Download The Leucine Binding Fluorescence Analysis of the Leucine Specific

Binding analysis of Leucine Specific Binding Protein
Andrea Fischer
Mentor: Dr. Linda Luck
Scope
The leucine-specific binding protein (LS) and the leucine-isoleucine-valine
binding protein (LIV) are the periplasmic components E. coli branched-chain amino acid
transport system. These two proteins are nearly identical in tertiary structure and share
about 80% of their amino acid content. These periplasmic binding proteins serve as initial
receptors of active transport and chemotaxis for many substrates. Although they are very
similar in both structure and function they differ in substrate specificities. The LS protein
binds leucine while the LIV will bind to leucine, isoleucine, and valine and to a smaller
degree, serine, alanine, and threonine. There is an interest is finding the reason for the
difference of the specificity of the ligands for these two proteins. The LS protein will be
studied through three mutations of key amino acid residues. Specifically the LSC53S,
LSC53SC78A and the LCD1CC53SC78A mutants will be created and analyzed.
Background
Periplasmic binding proteins
These proteins are in the periplasmic space of bacteria such as E.coli. Bacterial
periplasmic substrate binding proteins act as initial receptors for transport, chemotaxis,
antibiotic resistance, and energy utilization. There are more than 40 proteins that are
classified as periplasmic binding proteins. The protein binds to the solute within the
periplasm and mediates its transfer across the inner membrane of the bacteria. The
transfer of the ligand is catalyzed by the interaction of the binding protein with the
hydrophobic membrane of a set of membrane bound proteins.
Periplasmic proteins have an ellipsoid shape with two domains. There is a hinge
region that serves to link the two domains together and is distinctive of the structure. The
ligand binds in the region between the two domains. The protein is thus found in tow
forms one the ligand bound closed and the other in the ligand free open form. In the open
form the protein is stabilized by the interaction of the two domains and the binding cleft
is available to acquire the ligand. The two domains contain residues from both the amino
and carboxyl portions of the chain and in general the N terminal lobe is larger then that C
terminal. Each of the domains contains a α/β topology in which a central β-sheet is
surrounded on each side by two or three α-helices. Three short peptide segments connect
the two domains and without a ligand they are approximately 18 Â apart.
Figure 1a. The tertiary structure of the
Figure 1b. The tertiary structure of
LIV protein.
The LS protein.
When a ligand is present in the binding site the two domains move closer and through the
formation of hydrogen bonds the structure is stabilized. The ligand is then forced into the
interior of the protein structure preventing further interactions between it and the
surrounding solvent. When the protein is in the closed from there is a low energy barrier
when the protein reaches the membrane proteins for removal of the ligand and
subsequent transfer of it across the inner membrane. The mechanism for the ligand
specificity and the subsequent transportation is worthwhile to study.
As mentioned previously there are many similarities in function, size, shape and
method of ligand interaction of the periplasmic binding proteins. Most of them have
vastly different homologies yet still exhibit the same tertiary structure. The receptors
vary in size yet still appear to fold to the same tertiary structure making them good
candidates to study the folding and unfolding characteristics. LIV and LS are a
convenient pair to study because they have similarities of their homology of about 80%.
Both their structure and the mechanism are similar yet their specificities vary vastly. It is
therefore important to determine why their specificities are so different and how it relates
to structural constraints.
Quartz crystal microbalance
We can study the binding characteristics of these proteins through the use of a
quartz crystal microbalance (QCM). In 1982 the discovery of thickness shear mode
resonators in fluids associated with chemical reactions lead to the use of the QCM
process in bioanalytical applications. The QCM may be applied for use as a biosensor.
A biosensor can be explained basically as an analytical device that combines the use of
specific biological and physical elements. The biological element creates the recognition
event and the physical element perceives the event.
QCM’s allow for the quantitative determination of the total amount of mass that
ban be adsorbed nonspecifically to the active surface of crystals. Biomolecules are
binding to a metal at the quartz crystal surface. In this case the metal used is gold (Au).
The advantages of using a QCM include its ability to adapt for different uses by forming
coatings that will respond specifically to various target molecules. Its versatility is
important along side of the machines ruggedness, low power, small size and capability to
show direct chemical sensing in liquids.
Figure 2. A schematic diagram of a quartz crystal microbalance.
QCM’s are piezoelectric thickness shear mode resonators The QCM detects
frequency shifts resulting from changes in on the surface of a piezoelectric crystal. The
resonant frequency changes as a linear function of the mass of material deposited on the
crystal surface. In this case the piezoelectric effect is the application of voltage across the
crystal that induces mechanical stress. The sensor response is influenced by surface
roughness, surface charges of absorbed molecules, viscoelastic properties of the adhered
biomaterial, and interfacial phenomena. Also included are changes in stiffness,
conductivity and the dielectric constant of the layer. The QCM follows the principles of
the Sauerbrey equation.
ΔF = ΔM / nc
c – mass sensitivityconstant
n – overtone number
Sauerbrey made the assumption that his equation assumed that acoustic impedance is
identical for the film and quartz. He also assumed that the frequency shift, which was a
result of the mass deposited on the center of the crystal, will be the same regardless of the
radial distance. This proportionality can only hold true if the ideal layer of external mass
is strongly coupled to the resonator.
Methods
In order to study the LS protein binding characteristics certain amino acids need
to be altered to see the effects on the binding of the protein. In this study three mutations
will be made. Two of the naturally occurring cysteines in the protein will be mutated.
