Banerjee

The Effect of Mutations in the

Ligand-Binding Pocket on the
Function of Ftz-F1

Sumona Banerjee
Niles North High School

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Table of Contents
Title..........1

Table of Contents.....2

Acknowledgements..........3

Purpose.........4

Hypotheses...........5

Variables.......6

Review of Literature........7

Materials....................................21

Procedure...27

Results........41

Data Analysis & Discussion .....53

Experimental Error.....58

Conclusion.....60

Impact................................62

References..........64

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Acknowledgements

I would like to express my profound appreciation towards Professor Ishwar

Radhakrishnan, Ph.D., at Northwestern University, for allowing me to conduct independent

research in his laboratory this year. Dr. Radhakrishnan is currently studying the structure and

biology of macromolecular complexes in transcription regulation, specifically the mechanisms

involved in the context of cAMP signaling mediated by CREB and its coactivators.

I would also like to thank graduate student, Mr. Nicolas Daffern, for providing a large

portion of his time to mentor me and share his expertise in order to help me complete my

research. With his guidance and support, I was able to conduct a higher level of research than I

have completed in previous years.

In addition, I would like to thank Mrs. Christi Camel, my STEM Inquiry and Research

teacher for mentoring me throughout this project as well. Her encouragement and optimism has

helped foster my love in science. I would also like to thank Mrs. Georgia Taxakis for providing

insight in my statistical analysis and experimental design. Lastly, I would like to acknowledge

my friends and family for their assistance and encouragement.

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Purpose

Nuclear receptors are a family of transcription factors that have evolved to regulate a

broad range of fundamental biological processes. While most are regulated by ligands, a subclass

of nuclear receptors -- orphan receptors -- have been found to have no known ligands and instead

function by the active conformation of their ligand binding pockets. This project focuses on the

orphan receptor Drosophila Ftz F1.

In this project, there are two fundamental purposes. The first is to characterize what

effects do mutations in the ligand binding pocket have on the stability of Ftz F1. The second is to

examine whether certain mutations affect the way in which Ftz F1 binds to its coactivator Ftz

through its LxxLL motif. The implications of this study is greatly applicable to drug therapy.

Because nuclear receptors are controlled by a ligand, the way in which these ligand binds to its

nuclear receptor affects the way in which their target gene is expressed. Therefore, the

engineering of these ligands is highly advantageous as it allows for the down regulation of

certain genes correlated with disorders such as cancer, diabetes, arthritis etc.

Studying Ftz F1, specifically, would help create a prototype for ligand binding pocket

engineering as this orphan receptor does not function by a ligand. Determining the way in which

Ftz F1 destabilizes and thus down regulates transcription would also translate into creating a

synthetic binding pocket to down regulate abnormal steroidogenesis, which is a biological

process by which vital steroids are generated from cholesterol. It would also gain insight as to

how the pocket may be synthetically mutated to down regulate intestinal, liver, ovarian and

pancreatic cancer.

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Hypotheses

Hypothesis I: If Ftz F1s ligand binding domain is mutated, then the generated mutations will be

able to bind to its coactivators LxxLL peptide and remain stable. This is because van der Waals

forces play key roles in protein to protein recognition when complementary macromolecules are

involved. Although mutations of Ftz F1s ligand binding domain are being studied, these forces

should still allow for complementary proteins such as Ftz F1 and Ftz to be more stable when

bound together.

Hypothesis II: If the stability of the mutant protein plus peptide samples are compared to the

wild type plus peptide samples, then the G913K + peptide, G913Y + peptide, and L907F +

peptide samples should be more stable than the wild type + peptide sample. This is because the

increase in amino acid mass from glycine to lysine (G to K mutation) and glycine to tyrosine (G

to Y mutation) should decrease the speculated movement of helix 6 -- in and out of its ligand

binding pocket -- and stabilize the area. The increase in amino acid mass from leucine to

phenylalanine (L to F mutation) should slow down the dynamic behavior in the ligand binding

pocket and increase the stability of the protein.

Hypothesis III: If the stability of the mutant protein plus peptide samples are compared to the

wild type plus peptide samples, then the L907V + peptide samples should be less stable than the

wild type + protein sample. This is because the decrease in amino acid mass from leucine to

valine (L to V mutation) should increase the dynamic behavior in the ligand binding pocket and

thus destabilize the area.

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Variables

Independent Variable: Mutations of Ftz F1s ligand binding domain

G913K
G913Y
L907W
L907F
L907V
L907A
Dependent Variable: Melting point of individual mutant proteins, melting point of mutant

proteins bound to coactivator Ftz (measured in Tm through a thermal shift assay)

Control: Wild Type Ftz F1

Constants: 200 Microliters (L) of 1.8M protein sample, 2L of 100x loading dye into each

protein sample, 1 L of 9.2M LxxLL, plasmid composition, equipment used, experimental

environment, time and temperature in centrifuge, time and temperature in thermal shift assay.

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Review of Literature

As scientific research progresses, society has experienced a myriad of breakthroughs in

medicine, technology, engineering and beyond. Although these innovations have resulted in

significant societal progress, many fundamental questions regarding molecular substances are

still left unanswered. Nuclear receptors are a class of proteins that play key roles in the growth,

development, and homeostasis of an organism. Although ligands, which aid in the regulation of

gene expression by nuclear receptors, greatly affect the receptors behavior, about half of the

known nuclear receptors in humans have no known ligands (Taubert et al., 2011). The ancestral

nuclear receptor is widely thought to be ligand independent and ligands later evolved as a mean

of tuning the process of transcription. Studying the functionality of ligand deficient nuclear

receptors is necessary in order to gain insight on how these receptors aid in various

macromolecular processes such as cell transcription and translation and how they are targeted for

human benefit such as medicine.

DNA, RNA and Transcription Factors

Deoxyribonucleic acid, or DNA, is a genetic material found in humans and almost all

other organisms. Most of the DNA is located in the nucleus and is composed of four chemical

bases: adenine (A), guanine (G), cytosine (C), and thymine (T). The sequence of these bases

specify unique information being coded for such as hair or eye color, for example. When

sequencing DNA, each base is paired with each other, A with T, and G with C, and is supported

by a sugar-phosphate backbone. Together, the base, sugar, and phosphate is called a nucleotide.

Furthermore, several nucleotides bound together form a double helical structure with the

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nitrogenous bases pointed inward as the rungs of the ladder and the sugar-phosphate bonding

creating the vertical sidepieces. (Mandal, 2014)

DNA replication is a vital process in the cell cycle allowing for the formation of two

identical strands of DNA. During DNA replication, the parent molecule is separated by an

enzyme called helicase. Once unraveled, the two strands, called the three prime and five prime,

serve as a template for the complementary set of bases being synthesized onto each strand. As

shown in figure 1, copying occurs at a localized region called the replication fork and is aided by

DNA polymerase. When separating from the helix shape, the three prime strand is diverted to a

DNA polymerase where the enzyme guides the replication of the first daughter DNA helix by

adding on complementary base pairs to the original strand. The five prime strand, however,

emerges from the separation as sections called Okazaki fragments. DNA polymerase directs the

replication while DNA ligase I stiches the segments together. When the process is complete, two

identical daughter strands have been assembled and the next loop is drawn back for replication.

(Kimball 2015)

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While DNA is being replicated, a single strand of messenger RNA or mRNA is

simultaneously created in order to used in the process of translation. RNA polymerase binds to

the three prime end of a DNA strand and assembles ribonucleotides in a separate growing RNA

strand. The ribonucleotides are inserted following the base pairing mechanism. However, T is

substituted with U or uracil. This process is called transcription and results in a reverse

complement of a DNA strand. (Transcription, n.d.) Once the RNA strand is created, it is edited

through RNA splicing. Non-coding regions, or introns, are removed by an enzyme called the

spliceosome and coding regions, or exons, are linked together. (RNA Splicing, n.d.)

