Understanding the mechanism of
ALDC (Alpha Aceto-lactate decarboxylase)
By
Amit Anand
Supervisor: Prof. Vilmos Fulop
Biological Sciences
University of Warwick
Thesis
Submitted to the University of Warwick
in partial fulfilment of the requirement of
Master Of Science
Mco
A
MOAC Doctoral Training Centre
September 2005-07-18
1
Acknowledgements
I would like to thank the all the members of Prof. Vilmos’s Structural biology lab;
without whose help I would have not been able to complete this project.
I would like to offer special thanks to the following people for their help and assistance:
•
Mr Ian Portman whose experienced guidance and willingness to help saved my day.
•
Dr. Dean Rea for helping me with SDS-PAGE experiments
•
Dr. Helena for the help with the buffer problem.
•
Dave, Tom and Gian Franco for their help on Bio-Rad assay.
•
The company NOVOZYME for the gift of ALDC
Finally a huge thank you to my supervisor, Prof. Vilmos Fulop, whose patient
supervision had made this project possible
2
Content:
List of Figures: ......................................................................................................................4
Summary ...............................................................................................................................6
Chapter 1 Introduction ........................................................................................................7
1.1 Background:................................................................................................................7
1.3 Project one summary (What has been done before.): .............................................9
1.4 Summary of project two (What we have achieved in this project):.....................10
1.5 ALDC protein structure: .........................................................................................11
1.5.1 Protein structure: ..............................................................................................11
1.5.2 ALDC Structure ................................................................................................12
1.6 Crystal and Crystalline state:..................................................................................15
1.7 Mechanism of Crystal growth:................................................................................20
Chapter 2 Methods and Techniques:................................................................................22
2.1 Principle of X-ray crystallography: ........................................................................22
2.2 Crystallization Techniques: .....................................................................................25
Chapter 3 Materials and Methods:...................................................................................27
3.1 Materials: ..................................................................................................................27
3.2 Methods: ....................................................................................................................27
Experiment No 1 .........................................................................................................27
Experiment No 2 .........................................................................................................32
Experiment No. 3 ........................................................................................................34
Chapter 4 Future work: .....................................................................................................37
Chapter 5 Conclusion: .......................................................................................................38
References: ..........................................................................................................................40
Appendix A..........................................................................................................................43
Appendix B: ........................................................................................................................44
Appendix C..........................................................................................................................45
Appendix D..........................................................................................................................47
Appendix E..........................................................................................................................51
Appendix F ..........................................................................................................................58
3
List of Figures
List of Figures:
1.1 Background:
Figure 1.1. 1............................................................................................................................................................................................................. 7
Figure 1.1. 2............................................................................................................................................................................................................. 7
Figure 1.1. 3............................................................................................................................................................................................................. 8
1.5 ALDC protein structure:
Figure 1.5. 1........................................................................................................................................................................................................... 12
Figure 1.5. 2........................................................................................................................................................................................................... 12
Figure 1.5. 3........................................................................................................................................................................................................... 13
Figure 1.5. 4........................................................................................................................................................................................................... 13
Figure 1.5. 5........................................................................................................................................................................................................... 14
1.6 Crystal and Crystalline state:
Figure 1.6. 1........................................................................................................................................................................................................... 16
Figure 1.6. 2........................................................................................................................................................................................................... 16
Figure 1.6. 3........................................................................................................................................................................................................... 17
Figure 1.6. 4........................................................................................................................................................................................................... 17
Figure 1.6. 5........................................................................................................................................................................................................... 18
Figure 1.6. 6........................................................................................................................................................................................................... 18
Figure 1.6. 7........................................................................................................................................................................................................... 19
2.1 Principle of X-ray crystallography:
Figure 2.1. 1........................................................................................................................................................................................................... 22
Figure 2.1. 2........................................................................................................................................................................................................... 23
Figure 2.1. 3........................................................................................................................................................................................................... 23
Figure 2.1. 4........................................................................................................................................................................................................... 24
Figure 2.1. 5........................................................................................................................................................................................................... 24
2.2 Crystallization Techniques:
Figure 2.2. 1........................................................................................................................................................................................................... 25
Figure 2.2. 2........................................................................................................................................................................................................... 26
3.2 Methods:
Figure 3.2. 2........................................................................................................................................................................................................... 31
Figure 3.2. 3........................................................................................................................................................................................................... 31
Chapter 4 Future work:
Figure 4. 1 .............................................................................................................................................................................................................. 37
Appendix A
Figure A 1 .............................................................................................................................................................................................................. 43
Appendix B:
Figure B 1 .............................................................................................................................................................................................................. 44
Appendix C
Figure C 1 .............................................................................................................................................................................................................. 45
Figure C 2 .............................................................................................................................................................................................................. 45
Figure C 3 .............................................................................................................................................................................................................. 46
Figure C 4 .............................................................................................................................................................................................................. 46
Appendix D
Figure D 1 .............................................................................................................................................................................................................. 47
Figure D 2 .............................................................................................................................................................................................................. 48
Figure D 3 .............................................................................................................................................................................................................. 48
Figure D 4 .............................................................................................................................................................................................................. 49
Figure D 5 .............................................................................................................................................................................................................. 50
Appendix E
Figure E 1 .............................................................................................................................................................................................................. 51
Figure E 2 .............................................................................................................................................................................................................. 51
Figure E 3 .............................................................................................................................................................................................................. 52
Figure E 4 .............................................................................................................................................................................................................. 52
Figure E 5 .............................................................................................................................................................................................................. 52
Figure E 6 .............................................................................................................................................................................................................. 53
4
List of Figures
Table E 1: Shows all the seven crystal system. [30] ............................................................................................................................................... 55
Table E 2: Shows the conventional types of unit cells. [31].................................................................................................................................... 55
Table E 3: Shows all the known 230 three dimensional space groups known till now [29] .................................................................................. 57
Appendix
Table F 1 ................................................................................................................................................................................................................ 59
5
Summary
Summary
For the very first time a successful X-ray structure of ALDC (alpha acetolactate
decarboxylase) has been solved at 1.2 A (Angstrom) resolution in the structural biology lab of
department of Biological science at Warwick University. ALDC has been used in the Brewing
Industry for more than a decade. It reduces the maturation time from 10 weeks to less than 24
hours. However the mechanism of the enzyme is until now unknown as there was no atomic
structure of the enzyme available.
The first step in understanding the mechanism is to verify the proposed active site of
the enzyme. This is done by co-crystallizing the inhibitors along with the enzyme. In this
project we were successful in co-crystallizing 6 inhibitors. We received a new instalment of
the enzyme from the company NOVOZYME (based in Denmark). On the new sample we
carried out SDS-PAGE and Bio-Rad assay to finds its relative molecular mass and
concentration in comparison to the previous sample.
The application of the whole project is enormous. ALDC is a non-enantiomer specific
enzyme which makes it very important from bio-informatics point of view. Moreover creating
a mutant to increase the application of the enzyme is also a consideration. Acetoin is the final
product of the enzymatic reaction and it is considered as an evolutionary marker by many
microbiologists.
6
Chapter 1 Introduction
Chapter 1 Introduction
1.1 Background:
Enzyme Alpha-acetolactate decarboxylase (or ALDC) decarboxylates the natural
substrate (S) Acetolactate to Acetoin. Previous studies [1] have suggested that ALDC is also a
non-enantiomer specific enzyme. In other words ALDC coverts (R) Acetolactate to (S)
Acetolactate and then decarboxylates it to acetoin (Figure 1.1.1).
H3C
3
C
ALDC
2 HOOC
C (R)
OH 1
H3C
4
H3C
CH3
O
O
C
3C
ALDC
4
3
(S) C
COOH 2
1 HO
CH3
H
O
C (R)
OH 1
H3C
4
acetoin
Acetolactate
Figure 1.1. 1
Schematic diagram showing conversion of (R) enantiomer to (S) enantiomer and finally getting
converted to Acetoin with the help of the enzyme ALDC.