The first cysteine to be mutated occurs as the fifty third amino acid in the coding region.
The sequence will be mutated so that the nucleotides code for an alanine instead of a
cysteine. The second cysteine occurs at position seventy eight in the sequence and the
mutation will cause a serine to be coded instead. These two mutations areas will then be
duplicated in the D1C which has already been prepared previously. This mutant changes
the amino acid aspartic acid which occurs the first in the coding sequence to a cysteine.
These mutated LS proteins will then be analyzed.
In order to create these mutations in the sequence site directed mutagenesis needs
to be carried out. In order to do so primers are designed to be used in conjunction with
the polymerase chain reaction (PCR) technique. The wild type LS plasmid is used to
perform the site directed mutagenesis on. Two primers are created to anneal to the
double stranded DNA sequence. One needs to be created to anneal in the forward
direction and the other needs to be created to bind to the reverse sequence. Both the
mutagenic primers must contain the desired mutation and anneal to the same sequence on
opposite strands of the plasmid. The primers are rather short and should be only 23-45
base pairs in length. Other important characteristics that need to be taken into
consideration when designing the primers include that the desired mutation should be in
the middle of the primer, they should have a minimum GC content of 40% and should
terminate in one or more G or C bases. Finally the primers need to have a minimum
concentration of 25nm. The primers were designed manually with the following
sequences
LS C53S - 5’ GGA ATA TGA CGA CTC CGA CCC GAA ACA AGC – 3’
-
5’ GCT TGT TTC GGG TCG GAG TCG TCA TAT TCC – 3’
LS C78A – 5’ CGT TAT TGG TCA TCT CTG TTC TTC TTC TAC CC – 3’
-
5’ GGG TAG AAG AAG AAC AGA GAT GAC CAA TAA CG – 3’
PCR was developed in 1985 by Kary Mullis who was awarded the 1993 Nobel Prize in
chemistry for his work. The purpose of this technique is to make a large number of
copies of the DNA. There are three main steps in a PCR which are repeated for 30-40
cycles. This is done in an automatic cycler which can heat and cool the tubes containing
the reaction mixture in a very short time. The first step is detnaturation and occurs a very
high temperature ~94C. During the denaturation the double stranded DNA denatures
into single stranded DNA and all other reactions stop. The second step is the annealing
of the primers and occurs at ~54C. The primers are moved around in the mixture and
ionic bonds are constantly formed and broken between the single stranded primer and the
single stranded template DNA. The more stable bonds last longer then unstable ones.
Primers that fit exactly to the template will from the most stable bonds and there the
polymerase can attach to begin to copy the template. Once the polymerase has copied the
first few bases the ionic bonds between the template and the primer are very strong and
do not break. The third step is extension and occurs at ~72C. This temperature is ideal
for the polymerase to work. The primers have a stronger ionic attraction to the template
then the forces which would break this attraction. Primers that bind on positions that are
not the right match loosen at this higher temperature and no extension of the fragment is
created. The bases that are complimentary to the template are couple to the primer on the
3’ end. This process is summarized in the following diagram.
Figure 3. A diagram illustrating the three steps of a polymerase chain reaction.
Amplification of the template with the mutation occurs because the three steps are
repeated for between 30-40 cycles.
Figure 4. Illustrates the exponential amplification of a PCR reaction.
After a PCR has taken place the results can be confirmed by DNA gel electrophoresis. A
transformation can then be performed to place the mutated protein sequence into XL 1
competent cells.
Figure 5. An illustration of the steps involved in a transformation.
The wild type LS contains an antibiotic resistance gene to determine which colonies have
taken up the vector successfully. Once positive bacterial colonies are formed the DNA
can be sequenced to confirm that the mutations are present. One colony is taken from the
antibiotic containing plate and placed in a starter culture via a flame sterilized wire loop.
The starter culture is grown in LB media also containing an antibiotic, in our case
ampilicin, and is placed in a shaker incubator overnight. Contamination is an issue so a
control tube is prepared containing the media and antibiotic but no colony. A miniprep
DNA purification system can used to obtain the plasmid DNA and then it is sent out for
the sequencing confirmation. Once the presence of the mutation is confirmed protein
production can occur.
These steps are repeated for each of the desired mutations and in
the case of a double or triple mutation the plasmid already contain one or two of the
mutations are added along with the primers in the PCR phase to add the second or third
mutation to the plasmid. Once protein production occurs for all three mutations binding
analysis can begin. At this stage in the research the primers have been designed, as
shown above, and the site directed mutagenesis is in the stages of completion.
Timeline
March 25th – LSC53S mutant finished
April 15th – LSC53AC78A mutant finished, LSD1CSC53AC78A mutant finished
April 30th – Data collected for binding analysis
September 30th – Thesis outline complete
November 30th – Thesis 1st Draft complete
December 30th – Thesis final draft complete
Feb-March – Thesis presentation
References
1. Janshoff, Galla, Steinem, Piezolelectric Mass Sensing Devices as Biosensors – An
Alternative to Optical Biosensors? 2000
2. Nomura and Okuhara, Determination of lead absorption of the extracted 8
quinolinoluate on the electrodes of a piezoelectric quartz crystal, Anal. Chem, 1982
3. Sauerbrey, G.Z., 1959, Use of Quartz Vibration for Weighing thin Films on a
Microbalance
4. Kasemo, B. Lausma, J CRC Crit. Rev. Biocorpat, 1986