Transcription factors are proteins that bind to specific DNA sequences through a

DNA-binding domain. They are essential for the regulation of gene expression and include an

array of proteins that initiate and regulate the transcription process. (Transcription Factor, 2014)

Some transcription factors bind near the transcription start site and help form the transcription

initiation complex. Others, however, bind to regulatory sequences, such as enhancer or silencer

sequences and can either promote or repress the transcription of the relevant gene. (Rangarajan,

2011) General transcription factors are required for the initiation of RNA synthesis and

determine the site of initiation. Upstream factors are ubiquitous and increase the efficiency of

initiation. Inducible factors have a regulatory role and are activated at specific times and in

specific areas. (Huret, 2011)

There are several mechanisms in which a transcription factor may allow for the up- or

down- regulation of a gene. They may recruit a coactivator or corepressor protein in order to

enhance or diminish the effects of a gene. (Xu, 1999) They may also catalyze either the

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acetylation or deacetylation of histone proteins. Histones are positively charged proteins that

condense negatively charged DNA into units called nucleosomes. When acetylating histone

activity, the association between DNA and the histones is weakened allowing for the DNA to be

more accessible to transcription, thus up-regulating transcription. When deacetylating histone

activity, the opposite occurs, making the DNA less accessible and thus down-regulating

transcription. (Narlikar, 2002) The third most prevalent mechanism is stabilizing or completely

blocking the binding of the RNA polymerase to the DNA. (Gill, 2001)

After the process of transcription, translation is initiated by an AUG start codon. As the

mRNA is read in the elongation phase of translation, transfer RNA or tRNA carry amino acids

being specified by each frame of three bases

called a codon. Once the area of interest is done

being translated for, one of three possible stop

codons, UAA, UAG, or UGA, are employed.

The mRNA sequence is thus used as a template

to construct an amino acid chain held by peptide

bonds to later create a protein. (Clancy, 2008)

The resulting protein may have various

different functions. Structural proteins are

building materials, while transport proteins carry

substances such as oxygen rich blood to distinct

regions in the body. Enzymes are proteins that speed up a reaction by providing an alternative

path to overcoming activation energy. (Pal, n.d.) The particular sequence of amino acids in the

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chain creates distinction between individual proteins and determines the functionality of the

protein produced. (Freudenrich, n.d.) Furthermore, as shown in Figure 2, there are four possible

structures for a protein. The most basic structure is the primary structure and is made up of at

least thirty amino acids. These amino acids are covalently bonded by peptide bonds and are

supported by a backbone consisted of nitrogen, oxygen and hydrogen. They are arranged in a

linear fashion and are composed of a central carbon. The secondary structure includes two main

types dependent on hydrogen bonding. The -helix is composed of hydrogen bonds formed

between the oxygen of the C=O in each peptide bond and the hydrogen in the N-H group, as

shown in figure 2. This pattern of bonding pulls the amino acid chain into a helical structure that

resembles a curled ribbon. (Khan, 2015) In the -sheet two or more strands line up next to each

other forming hydrogen bonds between the strands rather than within. The hydrogen bonds form

between the same molecules as the -helix, however they shape the structure into a pleated sheet

that can be shown in figure 2. (Protein Structure, n.d.) The tertiary structure is the overall

three-dimensional structure of a protein which results from a large number of non-covalent

interactions between the amino acid chains. Many proteins are only made up of the first three

structure, however, some proteins are made up of the quaternary structure as well which binds

multiple polypeptides into a single, larger protein.

DNA Mutations and PCR

A mutation is a change that occurs in the DNA sequence resulting in changes in the

proteins that are made. These mutations can occur during DNA replication and may be caused

simply by a copying error or environmental factors such as UV radiation (DNA and Mutations,

2008). There are several types of DNA mutations that may occur, the most prevalent being

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substitution. A substitution is a mutation that exchanges one base for another such as switching a

T for a G. This change may result in a different amino acid being coded for. An insertion

mutation occurs when extra base pairs are inserted into the DNA strand and deletions are

mutations in which a section of the DNA is deleted. Since DNA is divided into codons that are

three base pairs long, an insertion or deletion mutation may shift the way the codons are parsed

and subsequently alter gene being coded for (DNA and Mutations, 2008).

Scientists often use mutations to analyze the

function and behaviors of proteins. Site directed

mutagenesis is a versatile technique that can be used

to introduce specific nucleotide substitutions using

customized primers. This may be done through a

polymerase chain reaction or PCR in which the PCR

protocol amplifies the entire plasmid template along

with the mutation. Primers are designed with the

point mutation and attach onto the two strands of

DNA once they are separated in the PCR reaction.

As PCR progresses, the mutated DNA generated is itself used as a template for replication, thus

resulting in a chain reaction in which the mutated DNA is exponentially amplified. As shown in

figure 3, once PCR is complete, enzyme DpnI is used to remove the parent template from the

generated DNA samples and the mutated DNA is then transformed using bacteria so that any

nicked plasmid may be patched together. Afterwards, the DNA is sequenced to confirm that the

desired modification is present in the generated DNA (Laursen, 2016).

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Protein Stability

Noncovalent bonding is a type of chemical interaction that aids in the bonding between

macromolecules, specifically proteins and nucleic acids. These interactions arise via three main

mechanisms: hydrogen bonding, van der Waals interactions and hydrophobic interactions

(Gorga, 2007). Hydrogen bonding is an electrostatic attraction between hydrogen and either

nitrogen, oxygen or fluorine. Its forces play an important role in the formation of the secondary

structure of a protein, as alpha helices are hydrogen bonded internally and beta strands are

hydrogen bonded to one another. Because hydrogen bonding results in dipole-dipole interactions

that are directional, bonds must be aligned in a certain manner in order to be favorable.

Consequently, this alignment plays a key role in determining the unique structures that different

proteins form and how proteins bind to other macromolecular structures (Protein Folding, 2006).

Van der Waals interactions exist between non polar substances forming temporary weak

dipoles. Although these interactions may be transient and weak, they provide an important

component to protein structure since they are abundant and packed sufficiently close together.

Van der Waals forces also play important roles protein-protein recognition when complementary

shapes are involved. These forces allow for complementary proteins such as ligands and

coactivators to be more stable when bound together (Marcey, 2001).

When the hydrogen bonding and van der Waal forces of proteins are studied together and

are surrounded by water, this creates a hydrophobic effect. Since it is unfavorable for water

molecules, containing hydrogen bonds, to organize themselves around a nonpolar surface,

nonpolar side chains of proteins bury themselves in the interior of their protein structure. This

burying allows for the water molecules to bond without becoming excessively ordered, thus

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decreasing the energy of the system and restoring equilibrium. Therefore, an important factor

governing protein structure and they way a protein folds is the distribution of its polar and

nonpolar amino acids (Protein Folding, 2006).

The stability of a protein is also related to its melting point; a protein with a higher

melting point will be more stable than a protein with a lower melting point due to the idea of

bond enthalpy. A protein that is more stable will require a greater amount of bond enthalpy

which is the energy required to break a chemical bond. Thus, the point at which its bonds break,

its melting point, will be higher as well. This may be studied through a Thermal Shift Assay in

which a dye is reacted the hydrophobic core of a protein while the temperature of the

surroundings are gradually increased. As the temperature increases, the protein should unfold

and the amount of fluorescence should increase. The midpoint of the fluorescence versus

temperature curve should signify the proteins melting point and correlate to its stability (Maksel,

n.d.).

Nuclear Receptors and Orphan Receptors

As mentioned earlier, nuclear receptors is a class of transcription factors that play many

important roles in eukaryotic development. Primarily, they help regulate the expression of

specific genes by directly binding to DNA. Proteins of the nuclear receptor super-family are

single polypeptide chains with two major structural domains: a DNA-binding domain and a

ligand-binding domain (Hooper, n.d.). The N-terminal region of the nuclear receptor contain

transcriptional activation functions, known as activation function-1 (AF1). The DNA binding

domain, responsible for targeting the receptors to the mRNA sequence, and the Ligand Binding

Domain are linked via a flexible hinge region, as shown in figure 4. The hinge region influences

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intracellular trafficking and localized nuclear signals. Nuclear receptors also contain a C-terminal

which differ in function depending on the specific receptor (Kosztin, 2006).

