv'ithout a ligand present is shown.
The results show that wild-type SCA (red boxllop) required a higher amount of heat
(900 kJ) to denature the crystal structure. Per-acetylated SCA-(Ac)18 (green
trianglelbollom) required only 300kJ of heat to denature the crystal. The nexl two
columns compared the presence oftvvo different ligands (STA or TA) in the SCA or
SCA-(Ac)18 crystals. Figure _ shoVv"S the molecular structure of the TA and STA
ligands. In the data presented, we are able to note that the SCA-Ac crystal structure is
less stable than SCA, Vv'here the SCA-Ac unravels at a lower melting temperature. The
presence of the ligands stabilizes acetylated SCA (melting temp increases), but
destabilizes the wild-type SCA. This phenomenon is still unexplained.
6H of Unfolding ,�
� •
� •
• '00
- • SCA <; � E
-� .. SCA-Ac ;2 .. -
% � ..
..
� ..
'00
'00
" BCA BCA+ TA BCA+ BTA
Figure 8: DSC data comparing the thermostability of Vv'ild-type SCA
(SCA) and per-aceyllated SCA (SCA-Ac). The difference of melting
temperature is compared with respect to ligand presence. The x-
axis depicts the presence of a ligand; TA or STA.
Cuddcmi 27
In Figure 9, the molecular structure of the ligands used in DSC are depicted.
BTA is the bulkier ligand, resulting in the decreased thermostability of wild-type BCA
when bound. TA shows a slightly increased thermostability in BCA than the BTA ligand.
But when examining the results of acetylated BCA-(Ac)18, the results are opposite.
When the ligands are bound, there seems to be an increase in protein thermostability,
shown by the higher temperature that it takes to denature the crystal. As mentioned
above, this is still not well understood.
o N II
}-S-NH2 S II
o
o eN II I }-S-NH2
S II o
Figure 9: Molecular structure of ligands
used; BTA (top), TA (bottom).
Cuddemi 28
4 Discussion
4.1 Summary of Results
4.1.1 Acetylation of BCA and Crystallization
In this study, we were able to successfully acetylate the eighteen lysine residues
on BCA (as shown in Figure 5 and Figure 6). The acetylation of the lysine residues
resulted in an overall negative charge to the BCA-(Ac)18 protein. When a protein is not in
solution, we found that acetylation does not affect the secondary or tertiary structures of
the protein, but it does affect the protein-protein interactions within the crystal structure.
As previously mentioned, a BCA crystal consists of eight lysine interactions, while a
BCA-(Ac)18 crystal consists of twenty-nine lysine interactions. I t seems that the presence
of a negative charge in the per-acetylated crystal structure helps the overall stability of
the solid crystal, when not in solution. A wild-type BCA crystal is more likely to denature
because there are less interactions between protein structures. This causes the crystal
to become less mechanically and less thermo stable (dissolving at a lower
temperature). The wild-type BCA crystal is also less dense and less favorable. But all of
this is only possible when the crystal structures are not in solution.
4.1.2 Thermostability
We determined that the thermostability in solution of wild-type BCA is high than
that of per-acetylated BCA-(Ac)18. I t seems that the positive charge of the Iysines
present on the wild-type BCA crystal stabilize the protein when in a solution of water.
The charge-charge interactions with other groups on the BCA (such as COO- groups)
that are present will interact with the positively charged lysine groups (N H3 +) , causing
Cuddemi 29
stronger bonds in the crystal. There are also lysine-water interactions that occur, which
also stabilize the crystal. These interactions are unable to occur in the per-acetylated
BCA-(Ac)18 crystals because of the presence of a negative charge.
We were able to disprove our hypothesis regarding the idea that altering the
overall charge of BCA would alter the secondary or tertiary structure of the protein. But
we were able to show that BCA-(Ac)18 is a more stable, interactive crystal when not in
solution, though wild-type BCA is the more stable crystal in solution. But as mentioned
previously in the thermostability results section, we are still unable to explain how the
presence of either the TA or BTA ligand in the acetylated BCA-(Ac)18 crystal increases
the thermostability. To test this, we could perform experiments that used different
ligands for interaction. This may allow insight into the binding of a ligand to acetylated
BCA-(Ac)18.
During the acetylation procedure, we ran into a few problems. The first few times
that we attempted to acetylate BCA, we were unsuccessful. Troubleshooting allowed us
to realize that our amount of reagents used was too large. In the small scale of our
research, we needed to avoid denaturing the protein during the reaction. But we learned
that the heat given off during the acetylation reaction was able to denature the proteins.
Therefore, we decreased the amount of acetic anhydride from 2mL to 200l-iL in the
reaction tubes. This allowed the acetylation to occur without disruption from the natural
heat given off.