A simple conversion of (R) form to (S) form, without the enzyme, takes place at a very high
pH of 12-13 (Fig 1.1.2) whereas ALDC does it at a normal pH of 5.5-6.5 (Fig 1.1.3).
CH3
2 HOOC
3
C
CH3
4
C
pH 12-13
(R)
C
OH 1
CH3
O
O
(S) C
1 OH
3
COOH 2
CH3
2-Hydroxy-2-methyl-3-oxo-butyric acid 4
(Acetolactate)
Figure 1.1. 2
Schematic representation of conversion of (R) form of acetolactate to (S) form at
very high pH of 12-13
7
Chapter 1 Introduction
H3C
3
C
H3C
4
C
3
ALDC pH 5.5-6.5
(R)
2 HOOC
C
CH3
O
O
(S) C
OH 1
1 HO
COOH 2
CH3 4
2-Hydroxy-2-methyl-3-oxo-butyric acid
(Acetolactate)
Figure 1.1. 3
Representation of reaction in which (R) acetolactate is converted to (S)
acetolactate.
This enzyme has been used in the brewing industry for more than a decade. It reduces
the maturation time of the beer from around 10 weeks to 24 hours (see appendix A for
details). The real mechanism is still unknown. Recently a successful X-ray crystal structure of
the enzyme has been reported [2].
1.2 Motivation behind the project:
There are a series of considerations which make this project very important.
Acetoin, the final product of the enzyme, has been considered important by
microbiologist for over more than a century. The Voges-Proskauer test
[3]
has been used to
test the production of acetoin by bacteria. By the help of this test bacteriologist use to
distinguish between Escherichia coli (which do not produce acetoin) and other bacteria like
Enterobacter aerogenes (or Aerobacter aerogenes), which produces acetoin enzymatically
(from pyruvic acid to alpha acetolactatic acid followed by decarboxylation, see appendix A)
[4]
. Modified versions
[5, 6]
of Voges-Proskauer are still a common practise in microbiology
labs, especially in classifying [7, 8] the Enterobacteria.
Evolutionary significance of acetoin production has been reported by researchers [9, 10]
as decarboxylation of acetolactate is believed to be a mechanism for switching glucose
catabolism from acidic to neutral products. Moreover acetoin is also found [8] both in animals
and higher plants.
Biacetyl [8] (or butane 2, 3 dione, an intermediate in forming acetoin, see appendix A)
along with acetoin contributes to the aroma of beer, butter and milk fermentation products like
cheese.
Importance of Acetolactate in the biosynthesis of branched chain amino-acids like
valine was for the fist time reported by Strassman and co-researchers
8
[11]
.
Harlpern and
Chapter 1 Introduction
Umbarger
[9]
were the fist one to demonstrate that acetolactic acid, only at pH 8 undergoes a
different enzymatic reaction to form amino acid (see appendix B). Moreover they also gave a
hint that acetoin formation takes place only at pH 6. It was later conformed by Rathbone
[12]
that the optimum pH for ALDC to work is 5.5 to 6.5 and the maximum activity becomes 50%
at extreme pH of 4.3 and 7.2.
Non-enantiomer specific nature of ALDC is already discussed, which make it a rare
enzyme. From bioinformatics point of view a mutant enzyme could give us more choice of
substrates with the same enzymatic catalytic mechanism.
Thus this project is very important not only with regards to understanding enzymatic
catalysis mechanism and bioinformatics but also for knowing evolutionary significance of
acetoin formation and to study closely the biosynthesis of essential amino-acids.
1.3 Project one summary (What has been done before.):
In the first project we successfully made 8 compounds. They are as follows:
S.No.
Structure and IUPAC Name
Nature and
Quantity
1
H3C
3
C
CH3
O
O
C
3
Substrate
2 HOOC
(S)
C (R)
C
COOH 2
OH 1
HO
1
H3C
4CH3
4
2-Hydroxy-2-methyl-3-oxo-butyric acid
((R)(S)Acetolactate) Racemic mixture
2
CH3
COOCH3
OH
C
(R) C
(S)
C
CH3
O
O
~150mg
Expected
CH3
OH
Known
Inhibitor
COOCH3
3.0990g
C
CH3
2-Hydroxy-methyl-oxo-butyric acid
methyl ester
(R)(S)methyl Acetolactate Racemic mixture
3
H3C
2 HOOC
3
C
O
C
3
C (R)
C2H5
4
OH 1
CH3
O
(S)C
1 HO
substrate
COOH 2
4C2H5
2-Ethyl-2-hydroxy-3-oxo-butyric acid (racemic mixture)
9
Known
~87.5 mg
Chapter 1 Introduction
H3C
4
2 H3COOC
3
C
Expected
CH3
O
O
C
3
C (R)
OH 1
(S)
1 HO
C2H5
4
C
inhibitor
0.53g
COOCH3 2
4C2H5
2-Ethyl-2-hydroxy-3-oxo-butyric acid methyl ester
(racemic mixture)
COOH
5
OH
C(S)
OH
H
CH3
C (S)
CH3
C
(R)
OH C (S)
CH3
OH
C (R)
CH3
H
OH
CH3C (R)
H
HOOC
CH3
COOH
COOH
OH
OH C CH3
(R)
OH
C CH
3
(S)
H
Expected
inhibitor
~20mg
2,3-Dihydroxy-2-methyl-butyric acid (racemic mixture)
COOCH3
6
OH
OH
H
CH3
C
CH3 (R)
C(S)
OH C (S)
OH
CH3
OH
CH3C (R)
C (R)
CH3
OH
H
H3COOC
C (S)
H
CH3
COOCH3
COOCH3
OH C CH3
(R)
OH
C CH
3
(S)
H
Expected
inhibitor
1.0350g
2,3-Dihydroxy-2-methyl-butyric acid
methyl ester (racemic mixture)
COOH
7
OH
OH
(S)
C
H
CH3
C2H5 (R)C
C (S)
H
C2H5
OH
OH
OH
OH
C (R)
OH
OH
C (S)
C2H5 OH
C (R)
COOH
Expected
C C2H5
(R)
inhibitor
C CH
3
(S)
150+120mg
CH3
OH
H
H
HOOC
CH3
2-Ethyl-2,3-dihydroxy-butyric acid (racemic mixture)
COOCH3
8
COOH
(S)
C
C (S)
H
CH3
H
CH3
C
C2H5 (R)
C2H5 C (R)
H3COOC
OH
OH OH
OH
H
COOCH3
COOCH3
C (S)
C2H5
C (R)
OH C C2H5
(R)
OH
C CH
3
(S)
H
CH3
Expected
inhibitor
0.6040g
2-Ethyl-2,3-dihydroxy-butyric acid methyl ester (racemic mixture)
1.4 Summary of project two (What we have achieved in this project):
•
The department received a new instalment of ALDC enzyme from the company
Novozyme (based in Denmark). We did qualitative (SDS-PAGE) and quantitative (BioRAD) test on it.
10
Chapter 1 Introduction
•
We were successful in reproducing Type I crystals.
•
Moreover we also co-crystallized 6 different inhibitors with the enzyme and got Type I
crystals.
•
Finally we found that the molarity of the pH buffer was very vital for these crystals. This
means in future we will be able to get high resolution Type II crystals.
1.5 ALDC protein structure:
A brief discussion on peptide bonds is given in Appendix C.
1.5.1 Protein structure:
The simplest definition of a protein is that it is a molecule composed of amino acids
linked together by peptide bonds. A protein structure can be divided into four main types via
primary, secondary, tertiary and quaternary structures.