Ligands are small structures used to regulate nuclear receptors. These ligands tend to bind

to the ligand-binding domain and control the interactions of the receptors with coactivators and

corepressors thereby influencing the conformation or the spatial shape of short helical structures

near the C-terminal end (AF-2 domain). Ligand binding simultaneously displaces corepressors

and allows for the recruitment of coactivators. As ligands create conformational change when

regulating the nuclear receptor, this change allows for the recognition of a specific motif

contained within the coactivator protein (Savkur, 2004). A structural motif is a pattern in a

protein structure formed by the spatial arrangement of amino acids. This motif is known as the

NR box or LXXLL. L can be defined by an amino acid called leucine and X can be defined

by any amino acid. The recruitment of coactivator complexes and thus the process of

transcription is dependent on the interaction of the ligands with the AF-2 domain and LXXLL

motifs in the coactivators (Plevin, 2005).

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Moreover, ligands function as gene-specific modulators by mediating the nuclear

receptors recruitment of coactivators and corepressors. The chemical structures and

functionalities presented by ligands can be translated into a broad spectrum of agonist, partial

agonist and antagonist outputs in terms of gene responses. As an agonist, ligands push to

up-regulate or increase the expression of the gene through the nuclear receptors recruitment of

stronger coactivators. As an antagonist, the ligand blocks the expression and thus down-regulates

in the same way. The mechanism underlying this type of gene selectivity is thought to be

cell-specific and reflective of the ratio of coactivators and corepressors present near the site

(Huang et al, 2013).

Orphan receptors is the third class of nuclear receptors which do not contain an apparent

ligand. In orphan receptors, the activation of gene expression and transcription are performed

independent of a ligand. When looking at the phylogenetic tree of the nuclear receptor

superfamily, orphan receptors are not clustered in a specific subfamily. They are instead spread

throughout the whole tree. This can be explained by the increase of ligand-binding capacity

several times during nuclear receptor evolution. However, this may also imply that the ancestral

nuclear receptor was orphan and thus a ligand was not necessary for its activation (Escriva et al,

2004). Although it is now well accepted that signals other than ligands may regulate nuclear

receptors, it remains unclear how many orphan receptors are actually ligand-independent and if

there are ligands yet to be identified.

Ftz F1 and Nuclear Receptor 5A Family

The Drosophila melanogaster is a fruit fly that has been used as a model organism for

research for several years. It also is one of the most valued organisms used in biological research,

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particularly in genetics and developmental biology (Manning, 2008). The Drosophila fushi

tarazu transcription factor 1 (Ftz F1) is an orphan receptor belonging to the Nuclear Receptor

5A family (NR5A), a family whose receptors are found in a diverse range of invertebrates and

vertebrates. The NR5A family binds to DNA as monomers and contains a C-terminal extension

to their DNA binding domains allowing for DNA-binding specificity. Although the receptors

stem from the same sub family of receptors, their individual interactions with other proteins

influence their in vivo functionality (Pick et al, 2006).

Studies on Ftz-F1 have revealed its protein-protein interaction with Ftz. The Ftz/Ftz-F1

interaction imitates the interactions between other nuclear receptors and standard coactivators as

Ftz contains an LXXLL motif. In addition, this protein-protein interaction plays a key role in the

embryonic development of the Drosophila (Pick et al, 2006). As shown in the research of Heffer

et al, this interaction promoted the evolution of Ftz from a homeotic gene, a gene that regulates

the development of anatomical structures, to a segmentation gene (Homeotic Genes and Body

Patterns, 2007). As a result, Ftz-F1 in the modern day Drosophila acts as an encoder for

segmentation expressions such an engrailed (en), wingless (wg), etc (Heffer et al, 2013).

Orthologs are genes in different species that evolved from a common ancestral source

(Lewis, n.d.). Two mammalian orthologs of Ftz-F1 are SF1 (steroidogenic factor 1) and LRH1

(liver receptor homolog 1), and they are most commonly found in mice and humans. SF1 is

normally expressed in reproductive organs and influences the development of steroidogenesis,

the synthesis of cholesterol, and sexual differentiation. LRH1, which is usually expressed in the

ovaries and the digestive system, aids in cell proliferation and embryonic development. It also

helps control bile acid levels and the transportation of cholesterol (Yussa et al, 2001).

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As shown in figure 5, the structure of the pocket differs in the mouse model, human

model, and Ftz-F1 model. In the mouse model, the ligand binding pocket is empty but the AF-2

helix is stabilized in an active conformation. AF-2 already being locked into an active

conformation verifies the idea that a ligand is not necessary for LRH1 regulation; being

ligand-deficient, the structure is able to signal spatial conformation through its own mechanism.

When looking at the human model of the LRH1, there is a significant amount of phospholipids

present in its lipid binding pocket.

Recent studies have revealed that the lipid binding pocket of the Drosophila Ftz-F1

contains a segment of a protein called helix 6, preventing any ligand from binding to the pocket

and allowing the AF-2 helix to be locked in active conformation as well (Yussa, 2001). Although

these features are consistent with an orphan receptor, it is unclear what role helix 6 exactly

performs for Ftz-F1 and what effects do mutations on the ligand binding pocket have on its

transcriptional activity. Since macromolecules, including proteins, are not static substances, their

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ability to conform and engage with other macromolecules is a direct implication in their

biological function. Through nuclear magnetic resonance spectroscopy (NMR), scientists are

able to study the dynamics of macromolecules and compare the changing dynamics in similar

structures (Yussa 2001). Recent NMR studies suggest that the helix 6 in Ftz-F1 located in the

ligand binding pocket exhibits both fast and slow dynamics. There is also speculation that a

certain area in helix 6 may be popping in and out of the ligand binding pocket. However, it is

unsure how this dynamic behavior affects transcriptional activity (Radhakrishnan 2016).

Currently, nuclear receptors are widely used as a drug target as they are controlled by a

ligand. Creating mutations of ligands have been shown to affect the way in which binding occurs

in its nuclear receptor and thereby the expression of its target gene. As of now, thirty four of the

top two hundred most prescribed drugs target nuclear receptors, however, there is minimal

research being done to study orphan receptors. It is unclear how to synthetically mutate their

ligand binding pockets and what effect do these mutations have on their natural stability (Moore

et al. 2006).

Both LRH1 and SF1, the orthologs of Ftz F1, regulate steroidogenesis, Steroidogenesis is

a vital biological process that regulates the creation of steroids from cholesterol. These steroids

may include progestogens, which helps maintain pregnancy in women, corticosteroids, which

function as an immunosuppression, and androgens, which contribute to the development and

maintenance of female and male secondary sex characteristics. Often times, the body may over-

or under- produce these hormones, resulting in fatal disorders such as polycystic ovary syndrome

or depression (Fayard et al., 2004). LRH1 is also involved in the expression of cell proliferation

in the intestine, liver, endocrine pancreas as well as the ovaries. Cancer occurring in any of these

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areas may be the result of erroneous cell proliferation, and there is little readily available

treatment for cancer of any of these sorts (Fayard et al., 2004).

The goal of this study is to create mutations of Ftz-F1 by changing the dynamics of the

ligand binding pocket through site directed mutagenesis and determine how do these mutations

affect the stability of Ftz F1 and its binding ability to its coactivator Ftz. By creating mutations of

the structure, we are able to study the relationship between the ligand binding pocket and

transcriptional activity and apply these finding to larger scale functions such as drug therapy and

cancer treatment. Specifically, we are able conclude ways in which the ligand binding pocket of

Ftz F1 may be mutated to down regulate the expression of its target genes. This may then be

translated onto the human model, LRH1, to create a drug specific and inexpensive method to

treat these various cancers and steroid dependent disorders with.