Cuddemi 30
Crystallizing the structures of SCA and SCA-(Ac)18 was completed by X-ray
crystallography, which allows us to obtain a picture of the solid-state, lowest energy
conformation of the protein. An alternative method to evaluate the structure of SCA
would be to use nuclear magnetic resonance (NMR). NMR give a picture of the protein
in solution, however there is no automated method for converting the peaks produced
by NMR into a 3D structure. In X-ray crystallography, there are software programs that
use the diffraction pattern and standardized methods to evaluate the patterns,
producing a 3D structure. Though we would have the ability to look at the structures of
SCA and SCA-(Ac)18 in solution with NMR, it would require us taking that average of
many time snapshots during the process. The final picture would be at a lower
resolution than X-ray crystallography provides. Figure 10 shown below, depicts an
NMR spectrum of human carbonic anhydrase (HCA). The dots represent the separate
points at which one would have to record the average. A 3D structure is difficult to
retrieve from the information provided by NMR.
12 11 10 9 8 7 6
10
11
.........
E 11 0..
0.. �
Z Lf) �
?: 12
13
13
12 11 10 9 8 7 6
Figure 10: NMR of human carbonic anhydrase. The dots are the average points that
are recorded during the NMR process. A 3D structure is extremely difficult to interpret frnfn th ic ri <>t<>
Cuddemi 31
4.2 Future Directions
The next step of our research is to test the binding affinity of arylsulfonamide
ligands to wild-type SCA and per-acetylated SCA using isothermal titration calorimetry.
We want to compare the difference in stability of the SCA and SCA-(Ac)18 protein crystal
structures in solution. It is noted that a SCA crystal is stable in solution, while SCA
(Ac)18 is less stable. When a ligand binds to a protein, heat is released. The question
asked in our research is if the heat released in the binding of a ligand is enough to
denature the crystal structure.
The long-term goals of our research are to aid in medicinal chemistry and
pharmaceuticals research. Since SCA is a model protein system, once we understand
the implications of protein-protein interactions, protein-ligand interactions and its
stability, we will be able to apply the knowledge to something more specific. In the area
of pharmaceuticals, protein-ligand interactions are a large part of research. Insight into
this phenomenon will guide research in diseases such as cancer. An important target in
cancer research is matrix metalloproteinases (MMPs), which degrade extracellular
matrices in the body. If we are aware of how a ligand interacts with a protein through
our SCA model, the knowledge may be applied to MMPs and their interactions.
Identifying the capability of molecules that can make proteins less stable could be used
to degrade a cancer cell in the body. This ability to apply our system to pharmaceutical
drugs in crucial in fostering the process of drug discovery. Though the long-term goals
of our research are far advanced, the relevance of less complex knowledge is largely
needed.
Cuddemi 32
5 References
[1] Leunissen, Mirjam. "Protein Crystallization." An essay on several aspects of protein
crystallization research. United Kingdom: Department of Solid State Chemistry, October
2001. http://people.ds.cam.ac. uk/ml527 Ipublications/assets/le unissen-
literature research .pdf.
[2] Krishnamurthy, Vijay M., et al. "Carbonic Anhydrase as a Model for Biophysical and
Physical-Organic Studies of Proteins and Protein-Ligand Binding." Chemistry
Review (September 2009): 946-1051.
http://www.ncbi.nlm.nih.gov/pmclarticles/PMC27407301.
[3] Saito, Ryuta, et al. "Structure of bovine carbnic anhydrase II at 1.95 A resolution."
Acta Crystal/ographica (February 2004): 792-795.
[4] Sarraf, B. S., et al. "Structural and functional changes of bovine carbonic anhydrase
as a consequence of temperature." Acta Biochimica Polonica (November 2003):
665-671.
[5] Carbeck, Jeffrey D., et al. "Protein Charge Ladders, Capillary Electrophoresis, and
the Role of Electrostatics in Biomolecular Recognition." Accounts of Chemical
Research (1998): 343-350.
[6] Tagliaro, F., et al. "A Brief Introduction to Capillary Electrophoresis." Forensic
Science International (1997): 75-88.
[7] Jelesarov, lIian and Hans Rudolf Bosshard. "Isothermal Titration Calorimetry and
Differential Scanning Calorimetry as Complementary Tools to Investigate the
Energetics of Biomolecular Recognition." Journal of Molecular Recognition
(1999): 3-18.
[8] Lockett, Matthew R. and George M. Whitesides. "The Surface Chemistry, and not
Shape Determines Differences in the Crystal Structures of Bovine Carbonic
Anhydrase and Per-acetylated Bovine Carbonic Anhydrase." (2011).
[9] Anderson, Janelle R., et al. "Analysis by Capillary Electrophoresis of the Kinetics of
Charge Ladder Formation for Bovine Carbonic Anhydrase." Analytical Chemistry (April
15, 2002): 1870-1878.
[10] Durbin, S. D. and G. Feher. "Protein Crystallization." Annual Review Physical
Chemistry (1996): 171-204.
Cuddemi 33
[11] Gudiksen, Katherine L., et al. "Eliminating Positively Charged Lysine E-NH3+
Groups on the Surface of Carbonic Anhydrase Has No Significant Influence on
Its Folding from Sodium Dodecyl Sulfate." Journal of the American Chemical
Society (October 11,2004): 4707-4714.