The amino acid sequence of a protein’s polypeptide chain is called its primary
structure (without regard to spatial arrangement). For Example the ALDC primary structure is
as follows:
1
11
|
21
|
31
|
41
|
51
|
|
1 MKKNIITSIT SLALVAGLSL TAFAATTATV PAPPAKQESK PAVAANPAPK NVLFQYSTIN
60
61 ALMLGQFEGD LTLKDLKLRG DMGLGTINDL DGEMIQMGTK FYQIDSTGKL SELPESVKTP
120
121 FAVTTHFEPK EKTTLTNVQD YNQLTKMLEE KFENKNVFYA VKLTGTFKMV KARTVPKQTR
180
181 PYPQLTEVTK KQSEFEFKNV KGTLIGFYTP NYAAALNVPG FHLHFITEDK TSGGHVLNLQ
240
241 FDNANLEISP IHEFDVQLPH TDDFAHSDLT QVTTSQVHQA ESERK
Primary structure sequence of ALDC [17]
Secondary structure generally refers to how peptide backbones in a protein are
connected to each other in 3-D space (but without regard to the conformation of its side
chains or to its relationship with other segments)
[19]
. They usually form regular, repeating
structures held together by the attractions between peptide bonds along it [20]. They are mainly
of three types namely alpha helices, beta sheets and turns. Moreover those secondary
structures which cannot be classified as the three types mentioned above are termed as
"random coil"
[21]
. Alpha helices and beta sheets are the most commonly found important
secondary structures. Some of their main properties are described in the appendix D.
Tertiary structures are formed by the packing of many secondary structural proteins
into one or several compact globular units called domains [25]. In other words we can say they
are spatial relationship of the secondary structural motifs to one another. One very important
11
Chapter 1 Introduction
feature of the tertiary stucture is that it is achieve by folding [26]. The final quaternary protein
structure may contain many tertiary structural domains (as shown in the figure 1.5.1 below).
Figure 1.5. 1
Diagram showing all the types of complex structures of a protein [25].
1.5.2 ALDC Structure
The following piece of work was done in Prof. Vilmos’s lab at Warwick University.
(The information is unpublished)
α-Acetolactate decarboxylase has no significant structural similarity to any other
protein. The enzyme is a 2 domain metallo-protein. Figure 1.5.2 shows the first (N-terminus)
domain which comprises of a 7 β-strand mixed β-sheet (in blue colour). Pink ball is Zinc
metal ion bound to histidine residues (seen more clearly in the complex picture).
N
Terminus
Figure 1.5. 2
N-terminus domain of ALDC
12
Chapter 1 Introduction
Figure 1.5.3 shows the starting part of C-terminus domain which is a β-cylinder
comprising 5 anti-parallel β-strands and a long α-helix (in green). It also contains the three
histidines which coordinates the zinc metal ion (explained much better in later sections). The
orange strand (shown in all the figures) is 14 β-strand β-sheet which is involved in dimeric
interaction with another molecule.
Figure 1.5. 3
Starting part of C-terminus domain of ALDC
Ending Cterminus
domain
Figure 1.5. 4
Ending part of C-terminus domain of ALDC
The coordination of the metal is completed by a conserved glutamate from the ending part of
C-terminus (Figure 1.5.4).
The complete structure looks is shown in figure 1.5.5
13
Chapter 1 Introduction
Figure 1.5. 5
Complete structure of ALDC monomer
14
Chapter 1 Introduction
1.6 Crystal and Crystalline state:
Researchers have been studying Crystals as early as eighteenth century (R. J. Havy). A
Crystal is defined as “a regular repetition in three dimensional space of an object made of
molecules”. [27]
Some exceptions to crystal definition as mentioned above are as follows:
1. Absence of three dimensional periodicity because crystal periodicity is modulated by
periodic distortions.
2. Presence of only bi-dimensional periodicity example some polymers. Moreover most
fibrous materials are ordered only along the fibre axis.
3. Presence of intermediate between solid and liquid state, example some organic
crystals upon heating show the presence of liquid crystal state, also called
mesomorphic crystals.
Generally looking at states of matter we come across gaseous, liquids and solids.
Gases occupy all the available volume evenly, but can change their position depending on
change in pressure. Similarly in liquids also particles are not linked together permanently and
thermal motion has sufficient energy to move the molecules away. By reducing the thermal
motion in a liquid, the links between the molecules can be made more stable giving rise to a
rigid body made up of clusters of molecules. These clusters of molecules are more likely to be
ordered as they correspond to lower energy state. This type of ordered pattern of molecules
with lower energy state is called crystalline state. [29]
This crystalline state is a type of solid state. Other form of solid is amorphous solid
state which are also non fluid material but with very high degree of disorder. However there
are many other types of states which could be misleading like glass, also referred to as over
cooled liquids [29].
Some general discussion on geometry of crystals, are described in Appendix E.
Figure 1.6.1 (a) and (b) shows type I and type II crystal’s external morphology. Type I, II, and
III crystal’s unit cell symmetry is described in figure 1.6.2, 1.6.5, and 1.6.7 respectively.
15
Chapter 1 Introduction
Figure 1.6. 1
(a) type I bi-pyramidal crystals
(b) type II Rectangular crystals [2]
Type I crystal has a bi-pyramidal external morphology (fig 1.6.1 (a)). The unit cell
symmetry of the cell is P 4222 (figure 1.6.3) and it is tetragonal. Here P referrers to Primitive
unit cell type (see appendix E, table E2).
Figure 1.6. 2
Type I crystal’s unit cell symmetry [39]
16
Chapter 1 Introduction
The point groups for this system are 422 (see third diagram in figure 1.6.3). A more detailed
explanation of point groups is given in appendix E.
Figure 1.6. 3
Showing different point groups and their orientation [29]
The space group 4 with subscript 2 is represented in the second diagram of figure
1.6.4. Here the distance between the first and the second repetitive groups is 2/4 times the
total length of the unit cell.
Figure 1.6. 4
Representation of space group 4 with subscript 1, 2 and 3. [29]
In type II the external crystal symmetry was rectangular (figure 1.6.1 (b)). Whereas
the unit cell symmetry was trigonal (figure 1.6.5). In this case the asymmetric unit was single
monomers. This type of crystal system was of the highest resolution (1.2 Angstrom).
17
Chapter 1 Introduction
Figure 1.6. 5
Type II crystal’s unit cell symmetry [39]
The space group symmetry is P3221, where P again refers as primitive and 321 are the
point groups. The space group 3 with subscript 2 can be explained by the figure 1.6.6, which
again means that the distance between the first and the second repetitive group is 2/3 of the
total distance between the plains.
Figure 1.6. 6
Representation of point groups 2 and 3 with subscripts 1and 2 [29]
18
Chapter 1 Introduction
The unit cell symmetry for type III is orthorhombic (Figure 1.6.7) and it had the
asymmetric unit as trimer. Space group symmetry is I222. Here “I” stands for body centred
(see appendix E, table E2) and representation of point groups 222 can be seen in figure 1.6.3.
This crystal type was of the lowest resolution. [29, 39]
Figure 1.6. 7
Type II crystal’s unit cell symmetry [39]
19
Chapter 1 Introduction
1.7 Mechanism of Crystal growth:
It is believed that for normal crystallization a supply of surface tension energy is
required for spontaneous formation of nuclei. A higher level of super-saturation is required to
cross this energy barrier and once that happens crystal growth begins. It is assumed to be true
for protein crystallization as well. [35]
There are four important things to be considered for protein crystallization:
1. High purity of the protein is very essential.
2. Solvent from which it must be precipitated in crystalline form. Solvent usually
contains buffer, precipitant, salt (in case of poor solubility) and in some cases
detergent (e.g. for membrane proteins which are insoluble in water-buffer or waterorganic solvent).
3. Solution should be brought to a super saturation for nuclei to form (mechanism
described above).
4. Once the nuclei have formed, actual crystal growth takes place. This happens usually
at the surface of the nuclei.
Other important considerations are as follows:
•
Very high super-saturation should be avoided as it could lead to many nuclei
formation and therefore resulting in too many small crystals.
•
To attain maximum degree of order crystals should grow slowly.
•
Easiest way to change the degree of super-saturation is sometimes changing the
temperature.
•
Common precipitation methods involve using salt (salting-out) or polyethylene glycol
(PEG). This usually immobilizes water and thereby increases the effective protein
concentration.