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Materials
Mutants:
Ftz F1 Ligand Binding Domain (785-1027)
G913K
G913Y
L907W
L907F
L907V
L907A

Primers
G913K Sense
G913K Antisense
G913Y Sense
G913Y Antisense
L907W Sense
L907W Antisense
L907F Sense
L907F Antisense
L907V Sense
L907V Antisense
L907A Sense
L907A Antisense

PCR Reaction Materials:

Equipment: Perkin Elmer GeneAmp PCR System 2400
6 thin-walled PCR tubes
0.5 ug of each primer
0.5 of each antisense primer
1uL of Ftz F1 Ligand Binding Domain Template
5 uL 10X thermopolymerase reaction buffer
1 uL pfu turbo DNA-polymerase
37 uL sterile-filtered UV/UF H2O
5 uL 2.5 dNTPs

Gel Electrophoresis Materials:

Equipment: Electrophoresis chamber and power supply

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Transilluminator (ultraviolet light box)

Hot plate
0.5 g 1% agarose
50 mL 1X TAE buffer (Tris-acetate-EDTA)
5 uL ethidium bromide (10000x dilution)
5 uL of each PCR samples
8 uL 6X loading dye
Gel casting trays
Sample combs
Loading buffers
1X Anode
1X Cathode
6 epi tubes
5 uL 1Kb Ladder
5 uL 100 base pair Ladder

DPN 1 Cleavage Materials:

Equipment: Perkin Elmer GeneAmp PCR System 2400
10.8 uL DPN I enzyme
45 of each PCR sample

Ethanol Purification Materials:

Equipment: Tabletop Centrifuge
Incubator
Vortex Genie 2
150 uL 7.5M NH4CH3Co2 (NH4OAc)
47 uL reaction sample from DPN 1 Cleavage
6 epi tubes
900 uL 100% isopropanol
6 mL 80% Ethanol (EtOH)
Argon gas tank
60 uL UV/UF H2O

QIAprep Spin Miniprep Materials:

Equipment: Tabletop Centrifuge
Qiagen QIAprep Spin Miniprep Kit
250 uL Buffer P1
250 uL Buffer P2
350 uL Buffer N3

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0.5 mL Buffer PB
0.75 mL Buffer PE
50 uL Buffer EB (10 mM Tris-Cl, pH 8.5)
Microcentrifuge tube
QIAprep 2.0 spin column
5 mL DH5a cells

Sequencing Materials:
3 uL of each bacterial sample
2 uL of each mutant primer
42 uL UV/UF H2O
6 epitues

Transformation into BL21 E. coli Cells Materials:

Equipment: Heat Block
Sorvall Incubator/Centrifuge
Incubator
BL21 E. coli cell line
900 uL S.O.C. media (2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10
mM MgCl2, 10 mM MgSO4, and 20 mM glucose)
1.5 uL of each mutant plasmid
6 LB Agar plates with Carbenicillin
Ice

Liquid Media Culture Preparation Materials:

Equipment: Autoclave
Sorvall Incubator/Centrifuge
25g premixed LB agar powder
Antibiotic
Aluminum foil
Bacterial colonies grown on LB Agar plates with Carbenicillin
6 graduated cylinders
6 mL Carbenicillin

Cell Optical Density Measurement Materials:

Equipment: Jasco Spectrophotometer
Sorvall Incubator/Centrifuge
100 uL cuvette
100 uL UV/UF H2O

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100 uL of each bacterial sample in liquid media culture

6mL Isopropyl -D-1-thiogalactopyranoside (IPTG)

Cell Lysis Materials:

Equipment: pH Meter
Digital Sonicator
Sorvall Incubator/Centrifuge
23.88g 200mM NaCl
12.11g 50mM Tris
0.29g of 0.5mM TCEP
140 uL 1mM BME (2-Mercaptoethanol)
2L UV/UF H2O
6N HCl
39mL pre-lysis buffer
0.01 g Imidazole
40uL 0.1% Triton-X
Protease Inhibitor
Microcentrifuge tubes

Wash and Elution Buffer Dilution Materials:

Equipment: pH Meter
0.0136g 5mM Imidazole
0.6808g 250mM Imidazole
80 mL pre lysis buffer
HCl

Protein Purification Materials:

2 mL chemical resin
Ring stand
10mL mL of each soluble supernatant
20 mL Wash Buffer
40 mL Elution Buffer
4 flow through tubes
16 tubes
GnHCl

Gel Electrophoresis Materials:

Equipment: Electrophoresis chamber and power supply
Hot plate

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Scanner
32 uL 6X loading dye
Gel casting trays
Sample combs
Loading buffers
1X Anode
1X Cathode
32 epi tubes
12 uL Tricine Ladder
20 uL of each total cell lysate sample
20 uL of each soluble supernatant sample
20 uL of each flow through sample
8 uL of each wash buffer sample
4 uL of each elution 1 sample
4 uL of each elution 2 sample
4 uL of each column sample
Coomassie Stain

His Tag Cleavage and Subtractive Purification Materials:

Equipment: Electrophoresis chamber and power supply
20M NaPO4
50M NaCl
1 mM TCEP
Resin
Dialysed Buffer
Molecular Weight Ladder
Tubes

Thermal Shift Assay Dilution Materials:

12.8 uL GY sample
10.9 uL GK sample
16.4 uL LV sample
41.9 uL LF sample
11.2 Wild Type sample
933.8 uL dialysis buffer
3uL loading dye
5 uL of 9.2uL LxxLL peptide
Tubes

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Thermal Shift Assay Materials:

Equipment: Biorad 600 Instrument
Well Plate Centrifuge
96 well plate
10 of each sample prepared in dilution step

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 Banerjee

Procedure

1. Use PCR mutagenesis to introduce mutations in Ftz F1 ligand binding domain

a. For each of the mutants, combine in a thin-walled PCR tube the following: 0.5g

of the appropriate sense primer, 0.5g of the appropriate antisense primer, 1L

template, 5L 10X thermopolymerase reaction buffer, 1L pfu turbo,

DNA-polymerase, 37L sterile-filtered UV/UF H2O to bring total volume to 45

b. Place tubes in PCR box. Once thermocycler heats up to 95oC, pause program and

add 5L 2.5 dNTPs

c. PCR settings:

Cycles 18

Dissociation Temperature and Time 95C / 50sec

Annealing Temperature and Time 55C / 50sec

Elongation Temperature and Time 68C / 6min

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 Banerjee

2. Run Gel Electrophoresis to see if length of all mutant plasmids are at the same length as

original plasmid.

a. Prepare an agarose solution with 0.5g agarose in 50 ml TAE. Heat the solution to

boiling in a hot plate and stir until agarose is dissolved. When molten gel has

cooled, add 5L of ethidium bromide into the solution. Mix the gel solution by

gentle swirling.

b. Gently pour the solution into the gel plate. The gel should be between 3-5 mm

thick. Press two sample gel combs onto each sides of the plate. Allow the gel to

set completely for 20-30 minutes.

c. While waiting, prepare 5L samples of PCR samples with 1L 6x loading dye.

Load prepared samples into 6 epi tubes (1 tube/protein).

d. When gel has hardened, carefully remove the combs and mount the plate into the

electrophoresis tank. Add just enough of the loading buffers to cover the gel to a

depth of approximately 1 mm.

e. Slowly pipet the protein samples into the submerged gel along with a 5L 1Kb

Ladder and 5L 100 base pair Ladder into gel plate using a micropipette. 1L of

6x loading dye should be mixed with ladder samples as well creating a total

volume of 6L.

f. When samples are loaded, close the lid of the gel tank and attach the electrical

leads so that DNA will migrate towards the positive anode. Apply a voltage of

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 Banerjee

90V/cm for two hours. If the leads have been attached correctly, bubbles should

be generated at the anode and cathode.

g. After two hours, remove the electrical leads and gel plate. Image the gel plate

with a transilluminator (UV imager).

Map of Gel Plate:

1Kb Ladder G913K G913Y L907W L907F L907V L907A 100 base pair Ladder
6 L 6 L 6 L 6 L 6 L 6 L 6 L 6 L

3. DPN I Cleavage: To extract Ftz F1 ligand binding domain mutant plasmid,

a. Add 0.9L of DPN to 45L of each protein PCR sample. Place samples into the

PCR machine at 37oC for 90 minutes

b. Add another 0.9L of DPN into each sample. Run the PCR for another 60

minutes at the same temperature.