[12] Gitlin, Irina, Katherine L. Gudiksen and George M. Whitesides. " Effects of Surface
Charge on Denaturation of Bovine Carbonic Anhydrase." ChemBioChem (2006):
1241-1250.
[13] Matulis, Daumantas, et al. "Thermodynamic Stability of Carbonic Anhydrase:
Measurements of Binding Affinity and Stoichiometry Using ThermoFluor."
Biochemistry (2005): 5258-5266.
[14] Mack, Eric T., et al. "Using Covalent Dimers of Human Carbonic Anhydrase II to
Model Bivalency in Immunoglobulins." Journal of the American Chemical Society
(2011).
[15] Shaw, Bryan F., et al. "Neutralizing Positive Charges at the Surface of a Protein
Lowers Its Rate of Amide Hydrogen Excahnge without Altering Its Structure or
Increasing Its Thermostability." Journal of the American Chemical Society (2010):
17411-17425.
Cuddemi 34
6 APPENDIX
The following is the procedure and results from the first acetylation method
described on page 12. These results helped us to determine which volume of BCA and
NaOH to use. As shown in the results below, Tube 2D, gave us the best results at that
time. From these volumes of reagents was how we carried out the preceding
experiments, until we determined that a smaller volume of acetic anhydride was
needed.
6.1 BCA CE Run
1. Things to note
a. we are using the CE machine to separate the BCA proteins based on their
charge.
b. should help us determine if the acetylation process worked correctly and at
which ratio of BCAacetic anhydride should be used.
2. Solutions
'Look at the procedure on February 1 for the calculations'
a. 5mL of 20l-lM BCA in HEPBS
b. 15mL of 200l-iM Acetic Anhydride in H20
3. BCA acetylation reactions
- we will prepare tubes will varying volumes of acetic anhydride with a constant volume
of BCA.
- this was done to check if the concentration of AA and NaOH were the cause for the
poor labeling with the last experiment.
- ten tubes were prepared with the following:
Tube 1A: O.5mL BCA
O.5mL AA
OI-iL NaOH
Tube 1B: O.5mL BCA
O.5mL AA
431-1L NaOH
Tube 2A: O.5mL BCA
2.0mL AA
OI-iL NaOH
Tube 2B: O.5mL BCA
2.0mL AA
431-1L NaOH
Cuddemi 35
Tube 1C: O.5mL SCA
O.5mL AA
86IJL NaOH
Tube 10: O.5mL SCA
O.5mL AA
172IJL NaOH
Tube 1E: O.5mL SCA
O.5mL AA
344IJL NaOH
Tube 2C: O.5mL SCA
2.0mL AA
86IJL NaOH
Tube 20: O.5mL SCA
2.0mL AA
172IJL NaOH
Tube 2E: O.5mL SCA
2.0mL AA
344IJL NaOH
- each of the tubes was then incubated in the cold room for 2 hours on the rocker.
- after 2 hours -7 O.5mL of NaOH was added to each tube to stop the reactions.
4. Spin column
- each of the solutions in Tubes 1A-2E needs to be placed into the correct buffer for the
CE run.
- this process will allow a buffer exchange.
A. Protein Desalting Spin Column Preparation and Sample Loading
*Look at the procedure described in the February 1 protocol.
*Run 07feb2013_SCA sequence on the CE machine*
- this was run with both a glycerol + H20 buffer and a 15% glycerol buffer (refer to the
previous experiment for buffer concentrations).
Cuddemi 36
6.1 CE Electropherogram Results
UV _ 21<1nm " _ 2Unm UV _ 214nm UV _ 214nm UV __ 2Unm
01fob2013 BCro01E.doi , eb2013 bcaOOI .'" 011002013 bcaOOI a.dlll 011002013 bcaOOlb.dat 01feb201J bcaOOlc.d"i
0_0015 0 0015
0 0010 {) 0010
0.0005 {) 0005
Tube IE
0 0000 0 0000
I Tube ID " " < <
-0 0005 -0 0005
Tube Ie -0 0010 - 0 0010
-00015 Tube tB - 0 0015
-0.0020 -0.0010
I Tube IA
-0.0025 -0 0025
" " ;0 " " " " " " " 70 " " "' " " ",
Minute.
UV - 214nm UV - 214nm UV - 214nm UV - 214nm UV - 214nm IJ7feb2013 Be 02E.dat 07feb2013 bcaQ()2d.dat ()71'eb2013 bcaOO2a.dat 07feb2013 bcaOO2b.dat 07'feb2013 �c.d;;Jt
0.00125 "''"'"
0 00100 0 00100
0_00015 "'''''
0""'" Tube2E
0000"'
"
0.00025 Tube 2D 0 00025 «
0= 0 00000
.0""'" .0""'"
.0""'" .0""'"
Tube2A
-000075 ,� -000075
" " " " .. , '.0 " " ., 70 " " " " " 10.0 10.5 '"
Minutes
Cuddemi 37