•
On the other hand by removing salt, the protein sometimes precipitates. This is also
called “salting in”. This is explained by Debye-Huckel theory for ionic solutions,
which states that “increasing ionic strength lowers the activity of the ions in the
solution and increases the solubility of ionic compounds.” Another way of explain
“salting-in” is that “it is a result of competition between charged groups on the surface
of the protein molecule and the ions in the solution”.
•
However protein precipitation takes place because of columbic attractions between
opposite charges on different proteins molecules in the absence of solvent ions.
20
Chapter 1 Introduction
•
Another method of protein precipitation is to reduce the repulsive forces or increase
the attractive forces between the protein molecules.
•
One milligram of protein can be used for 65 crystallization experiments, if we assume
a crystal of size 0.3 X 0.3 X 0.3 mm = 0.027 mm3 weighing about 15mg. [35]
21
Chapter 2 Methods and Techniques
Chapter 2 Methods and Techniques:
2.1 Principle of X-ray crystallography:
Generally to see small object we need microscopes, hence ideally speaking to see
atoms or molecules we need a “super-microscope”. This is true only in the case if light can be
focused on the molecules with the help of a lens. We cannot see the details of on object unless
the distance between the parts of the object (here in this case distance between atoms of a
molecules) are at least half the wave length of the radiation used to view them. Technically
speaking the resolution is not right enough for us to see the atoms of a molecule. The reason
of this resolution problem is that visible light has a wavelength in the range of 4000 to 8000 A
(Angstrom), where as atomic distances are of the order of 1 to 2 angstrom. Hence supermicroscopes should use X-rays rather that visible light, as they have wavelength in the range
of 1000 to 0.1 A. [37]
A new problem arises from the use of X-rays as they cannot be focused. In other
words they cannot form image. The solution lies within basic nature of electromagnetic
radiations.
Every electromagnetic wave makes a characteristic diffraction pattern, at half the
distance between the image and the lens, which has a mathematical relationship with its
object. The plane where these patterns are made is called focal or Fourier transform plane (as
the mathematical relationship is described exactly by Fourier transform). X-rays cannot be
focused to give an image but they do give diffraction pattern (figure 2.1 and 2.2) at the focal
plane which can be mathematically interpreted into an image of the object. [38]
Figure 2.1. 1
Shows Fourier transform (in the imaginary plane) of an object by a lens, f is the
focal and g is the angle with the plane of the scattering object.
22
[38]
Chapter 2 Methods and Techniques
Figure 2.1. 2
A typical diffraction pattern obtain at f
position in the reciprocal space
[40]
Problems are not yet over, diffraction patterns are also of two types:
1. Continuous diffraction pattern: formed by a single non repetitive object. (figure 2.3)
2. Discreet diffraction pattern: formed by object with many repetitive units. (figure 2.4)
Figure 2.1. 3
First figure is an object and second figure is a diffraction pattern of the same object.
[38]
23
Chapter 2 Methods and Techniques
Figure 2.1. 4
First figure is an object and the second figure is again a diffraction pattern.
[38]
It is the discreet diffraction pattern that we should be looking for, reason being that we
can find the coordinates (or indices), moreover intensity data can also be used and hence
mathematically it is possible to get back the original object.
A Crystal is defined as “a regular repetition in three dimensional space of an object
made of molecules” (discussed earlier in section 1.6). This definition of crystal perfectly
fulfils our requirement of repeated united which can give a discreet pattern of diffraction.
Moreover a crystal also serves as a half crystal (shown in the figure 2.1.5).
Figure 2.1. 5
Representation of a whole X-ray diffraction instrument
24
[41]
Chapter 2 Methods and Techniques
A brief discussion on X-rays and the instrumentation used in X-ray crystallography is given in
appendix F.
2.2 Crystallization Techniques:
A brief discussion on some common techniques used for crystallization is described in
this section. These are only the basic ideas and their further modifications are usually
employed.
1. Batch crystallization: This is one of the oldest techniques. Here precipitating agents
are instantaneously added to a protein solution which brings a sudden state of high
super-saturation and with luck crystals grow gradually.
2. Liquid-Liquid Diffusion: In this method protein solution is layered over precipitant
layer in a small-bore capillary. A sharp boundary usually forms between the two
layers and a gradual diffusion takes place. This creates a range of super-saturation in
the protein solution, high at the bottom and low at the top.
3. Vapor diffusion: They are of two types
(a) Hanging drop method (figure 2.2.1): Here we mix a known amount of protein
solution with the precipitant solution on a cover slip and mount it upside down
on a well of mother liquor. The cover-slip and the well are sealed with the high
vacuum grease to facilitate super-saturation which leads to nuclei formation
and subsequently crystal growth takes place.
Figure 2.2. 1
Showing hanging drop method [36]
(b) Sitting drop method (figure 2.2.2) : This method is preferred when the protein
solution has a low surface tension and it spreads over the cover slip in the
hanging drop method.
25
Chapter 2 Methods and Techniques
Figure 2.2. 2
Showing sitting drop method [36]
4. Dialysis: In this method protein solution is kept in a tube covered with a dialysis
membrane at one end. This tube at the membrane end is dipped in the mother liquor.
The advantage is that the precipitating mother liquor solution can be removed and
changed any time. [35]
26
Chapter 3 Materials and Methods
Chapter 3 Materials and Methods:
3.1 Materials:
All the chemical materials are described in the individual experiments. They were
analytical grade chemicals, purchased from Sigma Aldrich and were used as received.
3.2 Methods:
Experiment No 1
Aim: To carry out SDS-PAGE (Sodium dodecyl sulphate poly-acrylamide gel
electrophoresis) for estimating the relative concentration of three different protein samples.
Apparatus and chemicals: A single cassette (two clean plates with two Teflon spacers),
comb, buffers (described in discussion section), SDS and Acrylamide stock solutions, and
coomassie blue dye.
Principle: Electrophoresis is a technique used to separate macromolecules in an electric field.
SDS (Sodium dodecyl sulphate, also called lauryl sulphate) is an anionic detergent. SDS not
only destroys the polypeptide complex structure but also provides a net negative charge which
attracts the whole complex towards an anode (positively charged electrode) in an electric
field. Since the polypeptide binds to SDS in proportion to its relative molecular mass, the
final separation depends entirely on the differences in relative molecular mass of the
polypeptides.
Procedure:
1. Resolving gel: To a 5ml, 12% acrylamide solution (already made up), 50 ml of 10%
ammonium per sulphate was added.
2. Stacking gel (purple colour): To a 5ml, 12% acrylamide solution (already made up),
36 ml of 10% ammonium per sulphate was added.
3. Stacking the cassette: Cassette was stacked upright in the stand with the bottom of the
cassette tight to the bottom of the stand. Screws were used to create a water-tight
chamber.
4. A comb was inserted between the plates of the cassette and a fill line was marked one
cm below the bottom of the comb. This will serve as the separating line for resolving
and stacking gel.
5. 5ml of TEMED was added to resolving gel prepared in step 1. This mixture was mixed
well and poured between the plates of the cassette.
6. Butanol was added on the top of the gel and the whole assemble was kept aside for 15
minutes.
27
Chapter 3 Materials and Methods
7. Once the acrylamide has polymerised, butanol was removed and the top of the gel was
rinsed with water several times.
8. Stacking gel (prepared in step 2) was also mixed with 5ml of TEMED and mixed well.
It was poured on the set resolving gel. A comb was inserted and the whole assemble
was left aside for 15 minutes.
9. Ones the stacking gel has also polymerised, the comb is removed slowly.
10. Now we had two compartments upper and the lower (above and below the gels
respectively). They were filled with electrode buffer or running buffer.
11. Sample preparation:
a. 10ml of protein ladder was mixed with 10ml of sample buffer.
b. 10ml of old ALDC, new dilute ALDC and new concentrated ALDC were
mixed with 10ml of sample buffer in three separate eppendrofs.
The above mentioned mixtures were heated to 60 degree Celsius for few minutes.
12. 5ml of the sample mixtures were added on to the stacking gel in separate sections.