4. Ethanol purification: To separate protein from cleaved mixture,

a. Add 25L of 7.5M NH4CH3Co2 (NH4OAc) into each 47L DPN I reaction

mixture. Gently mix the liquid and transfer each solution into a 1.5mL epi tube,

preferably clear, not frosted for maximum visibility. Pipet 150L of 100%

isopropanol to each epitube.

b. Incubate the samples at room temperature for 10 minutes.

c. Centrifuge all the samples at 14000 revolutions per minute for 10 minutes in

tabletop centrifuge. While visualizing the pellet on the bottom of the tube,

carefully pipet off the supernatant.

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 Banerjee

d. Wash the pellet by adding 1 ml of 80% of EtOH. Vortex gently to dislodge the

pellet.

e. Centrifuge all the samples again at 14000 revolutions per minute for 10 minutes.

Discard of supernatant and last traces of 80% EtOH as before until 5-10L

remain.

f. Gently air dry the the remaining supernatant for 5 minutes with argon gas.

g. Add 10L of sterile filtered UV/UF H2O into each tube and resuspend the pellet.

5. QIAprep Spin Miniprep: Transformation of DNA into DH5a cells.

a. Pellet 5 ml bacterial overnight culture by centrifugation at 8000 rpm for 3 min at

room temperature (1525C). Resuspend pelleted bacterial cells in 250l Buffer

P1 and transfer to a microcentrifuge tube.

b. Add 250l Buffer P2 and mix thoroughly by inverting the tube 46 times until the

solution becomes clear. Do not allow the lysis reaction to proceed for more than 5

min.

c. Add 350l Buffer N3 and mix immediately and thoroughly by inverting the tube

46 times.

d. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge.

e. Apply 800l supernatant from step 5e to the QIAprep 2.0 spin column by

pipetting. Centrifuge for 3060 seconds and discard the flow-through

f. Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for

3060 seconds and discard the flow-through.

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 Banerjee

g. Wash the QIAprep 2.0 spin column by adding 0.75 ml Buffer PE. Centrifuge for

3060 seconds and discard the flow-through.

h. Centrifuge for 1 min to remove residual wash buffer.

i. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube.

j. To elute DNA, add 50l Buffer EB (10 mM Tris-Cl, pH 8.5) to the center of the

QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min.

6. Sequence: Sanger Method

a. Mix 3L of bacteria samples prepared in step 5, 2L of its appropriate primer and

7L of UV/UF H2O in an epi tube.

b. Package and send these samples to an outside lab in order to sequence their DNA.

7. Transformation into BL21 cells: Insert protein into bacteria cells by shocking the cells

a. Carefully remove BL21 cells from -80oC freezer and thaw on ice for

approximately 20-30 minutes. Remove SOC media from storage and let it thaw to

room temperature (1525C).

b. Pipet 1.5L of each mutant plasmid into each aliquot. Gently mix by flicking the

bottom of the tube a few times. Then place back on ice for approximately 30

minutes.

c. Heat shock each transformation tube by placing the bottom of the tube into a

heat block at 42oC for 45 seconds. Place the tubes back in ice for 2 minutes.

d. Add 150L of SOC media into each tube and stir gently.

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 Banerjee

e. Place tubes in 37oC shaking incubator at 225 revolution per minute for

approximately 60 minutes. While waiting, remove the LB Agar plates with

Carbenicillin and allow them to warm to room temperature.

f. Remove the tubes from the shaking incubator and pipet 80L of each tube onto a

separate agar plate. Gently spread the transformation using a sterile metal

spreader.

g. Incubate plates inverted at 37oC overnight.

8. Choose a colony and and prepare liquid media culture

a. To prepare for the liquid media culture, weigh out 25g premixed LB agar powder.

Loosely close the cap of the bottle and loosely cover the entire top of the bottle

with aluminum foil. Autoclave and allow to cool to room temperature. After,

close the top of the bottle and store the LB at room temperature.

b. When ready to grow the culture, add liquid LB to a six graduated cylinders and

add 1ml of carbenicillin into each.

c. Using a sterile pipette tip, select a single colony from each agar plate prepared in

step 7. Drop the tip into the liquid LB and swirl. Loosely cover each tube with

sterile aluminum foil.

d. Incubate tubes at 37oC overnight in a shaking incubator. After incubation, check

for growth, which is characterized by a cloudy haze in the media.

9. Measure optical density of cells

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 Banerjee

a. Turn on the spectrophotometer using the switch in the back of the machine. Turn

on the monitor screen and printer. Wait for the machine to go through its startup

routine.

b. Open the lid on the top of the machine. Choose the large cuvette holder and place

it in the holder in front of the light source.

c. Click on the visible light key to turn the light source on.

d. Choose Single Wavelength mode from the menu that appears. Make sure the

wavelength selected is 600 nm.

e. Place a 100 mL blank sample in a cuvette. Place the cuvette in the holder. Shut

the lid of the machine and click Read Blank in the bottom left corner of the

screen.

f. After the machine has read the blank, remove the cuvette, replace it with a cuvette

containing 100 mL of each bacterial sample.

g. Record each bacterial optical density. If OD is not at 0.6, allow samples to

incubate for a longer period of time.

h. Stop incubating once OD reaches 0.6 and add 1ml IPTG into each sample.

10. Cell lysis

a. In order to make the pre-lysis buffer, mix the following: 23.88g 200mM NaCl,

12.11g 50mM Tris, 0.29g of 0.5mM TCEP, 140L 1mM BME, 2L UV/UF H2O

b. Add HCl into pre-lysis buffer under a fume hood until pH reaches 8.5.

c. From this stock solution mix the following to make the lysis buffer: 39mL

pre-lysis buffer, 0.01 g Imidazole, 40L 0.1% Triton-X, Protease Inhibitor

33
 Banerjee

d. Spin cells to be lysed down in 15 mL centrifuge tubes.

e. Pipet off LB liquid media, leaving around 1 mL in each sample examined in step

9. Transfer the 1 mL into a microcentrifuge tube and spin the cells to pellet (spin

for 5-10 seconds at max speed in the microcentrifuge).

f. Resuspend cells in 4 mL of lysis buffer. Place samples under a sonicator in ice

room for 10-15 minutes to ensure complete lysis.

g. Collect 20mL lysed sample and place in separate tubes labeled its mutant name

and total cell lysate.

h. Spin remaining of lysis samples at max speed for 10 minutes at 2-4oC to pellet

nuclei.

i. Pipet off 20mL each supernatant into separate tubes labeled its mutant name and

soluble supernatant. Store each pellet labeled with its mutant name.

11. Wash Buffer and Elution Buffer Dilution

a. To create the wash buffer, mix 0.0136g of 5mM Imidazole in 40 milliliters of the

pre-lysis buffer. In the fume hood, add HCl to solution created until it reaches pH

of 8.5.

b. To create the elution buffer, mix 0.6808g of 250mM Imidazole in 40 milliliters of

the pre lysis buffer. In the fume hood, add HCl to solution created until it reaches

pH of 8.5.

12. Protein Purification

a. Apply a 0.15 (later increased to .5) mL of resin onto the bottom of a each column.

Each tube should be designated towards a specific mutant for purification.

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 Banerjee

b. Obtain 10 mL each soluble supernatant and pipet it through the appropriate tube.

c. Close the cap and gently rock the tube side to side. Place the tube back onto the

ring stand and open the cap. Gently release the flow through into a separate fresh

tube labeled the appropriate mutants name along with flow through.

d. Obtain 5 mL of the wash buffer prepared in step 11 and pipet it through each tube.

e. Repeat step 12c for each mutants tube. But instead, label the tubes with wash

buffer and its appropriate mutants name.

f. Obtain 4 mL of the elution buffer prepared in step 11 as well and pipet it through

each tube.

g. Repeat step 12c for each mutants tube. However, label the tubes with elution 1

and its appropriate mutants name.

h. Repeat steps f and g for each mutants tube. Label the tubes with elution 2 and

its appropriate mutant name.

i. Obtain 4 mL of the elution buffer and pipet it through each tube.

j. Close the cap and gently rock the tube side to side. Place the tube back onto the

ring stand and open the cap. Without releasing the liquid, pipet out 20L of the

liquid and insert it into a separate tube. Label the tube its appropriate mutant name

along with column sample.