13. The anode (+ electrode) was connected to the bottom of the chamber and the cathode
to the top chamber. 150 volts of current was passed though it.
14. When the dye front was near the bottom of the resolving gel then the current was
stopped.
15. The gel was removed from the cassette and kept in a staining dish. It was then first
washed with deionised water. Water was poured off and coomassie blue dye was
added for staining.
16. Staining was done overnight with agitation.
17. Excess dye was washed out by de-staining with acetic acid/ methanol.
18. The gel was dried, photographed and preserved in cellophane paper.
Discussion:
Acrylamide solution gets converted into a gel on polymerisation. This conversion
takes place spontaneously in absence of oxygen. In this experiment polymerisation is initiated
by adding 10% solution of ammonium per-sulphate (AP) followed by N, N, N’, N’ –
tetramethylethylenediamine (TEMED), which serves as a catalyst. Once the catalyst is added
polymerisation reaction takes place very quickly, hence it is necessary to have the assembly
ready before adding the catalyst.
There are two different types of gels used in this experiment.
28
Chapter 3 Materials and Methods
1. Lower layer or separating, or resolving gel – responsible for actual separation of
polypeptide by size. Separating gel buffer: 0.4% SDS, 1.5 M Tris-Cl, pH 8.8.
Gel composition:
•
A 7% acrylamide composition can easily separate 45 to 200 k Da polypeptides.
•
Polypeptide with less than 45 k Da molecular mass needs a denser gel (14%). ALDC
has a molecular mass of approximately 31 k Da. Hence we used 12% gel, which was
perfect for the experiment.
2. Upper layer or stacking gel- which contains the sample wells for initial sample
loading. This makes sure that loading of the sample is even and proper. Stacking gel
buffer composition 0.5M Tris-Cl, pH 6.8, with 0.4% SDS. Gel composition 3 to 4.5%
acrylamide is usually sufficient. Since our protein was only 31 k Da so we used only 3
% gel.
Water saturated butanol is added after adding the resolving gel between the plates of
the cassette. This produces a smooth and completely levelled surface on top of the resolving
gel, which will in-turn help in the formation of straight and uniform bands. Butanol used is
saturated with water so that the gel does not dry up. However using only butanol could make
the gel dry and using only water could make the gel dilute, week and irregular. If in 15
minutes the gel does not polymerizes then it means there is something wrong and the whole
process should be checked again.
Protein sample and its preparation: A sequence of single chain of amino acids of a poly
peptide is called its primary structure. Whereas presence of hydrogen bonds usually leads to
formation of secondary structures like helices, pleated sheets and turns. Moreover tertiary
structures results from the presence of hydrogen bonds, hydrophobic amino acids, Van der
Waal’s forces and disulphide bonds. Furthermore quaternary structures of protein are a result
of interactions of individual polypeptide chains, which are linked by covalent bonds and /or
non-covalent forces.
Main aim of sample preparation is to denature all the complex structures in a
polypeptide to its primary structure. Sample buffer is mixed with the protein solution in 1:1
ratio. Sample buffer usually consist of 2% SDS, 20% glycerol, 20mM Tris-Cl, pH 8, 2mM
ethylene di-amine tetra-acetic acid (EDTA), a reducing agent like dithiothreitol (DTT) or 2mercaptoethanol, and a small amount of bromophenol blue to serve as a tracking dye
(0.1mg/ml).
EDTA chelates divalent cations and thus reduces the activity of the protein (which
may have metal ions as cofactors). Tris is a buffer and is important as we need specific pH for
29
Chapter 3 Materials and Methods
the electrophoresis. Glycerol makes the sample denser than the running buffer and hence the
sample stops floating around. The dye helps to keep track of the progress of electrophoresis.
SDS breaks up the complex structure of polypeptide and gives a net negative charge to
it. DTT’s role in denaturing is that it helps in breaking the disulphide bonds as it is a strong
reducing agent. Heating to at least 60 degree Celsius shakes up the molecules and allows SDS
and DTT to react and complete the denaturation. Finally the whole polypeptide gets negative
charge and since like charges repel so the polypeptide more or less straightens up.
Staining and De-staining of the gel:
After the electrophoresis is complete staining of the gel is done, which usually takes
overnight with agitation. Staining is done with 0.1% Coomassie Blue dye in 50% methanol
and 10% glacial acetic acid. Agitation usually circulates the dye uniformly and helps in better
penetration. Moreover acidified methanol precipitates the proteins.
The next step is de-staining where excess dye is removed by washing with acetic acid
and methanol, again with agitation. Dye sticks permanently to the polypeptide and the rest of
it is removed during de-staining. [42]
Uses of SDS PAGE:
1. Finding the percentage purity of a protein: In a gel of uniform density the relative
distance travelled by the polypeptide (Rf) is negatively proportional to the log of its
mass. In a simultaneous run of known and unknown masses of polypeptide, the
relationship between Rf and mass can be plotted, and the mass of unknown
polypeptide can be estimated.
2. Estimation of relative molecular mass of a new unknown protein.
3. Different staining methods can be used to detect rare proteins and a lot of information
about their biochemical properties can be learnt. [42]
Observation: Figure 3.2.2 and 3.2.3 have the first column of protein ladder. Second column
is the old ALDC protein, third and fourth column are the new dilute and concentrated proteins
respectively.
30
Chapter 3 Materials and Methods
Figure 3.2. 1
SDS-PAGE gel with a negative (dark) background [42]
Figure 3.2. 2
SDS-PAGE with plain background [42]
Results: As shown in the figure 3.2.2 and 3.2.3 the old ALDC protein was intact even after
two years of storage. Moreover the new ALDC protein provided by the company was dilute
and the new concentrated protein is relatively less concentrated than the old ALDC protein.
This signifies that the new concentrated protein will require more % of PEG (Poly ethylene
glycol) for crystallization.
31
Chapter 3 Materials and Methods
Experiment No 2
Aim: To determine the relative concentration of two different ALDC protein samples using
Bio-Rad assay.
Apparatus and chemicals: Bio-Rad assay reagent consists of Coomassie Blue dye,
phosphoric acid and methanol. Perkin Elmer spectrophotometer 400.
Principle: Bio-Rad protein assay is based on Bradford’s method of quantitative protein
analysis. It involves the addition of an acidic solution of Coomassie Brilliant Blue G-250 dye
to protein solution, followed by measurement of absorbance with a spectrometer at 595 nm
wavelength. The Coomassie Blue dye binds to mainly basic and aromatic amino acid
residues, example arginine. With this interaction the absorbance maximum of Coomassie Blue
shifts from 465 to 595 nm. Hence Beer Lambert’s law can be applied and accurate protein
concentration can be determined by selecting an appropriate ratio of dye volume to sample
concentration.
Procedure:
1. 20ml of both protein samples were taken separately and diluted by adding 380 ml of
water in an eppendorf tube to make primary stock solutions.
2. From the primary stock solution 10 ml was taken in a new eppendorf tube for both the
samples. To this 790 ml of water was added along with 200 ml of Bio-Rad solution.
3. A blank was also prepared in a similar manner without the protein.
4. The solutions were mixed well.
5. After a fifteen minute wait absorbance was taken at 595 nm wavelength and value
were noted
6. The whole procedure was repeated three times and average values were taken for
concluding the results.
Discussion: Out of 285 residues ALDC has 29 positively charged residues (25 Lysine which
are basic and 4 Arginine which are both basic and aromatic). Hence Bio-Rad assay should
work for this protein. Although we could use bovine gamma globulin or bovine serum
albumin as standards but we want only relative protein concentration determination. Hence
we avoid using standards.
32
Chapter 3 Materials and Methods
Observation table: Since we are interested only in determining relative concentration so we
used the spectrometer values directly, i.e. without converting to any units using any formula.
1st trial
2nd trial
3rd trial
Average values
Old protein (S1)
0.330
0.352
0.347
0.343
Protein
0.160
0.156
0.152
0.156
0.271
0.267
0.262
0.266
provide
by the company
(S2)
New concentrated
Protein (S3)
Result: It can be conclude from the observation table that the old ALDC protein (S1) was
more concentrated then the new ALDC protein (S2) provide by the company. Moreover the
new concentrated protein (S3) was also less concentrated the old ALDC (S1). Hence while
using S3 for crystallization we will have to use more PEG concentration. [43, 44, 45]
33
Chapter 3 Materials and Methods
Experiment No. 3
Aim: Co-crystallizing type I and II ALDC crystals along with 6 different inhibitors. [2]
Chemicals used:
(1) Poly ethylene glycol 2000 monomethyl ether (PEG 2000 MME):
Procedure for making 50% W/V solution: 25 grams was weighed in a clean beaker
and then it was dissolved in 20ml of water. The mixture was then heated along with
stirring. The resulting solution was cooled to room temperature and the volume of the
solution was made to 50 ml.
(2) Cadmium Chloride solution:
Procedure for making 0.1M solution: Mol wt of CdCl2.2.5 H20 is 228.335 142.6 mg of
Cadmium salt was dissolved in 6.245ml of distilled water.
(3) Buffer TRIS 1M pH 7.0: Borrowed from technician.
(4) Distilled water
Procedure:
All the solutions mentioned above were filtered with micro-pore filters. Then
the solutions were transferred to crystallization plates in quantities as shown in the following
table. Total volume of every well was made to 200mL with distilled water. Protein
concentration was approximately 10mg/ml. Inhibitors were added to the protein solution and
incubated for 24 hours at 8 degree Celsius. The final concentration of the inhibitors in the
protein solution was about 65 to 80 milli-molar.
Crystals were grown using hanging drop method. 0.8mL of protein (along with the
inhibitor) solution was put on the plastic cover-slip; this was then diluted with same amount
of mother liquor. The cover-slip was sealed using high vacuum grease and the
crystallographic plate was kept at controlled environment for the super-saturation to take
place, leading to crystal formation. Table 1 is an example of how a crystallographic plate is
arranged.
34
Chapter 3 Materials and Methods
Table 1: Represents an example in which each constituent were added.
A
1
2
3
4
5
6
PEG-14% 56mL
PEG-16% 64mL
PEG-18% 72 mL
PEG-20% 80mL
PEG-22% 88mL
PEG-24% 96 mL
Tris-20mL 0.1M
Tris-20mL 0.1M
Tris-20mL 0.1M
Tris-20mL 0.1M
Tris-20mL 0.1M
Tris-20mL 0.1M
CdCl2 20mL 0.01M
CdCl2 20mL 0.01M
CdCl2 20mL 0.01M
CdCl2 20mL 0.01M
CdCl2 20mL 0.01M
CdCl2 20mL 0.01M
D. Water 104 mL
D. Water 96 mL
D. Water 88 mL
D. Water 80 mL
D. Water 72 mL
D. Water 64 mL
B
C
D
Calculation:
Inhibitor No. 1
Inhibitor Stock solution:
10ml = 11.5mg of inhibitor diluted to 200ml buffer.
Protein inhibitor mixture solution:
50ml of protein was mixed with 10ml of inhibitor stock solution and incubated for 24
hours at 8 degree Celsius.
Moles of the inhibitor in 10ml of stock solution = (11.5/1000) X (1/146) X (10/200)
Molarity of inhibitor in 60ml protein solution = 0.065 M or 65 milli-molar.
Similarly all the inhibitor stock solutions were made and they were mixed to the protein
solution in similar fashion. As the molecular weight is similar hence their molarity was also
similar.
Observations:
CH3
COOCH3
OH
C
(R) C
(S)
C
CH3
O
CH3
OH
O
COOCH3
C
CH3
2-Hydroxy-methyl-oxo-butyric acid
methyl ester
(R)(S)methyl Acetolactate Racemic mixture
35
Chapter 3 Materials and Methods
H3C
2 H3COOC
3
C
CH3
O
O
C
3
C (R)
OH 1
(S)
1 HO
C2H5
4
C
COOCH3 2
4C2H5
2-Ethyl-2-hydroxy-3-oxo-butyric acid methyl ester
(racemic mixture)
COOH
OH
C(S)
OH
H
CH3
C (S)
CH3
C
(R)
OH C (S)
CH3
OH
C (R)
CH3
H
OH
CH3C (R)
H
OH
HOOC
CH3
COOH
COOH
OH C CH3
(R)
OH
C CH
3
(S)
H
2,3-Dihydroxy-2-methyl-butyric acid (racemic mixture)
COOCH3
OH
(S)
C
OH
H
CH3
C
C2H5 (R)
C2H5 C (R)
C (S)
H
CH3
OH
OH OH
OH
H3COOC
H
COOCH3
COOCH3
C (S)
C2H5
C (R)
OH C C2H5
(R)
OH
C CH
3
(S)
H
CH3
2-Ethyl-2,3-dihydroxy-butyric acid methyl ester (racemic mixture)
COOH
OH
C(S)
OH
C (S)
H
CH3
H
CH3
CH3
C
(R)
OH
CH3C (R)
HOOC
COOH
COOH
OH
OH C (S)
CH3
OH
C (R)
CH3
H
OH C CH3
(R)
OH
C CH
3
(S)
H
2,3-Dihydroxy-2-methyl-butyric acid (racemic mixture)
COOH
OH
OH
(S)
C
C (S)
H
H
CH3
C2H5 (R)C
C2H5
C (R)
COOH
COOH
OH
OH
OH
OH
C (S)
C2H5 OH
C (R)
C C2H5
(R)
C CH
3
(S)
CH3
OH
H
H
HOOC
CH3
2-Ethyl-2,3-dihydroxy-butyric acid (racemic mixture)
Result: As seen in the observation table all the inhibitors were co-crystallized with Type I
crystals. They have been frozen with the cryoprotectant (15% ethylene glycol and mother
liquor containing 20% PEG). As soon the X-ray machine in the department is up and running
or if we manage to get a beam time booked on any synchrotron then we will be able to shoot
the crystals.
36
Chapter 4 Future work
Chapter 4 Future work:
The main aim of the whole project is to understand the mechanism of the enzyme
ALDC. X-ray crystal structure of the enzyme has been solved and we have a proposed active
site (figure 4.1). In this project we have been able to co-crystallize 6 inhibitors.
Our main future work will be to get the diffraction pattern and confirm that the
proposed active site is the correct one. We will then try to co-crystallize the substrate and
substrate analogue, which may be difficult as the enzyme is active at a very wide range of pH
range (pH 4 to 8). Using Computerised Molecular Simulation is an easier way to confirm a
complex between the proposed active site and the substrate in the lowest possible energy
state. Till now we have synthesised and co-crystallized only substrate analogue inhibitors.
Another future work would be to use transition state analogue inhibitors, which will turn over
but the remaining part of the product will get stuck in the active site.
Figure 4. 1
Proposed active site of ALDC enzyme. (Source:
unpublished data carried out previously in Prof. Vilmos’s
lab)
Once we identify the key residues responsible for the enzyme mechanism then we
could mutate the enzyme and prove the importance of the residue by doing enzyme kinetic
studies (using CD and/or NMR data). Moreover it is also important to know the nature of
inhibition done by each inhibitor (i.e. competitive or non competitive). The reasoning for the
nature of an inhibitor will hold vital clues to the future extension of the application of ALDC.
Understanding the mechanism of the enzyme will be very important from bio-informatics
point of view, in future as we can design enzymes based on this study.
37
Chapter 5 Conclusion
Chapter 5 Conclusion:
Getting an X-ray crystallography picture of the enzyme ALDC has been tried decades
ago, but it was in vain. For the very first time Division of Structural Biology at Biological
Science department of Warwick University, under the leadership of Prof. Vilmos Fulop has
been successful in getting a structure [2]. In the previous project (fist mini-project) we made 6
inhibitors, natural substrate and one substrate analogue.