13. Measure optical density of each elution sample

a. Turn on the spectrophotometer using the switch in the back of the machine. Turn

on the monitor screen and printer. Wait for the machine to go through its startup

routine.

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 Banerjee

b. Open the lid on the top of the machine. Choose the large cuvette holder and place

it in the holder in front of the light source.

c. Click on the visible light key to turn the light source on.

d. Choose Single Wavelength mode from the menu that appears. Make sure the

wavelength selected is 280 nm.

e. Place a 100 mL blank sample in a cuvette. Place the cuvette in the holder.

f. Shut the lid of the machine and click Read Blank in the bottom left corner of

the screen.

g. After the machine has read the blank, remove the cuvette, replace it with a cuvette

containing 32L of each elution sample diluted with 448 L GnHCL.

h. Record each optical density.

14. Run Gel Electrophoresis to determine in which tube (total cell lysate, soluble supernatant,

flow through, wash, elution 1 or elution 2) the protein lies in.

a. Prepare the gel samples as shown in the gel maps below. Along with the amount

of substance specified, 1L of 6x loading dye should be inserted to each tube as

well. For each mutant, a separate epitube should be used for separate samples.

b. Mount two prepared gel plate into the electrophoresis tank. Add just enough of

the electrophoresis buffers to cover the gel to a depth of approximately 1 mm.

c. Slowly load the protein samples into the submerged gel along with a Tricine

Ladder into gel plate using a micropipette.

d. When samples are loaded, close the lid of the gel tank and attach the electrical

leads so that DNA will migrate towards the positive anode. Apply a voltage of

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 Banerjee

90V/cm for two hours. If the leads have been attached correctly, bubbles should

be generated at the anode and cathode.

e. After two hours, remove the electrical leads and gel plate.

f. Stain gel with coomassie and image gel with a scanner.

LF --
20 L 20L 3L 20L 8L 4L 4L 4L
Total Lys Sol. Sup. Ladder Flow Wash Elution 1 Elution 2 Column
Through

LV --
20 L 20L 20L 3L 8L 4L 4L 4L
Total Lys Sol. Sup. Flow Ladder Wash Elution 1 Elution 2 Column
Through

GK --
3L 20 L 20L 20L 8L 4L 4L 4L
Ladder Total Lys Sol. Sup Flow Wash Elution 1 Elution 2 Column
Through

GY --
20 L 3L 20L 20L 8L 4L 4L 4L
Total Lys Ladder Sol. Sup Flow Wash Elution 1 Elution 2 Column
Through

15. His-Tag Cleavage

a. To remove other irrelevant proteins and to cleave his-tag from mutant proteins,

add 2 mL TEV protease into each elution 2 sample.

b. Leave overnight at 4 C in dialysis buffer. In order to dialyze, mix the following:

20M NaPO4, 50M NaCl, 1 mM TCEP.

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 Banerjee

16. Subtractive Purification

a. To remove TEV protease, the His-tag and uncut protein, pour the dialysed protein

into a small column containing 0.5 mL resin.

b. Close the cap and gently rock the tube side to side. Place the tube back onto the

ring stand and open the cap. Gently release the flow through into a separate fresh

tube. Make sure to keep and label the flow through as it contains protein with

His-tag removed. Repeat this for each dialysed sample

c. Repeat steps 15a-b again, once with wash buffer and once collecting column

sample, to ensure only mutated protein remains in the sample.

d. Run a gel with flow through samples to confirm that only mutated proteins

remain. Compare mutant protein lengths to the Molecular Weight Ladder.

Subtractive Purification Gel Map:

Ladder Sample Post Post Flow Wash Column

3 L before cleavage centrifuged Through 5 L 5L
cleavage sample sample 20 L
20 L 20 L 20 L

17. Thermal Shift Assay Dilutions

a. To obtain a 200L 1.8M protein sample, prepare two of each of the following

dilutions in separate tubes. Use the dialysis buffer prepared in step 14b

GY 12.8 L protein sample

187.2 L dialysis buffer

GK 10.9 L protein sample

189.1 L dialysis buffer

LV 16.4 L protein sample

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 Banerjee

183.6 L dialysis buffer

LF 41.9 L protein sample

158.1 L dialysis buffer

WT 11.2 L protein sample

188.8 L dialysis buffer

b. To obtain 100X dye, dilute 3L loading dye into 27.0L dialysis buffer

c. Add 2 L of 100x dye into each 200L protein sample. Add 1 L of 9.2M

LxxLL to 1 pair of protein samples. This should result in 4 samples of the protein

alone and 4 samples with the protein plus 9.2M LxxLL peptide

18. Thermal Shift Assay

a. In a 96 well plate, carefully pipet 10L of each sample into a separate well.

b. Cover the assay plate with a sheet of optically clear adhesive and carefully seal

each well. Centrifuge the assay plate at 800 g for 5 min at 25 C to collect

solutions in the bottom of the well

c. Place the assay plate into the Biorad 600 instrument and open Biorad software.

Under Experimental Properties, select the following parameters:

Set up: Fast 96-well block

Experiment Type: Melt Curve

d. Select the Assign tab on the left, then highlight 45 of the wells being used in the

assay plate

39
 Banerjee

e. Select the Run Method tab and set the following temperatures: an initial 2:00 hold

at 10C, ramping up in increments of 0.5C to a final temperature of 90C (with a

2:00 hold)

f. Click on all three cameras to activate fluorescence detection throughout the

experiment

g. Select total volume per well of 10l

h. Click on the RUN tab to initiate thermal denaturation.

i. Once the experiment is done (about 1 hour 45 minutes with the current set-up),

export data into a comma-separated value (csv) Excel file.

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 Banerjee

Results
PCR Samples Gel UV Image (Step 2)

Figure 6: UV Image presenting PCR samples. Brightness of the image is increased to show individual strands

Optical Density of BL21 E.coli Cell samples (Step 9)

Mutation Initial Volume Optical Density Transferred OD at 3:30 OD at 4:10

(mL) at 1:55 (OD) Volume (mL)

LV 10 0.7 100 0.48 0.75

LF 10 0.7 100 0.44 0.86

GY 10 0.7 100 0.47 0.80

GK 10 0.7 100 0.46 0.78

Table 1: Optical densities were measured to determine if BL21 E.coli Cell samples were at a concentration high
enough to proceed with rest of the experimentation

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 Banerjee

Optical Density of Elution Samples Post Purification (Step 13)

Mutation Elution 1 (OD) Elution 2 (OD) Elution 3 (OD)

LV 0.065 0.029 0.013

LF 0.019 0.009 0.006

GY 0.134 0.079 0.027

GK 0.420 0.000 ---

Table 2: Optical densities of only the elution samples used are included. For example, the OD for the first
purification experiment is listed for mutant protein GK, while the OD for the second purification experiment is listed
for mutant protein LF because those were the appropriate experiments that their elution was taken from for further
experimentation.

Protein Purification Gels (Step 14)

Ftz F1 G913K Purification Gel

Figure 7: G913K Purification Gel was stained with coomassie dye and scanned. Gel is annotated as displayed
above. Gel maps are also explicated in the procedure.

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 Banerjee

Ftz F1 G913Y Purification Gel (Purification 1)

Figure 8: G913Y Purification Gel was stained with coomassie dye and scanned. Gel is annotated as displayed
above. Gel maps are also explicated in the procedure.
Ftz F1 G913Y Purification Gel (Purification 2)

Figure 9: G913Y Purification Gel was stained with coomassie dye and scanned. Gel is annotated as displayed
above. Gel maps are also explicated in the procedure. This protein was purified and imaged again as a result of
experimental error.