The main aim of this second mini-project was to co-crystallize all the compounds,
with the enzyme. Second important part was to get an X-ray picture of the active site. Initially
it was reported that the enzyme can crystallize in three different crystalline forms (Type I, II,
and III) at different conditions. We successfully reproduced the type I crystals and we were
also able to co-crystallize all the 6 inhibitors. However we couldn’t try co-crystallizing the
natural substrate because of shortage of time.
Often it is found that proteins fail to crystallize in presence of inhibitors or substrates.
From this project we have found that the protein easily co-crystallizes with inhibitors. Cocrystallizing natural substrate will definitely take a lot of effort and time as the enzyme ALDC
is active at a very wide range of pH. ALDC is fully active at pH 5.5-6.5 and it is about 50%
active at even a very high pH of 8 and a very low pH of 4. Changing the pH will alter the
crystallizing conditions.
We received a second instalment of the enzyme from the NOVOZYME Company
(based in Denmark). We used SDS-PAGE technique to verify molecular weight of both the
old and the new enzyme. Even after two years of storage the old enzyme did not deteriorated
and it was as good as the new enzyme. This gives us confidence that the enzyme will keep
crystallizing at similar conditions as before. However the new sample of the enzyme was
more dilute. We concentrated it and carried out Bio-Rad assay to find out its relative
concentration in comparison to the old sample. After concentrating, it was still slightly less
concentrated then the old sample and we were able to crystallize it, but this time at a slightly
higher PEG concentration.
We were successful in crystallizing type I crystals. However our all attempts of
crystallizing type II high resolution crystals failed. We found needle like crystals for type II
which were long but very thin in size. Initially we could not find any reason for this. In order
to avoid wasting time we carried out co-crystallization with type I crystals which are slightly
lower in resolution. Towards the end of the project we found that molarity of the pH buffer
was very vital for the crystal growth which was never reported previously for this protein.
38
Chapter 5 Conclusion
Now we are confident that by taking this point in consideration we will be able to get high
resolution type II crystals in future.
The overall achievement of the research was good. We were able to co-crystallize six
different inhibitors with the enzyme. This proves that the enzyme can co-crystallize and in
future we will be able to see an inhibitor in the active site with high resolution type II crystals.
Unfortunately the X-ray diffraction instrument in the department was not working and the
country’s only working synchrotron at Daresbury had technical problems. However the
crystals were preserved for future use.
39
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41
References
[44] Bradford, MM. A rapid and sensitive for the quantitation of microgram quantitites of
protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72: 248-254.
1976.
[45] Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182: 50-69 (1990)
42
Appendix
Appendix A
Yeast extract fermentation
CO2
H+
H+ +
NADH
O
Glycolysis
Glucose
C
H3C
O-
NAD+
H2
C
O
Alcohol
Dehydrogenase
O
pyruvate
CH3
HO
Pyruvate Decarboxylase Acetaldehyde
C
Ethanol
Acetolactate Synthease
-O
HO
3 to 10 weeks
O
C
O
C
H3C
C
Slow Oxidation
C
CH3
O
[O]
CH3
CH3
butan- 2, 3-dione
Acetolactate
ALDC
Flavour Maturation Step
Yeast
Reductase
less than 24 hours
H3C
H3C
CH
HO
CH3
2, 3-dihydroxy butane
Figure A 1
Yeast extract fermentation
43
CH
OH
CH
Yeast
Reductase
O
C
H3C
C
O
acetoin
OH
Appendix
Appendix B:
Figure B 1
Shows branched chain amino acid Valine and isoleucine biosynthesis
44
Appendix
Appendix C
Peptide bond:
A peptide bond is a simple amide group formation between the carboxylic acid groups
of one amino acid and the amino group of another amino acid
[13]
(as show in the figure
below).
Figure C 1
Reaction showing Peptide bond formation[14]
One of the important characteristic of this bond is that is has a partial double bond
character (40%). This is because of the resonance of electrons which rapidly move between
the oxygen and nitrogen and as a consequence the C-N bond becomes a partial double bond
C=N. One of the proofs for this is that the C-N bond length of the peptide is 10% shorter than
that found in usual C-N amine bonds [17].
Figure C 2
Showing bond length and angles of a peptide bond [16]
45
Appendix
Another piece of evidence is that all the six atoms that are involved in the peptide bond lie in
a flat plane owing to the fact that C=N is much less flexible than a C-N bond.
Figure C 3
Diagrammatic representation of peptide bonds in a plane [15]
As a result of this flat plane geometry the only possible free rotation in the structure is
at the ends of the peptide i.e. C-C and C-N known as psi(y) and phi(f) respectively. However
the amount of rotation possible and the value of phi and psi are based on the R group of the
amino acids involved in the peptide bond formation.
Figure C 4
Sketch of peptide bond showing Phi(f) and Psi(y)
angles [15]
46
Appendix
Appendix D
Secondary structural proteins:
Alpha helices:
•
The structure repeats itself every 5.4 Angstroms along the helix axis. Alpha-helix has
a rise of 1.5 Angstroms per residue [22]
Figure D 1
Self explanatory diagram of alpha helix
•
Alpha-helices have 3.6 amino acid residues per turn, ie a helix 36 amino acids long
would form 10 turns. [23]
47
Appendix
Figure D 2
Self explanatory diagram of alpha helix
•
Mostly C=O group forms a hydrogen-bonded to N-H 4 residues away (ie O(i) to
N(i+4)).[24] (as shown in the diagram below)
•
Because of the previous point all C=O groups point in the same direction and all N-H
groups point the other way.
Figure D 3
Self explanatory diagram of alpha helix
•
Alpha helixes are generally of two types: left handed (moving anticlockwise) and right
handed (moving clockwise).
48
Appendix
Figure D 4
Diagram showing difference between
left and right handed helix
Beta sheets:
•
The basic unit of a beta-sheet is a beta strand
•
The beta strand is then like the alpha-helix, a repeating secondary structure.
•
The strands are held together and stabilized by hydrogen bonding [23]. The N-H groups
in the backbone of one strand establish hydrogen bonds with the C=O groups in the
backbone of the adjacent, parallel strand(s). consists of pairs of chains lying side-byside and
•
stabilized by hydrogen bonds between the carbonyl oxygen atom on one chain and the
-NH group on the adjacent chain
•
beta-sheets are found in two forms designated as "Antiparallel" or "Parallel" based on
the relative directions of two interacting beta strand (as shown below).
•
Hydrogen bonds in beta-sheets are on average 0.1 Å shorter than those found in alphahelices
•
The axial distance between adjacent residues is 3.5 Angstroms. There are two residues
per repeat unit which gives the beta-strand a 7 Angstrom pitch
49
Appendix
Figure D 5
Diagram showing parallel and anti-parallel beta sheets.
•
The beta-sheet is sometimes called the beta pleated sheet since sequential
neighbouring Calpha atoms are alternately above and below the plane of the sheet
giving a pleated appearance.
Turn is a U-shaped four residue segment of a protein. Turns are stabilized by hydrogen bonds
between their arms.
50
Appendix
Appendix E
Geometry of Crystalline state:
Reference Axes: In coordinate geometry when x and y axes are at right angle with each other
then any line AB as shown in the figure is referred to as rectangular axis. While describing the
geometry of a crystal sometimes it is appropriate to choose a different axes where x and y
axes are not at right angles, for example as shown in the figure E1 and E2 line AB is now
referred to as oblique axis. [27]
Figure E 1
Example of a rectangular axis (AB) [27]
Figure E 2
Example of an oblique axis (AB) [27]
Crystallographic Axes: Imagine a crystal to be a perfect rectangle like PQRS (assuming only
two dimensions, see figure E3) with line OA and OB as x and y axes respectively. In this case
a rectangular axis like AB is sufficient to describe the geometry. Now if we consider that the
line AB can be described as (1, 1) coordinates then the lines PQ, QR, RS, and SP can be
designated as (1, 0), (0, 1), (1-bar, 0) and (0, 1-bar) respectively. [27]
51
Appendix
Figure E 3
Showing rectangular axis (AB) in a 2 dimension crystal PQRS [27]
Unfortunately this is not the usual case when dealing with crystals geometry. Hence
we have to change from rectangular axis to oblique axis, for example in the figure E4 Line
AB is a rectangular axis where as in figure E5 Line AB is an oblique axis. [27]
Figure E 4
Showing rectangular axis (AB) in a 2 dimension crystal PQRS [27]
Figure E 5
Showing oblique axis (AB) in a 2 dimension crystal PQRS [27]
52
Appendix
Here if we take line AB’s coordinates as (1, 1) for rectangular axis (figure E4) then we
cannot describe the lines PQ, QR, RS, and SP as (1, 0), (0, 1), (1-bar, 0) and (0, 1-bar)
respectively, where as in the figure E5 using oblique axis we can designate them.