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 Banerjee

Ftz F1 L907F Purification Gel (Purification 1)

Figure 10: L907F Purification Gel was stained with coomassie dye and scanned. Gel is annotated as displayed
above. Gel maps are also explicated in the procedure.

Ftz F1 L907F Purification Gel (Purification 2)

Figure 11: L907F Purification Gel was stained with coomassie dye and scanned. Gel is annotated as displayed
above. Gel maps are also explicated in the procedure. This protein was purified and imaged again as a result of
experimental error.

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 Banerjee

Ftz F1 L907V Purification Gel (Purification 1)

Figure 12: L907V Purification Gel was stained with coomassie dye and scanned. Gel is annotated as displayed
above. Gel maps are also explicated in the procedure.

Ftz F1 L907V Purification Gel (Purification 2)

Figure 13: L907V Purification Gel was stained with coomassie dye and scanned. Gel is annotated as displayed
above. Gel maps are also explicated in the procedure. This protein was purified and imaged again as a result of
experimental error.

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 Banerjee

Subtractive Purification Gel (Step 16)

Figure 14: Subtractive Purification Gel was stained with coomassie dye and scanned. Gel is not annotated as streaks
are vague. Gel map is provided in the procedure.

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 Banerjee

Thermal Shift Assay Data (Step 18)

Graph 1: Raw data before sorting of all well samples. Graph displays temperature in degrees Celsius against
normalized fluorescence.

Graph 2: This graph displays the amount fluorescence at each 0.5oC increment for G913K and G913K bound to
LXXLL peptide. Standard melting curve displays protein is stable and point of inflection should signify melting
point of protein sample.

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 Banerjee

Graph 3: This graph displays the amount fluorescence at each 0.5oC increment for G913Y and G913Y bound to
LXXLL peptide. Standard melting curve displays protein is stable and point of inflection should signify melting
point of protein sample.

Graph 4: This graph displays the amount fluorescence at each 0.5oC increment for L913V and L913V bound to
LXXLL peptide. Standard melting curve displays protein is stable and point of inflection should signify melting
point of protein sample.

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 Banerjee

Graph 5: This graph displays the amount fluorescence at each 0.5oC increment for L913F and L913F bound to
LXXLL peptide. Standard melting curve displays protein is stable and point of inflection should signify melting
point of protein sample.

Graph 6: This graph displays the amount fluorescence at each 0.5oC increment for wild type protein FTZ F1 and the
wild type bound to LXXLL peptide. Standard melting curve displays protein is stable and point of inflection should
signify melting point of protein sample.

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 Banerjee

G913K and G913K + Peptide Melting Points (Tm)

Run Melting Point (Tm) Fluorescence

GK1 42.39 0.4305325

GK2 41.80 0.5038493

GK3 41.75 0.5109057

GK4 41.85 0.4968536

GK5 41.79 0.5049848

GK + Peptide 1 48.08 0.4521854

GK + Peptide 2 48.26 0.4293719

GK + Peptide 3 48.05 0.4542448

GK + Peptide 4 48.28 0.4261363

GK + Peptide 5 48.40 0.4090662

Table 3: Melting point (Tm) calculated by point of inflection. Tm<for each sample G913K and G913K+peptide
sample is displayed in table above.
G913Y and G913Y + Peptide Melting Points (Tm)
Run Melting Point (Tm) Fluorescence

GY1 44.50 0.4141998

GY2 44.79 0.4915255

GY3 45.21 0.4442951

GY4 44.99 0.4690875

GY5 44.94 0.4738643

GY + Peptide 1 49.59 0.5256454

GY + Peptide 2 49.67 0.5159733

GY + Peptide 3 50.13 0.4531551

GY + Peptide 4 50.03 0.4665341

GY + Peptide 5 49.86 0.4890936

Table 4: Melting point (Tm) calculated by point of inflection. Tm<for each sample G913Y and G913Y+peptide
sample is displayed in table above.

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 Banerjee

L907V and L907V + Peptide Melting Points (Tm)

Run Melting Point (Tm) Fluorescence

LV1 39.76 0.5077151

LV2 38.73 0.5056168

LV3 39.02 0.4703507

LV4 38.83 0.4931975

LV5 38.83 0.4945014

LV + Peptide 1 44.28 0.4328566

LV + Peptide 2 44.47 0.4099219

LV + Peptide 3 44.34 0.4262810

LV + Peptide 4 44.66 0.5153185

LV + Peptide 5 45.06 0.4630888

Table 5: Melting point (Tm) calculated by point of inflection. Tm<for each sample L907V and L907V+peptide
sample is displayed in table above.

L907F and L907F + Peptide Melting Points (Tm)

Run Melting Point (Tm) Fluorescence

LF1 28.56 0.5691102

LF2 28.97 0.4991316

LF3 29.43 0.4250529

LF4 30.27 0.4390370

LF5 30.55 0.5541453

LF + Peptide 1 37.21 0.4436630

LF + Peptide 2 38.50 0.3927573

LF + Peptide 3 38.62 0.5622670

LF + Peptide 4 38.15 0.4544860

LF + Peptide 5 38.83 0.5165044

Table 6: Melting point (Tm) calculated by point of inflection. Tm<for each sample L907F and L907F+peptide sample
is displayed in table above.

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 Banerjee

Wild Type Protein + Peptide Melting Points (Tm) (Control)

Run Melting Point (Tm) Fluorescence

WT + Peptide 1 45.88 0.4698107

WT + Peptide 2 47.49 0.4087802

WT + Peptide 3 47.62 0.5161245

WT + Peptide 4 48.63 0.5241219

WT + Peptide 5 48.28 0.4326318

Table 7: Melting point (Tm) calculated by point of inflection. Tm<for each sample wild type +peptide sample is
displayed in table above.

Average Melting Points and Standard Deviations of Samples

Sample Average Melting Point (Tm) Standard Deviation

G913K 41.916 0.2673574

G913K + Peptide 48.214 0.1465606

G913Y 44.886 0.2631159

G913Y + Peptide 49.856 0.2295212

L907V 39.034 0.4192016

L907V + Peptide 44.562 0.3141974

L907F 29.556 0.8439668

L907F + Peptide 38.262 0.6377068

Wild Type 45.050 0.9880123

Wild Type + Peptide 47.580 0.3894212

Table 8: The table above showed all average melting points of the samples as well as the standard deviations of
each sample.

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 Banerjee

Data Analysis & Discussion

PCR Samples Gel UV Image Analysis (Step 2)

When constructing the original Ftz F1 plasmid, the 729 base pair insert was ligated into a

5,286 base pair plasmid. This resulted in the final protein being a total of 6,015 base pairs long.

When mutating this plasmid, the resulting proteins should have been the same length since three

base pairs were being replaced and none were being added or removed. After constructing our

mutations through PCR, running a gel would be able to confirm whether the mutations were at

the same length as our original plasmid. The gel did indeed display that the six mutations were

all at the same length of 6,015 base pairs long.

Optical Density of BL21 E.coli Cell samples Analysis (Step 9)

After constructing the mutations, their resulting DNAs were transformed into a BL21 E.

coli cell line. This transformation would enhance the protein yield for examination through the

thermal shift assay. Due to this, an optical density of 0.6 needed to be obtained in order to

proceed with the rest of the experiment. According to the Beer Lambert Law (A=bc),

absorbance is directly proportional to the path length and the concentration of a substance. The

proportionality is affected by the molar extinction coefficient of the substance, in this case being

25,500 L mol-1 cm-1. Thus, an optical density of 0.6 should correlate to a cell concentration of

2.35x10-5M. After measuring the optical density of the mutant cell samples after approximately

two hours of incubation, all of the samples had exceeded a 0.6 OD. The calculated

concentrations of the LV, LF, GY, and GK cell samples were 2.94x10-5M, 3.37x10-5M,

3.14x10-5M, and 3.06x10-5M respectively.