Miller indices:
In the figure E6 line AB and PQ are planes in a crystal (shown in 2 dimensions). AB is
a rectangular axis and here we use it as reference or parametral line. AB intercepts x and y
axes at a and b respectively. The other line PQ intercepts the x and y axes at a/2 and b/3
respectively.
Figure E 6
Showing how reference line (AB) are used [27]
If reference line AB is designated (1, 1) coordinates then the line PQ can be identified
by two numbers h and k such that h is the ratio of the intercept made on x axis by the
parametral line to that made by the line PQ, and k is the corresponding ratio for the y axis.
h = a /( a / 2) = 2
k = b /(b / 3) = 3
Thus PQ can be described as the line (23) –two-three. These numbers can be extended
in three dimensions as h, k and l and they are known as miller indices (named behind the
famous mathematician who used the notation for the first time in 1839). A good choice of the
parametral plane leads to small numerical values for the miller indices of crystal faces. [27]
Symmetry of crystals:
Definition of Symmetry: “Symmetry is that spatial property of a body (or pattern) by which
the body (or pattern) can be brought from an initial state to another indistinguishable state by
means of a certain operation – a symmetry operation” [28].
Definition of a symmetry element: “A symmetry element is a geometrical entity (point, line,
or plane) in a body or assemblage, with which is associated an appropriate symmetry
53
Appendix
operation. The symmetry element is strictly conceptual, but it is convenient to accord it a
sense or reality” [27].
Definition of symmetry operations: “The symmetry operation corresponding to a symmetry
element, when applied to a body, converts it to a state that is indistinguishable from the initial
state of that body, and thus the operation reveals the symmetry inherent in the body” [28].
Symmetry operations corresponding to a symmetry element are as follows:
1. Inversion: A symmetry operation with respect to a point.
2. Reflection: A symmetry operation with respect to a plane.
3. Roto-inversion: The product of a rotation around an axis by an inversion with respect
to a point on the axis.
4. Glide plane is the product of a reflection by a translation parallel to the reflection
plane.
5. Roto-reflection is the product of a rotation by a reflection with respect to a plane
perpendicular to the axis. [29]
Point group:
Definition: “A set of interacting symmetry elements all of which pass through a single fixed
point, this point is taken as the origin of the reference axes for the body”. Apart from this
definition an important requirement is that during symmetry operations of a point group at
least one point should be unmoved. In some cases it is a line (symmetry axis) or a plane
(reflection plane) that remains invariant. [28]
The seven crystal systems:
Based on rotation axes and inversion axes crystals are broadly classified into seven
systems. Table 1 shows all the seven classes along with there respective sides and angles.
54
Appendix
Table E 1: Shows all the seven crystal system. [30]
Lattices:
Definition: “A lattice may be described as a regular, infinite arrangement of points in which
every point has the same environment as any other point. This description is applicable,
equally, in one-, two-, and three-dimensional space.” [27]
Unit cell:
Definition: “The smallest building block of a crystal, consisting of atoms, ions, or molecules,
whose geometric arrangement defines a crystal's characteristic symmetry and whose
repetition in space produces a crystal lattice” [31]. Conventional types of unit cells are shown
in the table E2 below.
Table E 2: Shows the conventional types of unit cells. [31]
Symbol
Type
Number of lattice points per
cell
P
Primitive
1
I
Body Centred
2
55
Appendix
A
A-face centred
2
B
B-face centred
2
C
C-face centred
2
F
All faces centred
4
R
Rhombohedrally centred
3
Bravais lattices: Definition: “A Bravais lattice is an infinite set of points generated by a set of
discrete translation operations. A Bravais lattice looks exactly the same no matter from which
point one views it”. They are of two type plane and space lattices. There are five possible
plane lattices and fourteen different space lattices based both on primitive and non-primitive
cells. [32]
Space groups: In simple words it is a mathematical term which describes the symmetry of a
structure in a crystal.
Definition: “A space group is an infinite set of symmetry elements, the operation of which
leaves the pattern to which it refers indistinguishable from its condition before the
operation”. [34]
There are only 230 three dimensional space groups known till now. Table E3 describes all of
them.
56
Appendix
Table E 3: Shows all the known 230 three dimensional space groups known till now [29]
57
Appendix
Appendix F
Instrumentation:
X-ray introduction:
X-rays were first discovered by Rontgen in 1895. Since the nature of the rays was unknown
he named them X-rays, still they are known as X-rays.
X-rays are electromagnetic rays similar to visible light. Some of the important properties
identified by Rontgen are as follows:
•
These rays were invisible (just opposite to ordinary light)
•
They travelled in straight line (similar to ordinary light)
•
They were more penetrating than the visible light. They could even pass human body,
metal, wood and other opaque objects where visible light could not.
The most important parts in X-ray instrumentation are the X-ray source and X-ray
detectors.
Rontgen in 1895 was the first one to discover X-rays. He realized that the nature of these rays
was unknown so he used letter X. Since then these rays are called X-rays.
Some of the important properties which he saw were:
•
These rays were invisible (just opposite to ordinary light)
•
They travelled in straight line (similar to ordinary light)
•
They were more penetrating than the visible light. They could even pass human body,
metal, wood and other opaque objects where visible light could not.
By 1912 many properties of x-rays were explored to study the nature of these rays
(including x-ray diffraction of crystals). [7]
Production of X-rays:
In an atom the nucleus is surrounded by electrons, which orbit around the nucleus in
their own shells. The shell nearest to the nucleus is called k shell and so the electrons in that
shell are called k shell electrons. In short X-rays are produced when any electrically charged
58
Appendix
particle of sufficient kinetic energy, knocks off or removes these k shell electrons.
Table F 1
Diagrammatic representation of a nucleus and its shells. Figure taken from: - Crystal Structure AnalysisW.Clegg.
Another electron enters the k shell. In k shell the electron’s energy should be lowest possible,
hence this new electron librates energy in the form of radiation (one of which will be X-rays).
As shown in the figure F1 if the electron comes form L shell the radiation is called ka and if
the electron comes from M shell then the radiation is called Kb [7].
The assembly for x-ray production usually consist of
(a) a source of electrons (b) a high accelerating voltage and (c) a metal target
An x-ray tube contains two electrodes and when a high voltage is applied across these
electrodes then electrons move towards the anode or the target. Electrons thus strike the
target at a very high speed and so x-rays are produced and radiated in all directions. Some of
metals used as targets are copper, cobalt, iron, and chromium.
Some other examples of x-ray sources are Gas tubes, Filament tubes, rotating anode
tubes, micro-focus tubes, pulsed (or flashed) tubes, miniature tubes, high voltage tubes and
linear accelerators and Synchrotron radiation. Synchrotron radiations are currently the best
source of X-rays. A synchrotron is a particular type of cyclic particle accelerator in which the
magentic field (to turn the particles so they circulate) and the electric field (to accelerate the
particles) are carefully synchronized with the travelling particle beam. While a cyclotron uses
a constant magnetic field and a constant-frequency applied electric field, and one of these is
varied in the synchrocyclotron, both of these are varied in the synchrotron. The principle is
based on the fact that acceleration of charged particles produces electromagnetic radiations
[7]
.
59
Appendix
Detection of X-rays:
Previous methods were fluorescent screens, photographic film, and electronic
detectors. Currently the most popular one image (storage) plates [7].
60