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 Banerjee

Optical Density of Elution Samples Post Purification (Step 13)

While purifying the protein samples, three elutions were conducted to ensure that a

maximum amount of protein was being extracted from the resin bed. To verify that the elutions

contained the proteins, the optical density of each elution sample was obtained. The data showed

that the absorbance decreased with each elution sample, confirming that the majority of the

protein had been collected during the first elution and the remaining elutions were collecting the

leftover protein on the resin; thus, with each sample there was less and less available protein to

elute resulting in a lower absorbance each time.

Protein Purification Gels Analysis (Step 14)

Although checking the optical density of

the elution samples would confirm the presence

of the proteins, a gel would be able to specifically

show if the appropriate mutant protein was being

eluted and/or if the there was more protein

available for elution in other purification samples

such as the wash sample or flow through sample

etc.

Running the purification samples along

with a tricine ladder, as shown in figure 6, would

allow for the comparison of the protein sample with the 28 kDa band. The original non mutated

plasmid would have displayed a kDa of approximately 28 and so should have the mutated

proteins. For each mutant gel, a heavy band was visible at around 28 kDa. This confirmed that

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 Banerjee

the appropriate mutant proteins were being displayed in the gels, and the bands at 28 kDa for the

elution samples indicated that the appropriate proteins were being eluted. However, for each of

the gels, there was a thick band present in the total cell lysate sample, soluble supernatant

sample, and the flow through sample. This indicated that a lot of the protein did not stick to the

resin bed and flowed through the column, so a greater amount of resin was needed to used and

possibly more elutions could have been conducted. Due to this, the protein purification step was

repeated only for the LV, LF and GY proteins since the optical density reading showed that there

was a high enough concentration of the GK protein eluted in the first purification experiment.

During the second purification experiment, the same results as the first experiment

occurred. However, there was a high enough concentration of the proteins eluted that the rest of

the procedure could be proceeded with.

Subtractive Purification Gel Analysis (Step 16)

To determine if the cleavage and subtractive purification removed all other irrelevant

proteins and the his-tag from mutant proteins, a gel was run along with a tricine ladder. All of the

wells other than the well including a pre-cleavage sample showed a lower kDa. This indicated

that the his-tag was cleaved since there was a difference in weight between the pre- and post-

cleavage samples. The post-cleavage sample, however, showed a thin line at the same kDa as the

pre-cleavage sample, but the band was not significantly thick thus indicating that the his-tag

stuck to the resin and was removed from the mutant protein

Thermal Shift Assay Data Analysis (Step 18)

In order to determine the stability of the mutant proteins alone and when binding to Ftz, a

thermal shift assay was conducted. The average melting points for the GK, GY, LV, and LF

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samples respectively were 41.916oC, 44.886oC, 39.034o, and 29.556oC. The average melting

points for the GK + Peptide, GY + Peptide, LV + Peptide, and LF + Peptide samples respectively

were 48.214oC, 49.856oC, 44.562oC, and 38.262oC. Overall, the melting points of the four

mutants bound to the peptide resulted in a higher average melting point compared to just the

mutant, thus indicating a higher stability.

When analyzing the data for the first hypothesis, for all mutant protein + peptide samples,

the protein folding curves were normally shaped as should a temperature versus fluorescence

graph be, and the curves were similar to the wild type + peptide sample graph. This supported

our first hypothesis and indicated that the mutations were binding to the peptide and were more

stable when doing so.

When analyzing the data for the second hypothesis, both the average melting points for

the GK + peptide and GY + peptide were higher than the wild type + peptide melting point.

However, in order to determine whether this difference was significant a two-sample t test was

performed. For the GK + peptide samples, the resulting t-value was -46.189 with a p-value of

2.064x10-9 and for the GY + peptide samples the resulting t-value was -31.828 with a p-value of

6.895x10-10. If there was no difference in the GK + peptide sample and the wild type + peptide

sample, then a difference this large would have occurred 2 times in 100000000 due to random

chance. If there was no difference in the GY + peptide sample and the wild type + peptide

sample, then a difference this large would have occurred 6 times in 1000000000 due to random

chance. Because these probabilities are too rare to believe, the difference may be attributed to a

higher stability in both the GK + peptide samples and GY + peptide samples than the wild type +

peptide samples.

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It was hypothesized that the LF + peptide samples should be more stable than the wild

type + peptide samples, however, this was not shown. Instead, the LF + peptide samples were

less stable along with the LV + peptide samples. A two-sample t test was also performed to

determine if this difference in melting point between the two mutants and wild type was

significant. For the LF + peptide samples, the resulting t-value was -18.403 with a p-value of

8.863x10-8 and for the LV + peptide samples the resulting t-value was -23.595 with a p-value of

1.506x10-8. If there was no difference in the LF + peptide sample and the wild type + peptide

sample, then a difference this large would have occurred 9 times in 10000000 due to random

chance. If there was no difference in the LV + peptide sample and the wild type + peptide

sample, then a difference this large would have occurred 2 times in 10000000 due to random

chance. Because these probabilities are too rare to believe, the difference may be attributed to a

lower stability in both the LF + peptide samples and LV + peptide samples than the wild type +

peptide samples.

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Experimental Error

Although multiple precautionary steps were taken, there were several areas in which

experimental error could have potentially occurred. An unknown error in the sequencing of the

DNA mutations may have led to a different mutation of Ftz F1 being studied. If this had

occurred, conclusions made on the certain mutation would not be credible and the PCR process

would have to be repeated.

Another potential point of error may have occurred when cleaving the his tag off the

mutated proteins. Although the post cleavage samples only showed a thin line at the same kDa as

the pre-cleavage sample, some of the his-tag or other irrelevant proteins may have been left in

the individual mutated protein samples. This may have affected either the individual melting

points of the mutants or the way they bound to the LxxLL peptide. In order to eliminate this type

of error, more resin should be used and more elutions should be performed to ensure the

complete cleavage of the his tag and unnecessary proteins.

Error in the purification steps of the experiment were corrected and explained in the data

analysis section of this paper. However, if the purification step was not repeated for the LV, LF

and GY proteins then this would have led to an overwhelmingly lower concentration of protein

as compared to the GK protein concentration. Using the original protein concentrations may have

resulted in either insufficient or insignificant data in the following steps.

When analyzing the data through a statistical approach, Type I error may have occurred

thus leading to a false rejection of a true null hypothesis (Ho= there is no significant difference

between the mutant + peptide melting points and the wild type + peptide melting points). If this

had occurred, then the higher/lower stabilization would have been falsely attributed by the

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mutations successfully increasing/decreasing the dynamics speculated in Ftz F1s ligand binding

pocket.

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Conclusion

Studying the functionality of nuclear receptors is necessary in order to gain insight on

how these receptors aid in vital processes such as cell transcription and translation. Orphan

receptors, specifically, have been a topic of great interest as there is little known information on

how these receptors function without a known ligand. The purpose of this study is to create

mutations of Ftz-F1 by changing the dynamics of the ligand binding pocket through site directed

mutagenesis and determine how do these mutations affect the stability of Ftz F1 and its binding

ability to its coactivator Ftz. We hypothesized that the mutations created of Ftz F1 would bind to

the LxxLL motif of Ftz in the same way as does the wild type and when comparing the mutations

stability alone to the mutations stability while bound to LxxLL, the bounded samples should be

more stable. This is because van der Waals forces should allow for complementary proteins such

as Ftz F1 and Ftz to be more stable when bound together. We also hypothesized that The G913K

+ peptide, G913Y + peptide, and L907F + peptide samples should be more stable than the wild

type + peptide sample. This is because the increase in amino acid mass from glycine to lysine

and glycine to tyrosine should decrease the speculated movement of helix 6 in and out of its

ligand binding pocket and stabilize the area. The increase in amino acid mass from leucine to

phenylalanine should slow down the dynamic behavior in the ligand binding pocket and increase

the stability of the protein. Also, L907V + peptide samples should be less stable than the wild

type + protein sample. This is because the decrease in amino acid mass from leucine to valine

should increase the dynamic behavior in the ligand binding pocket and thus destabilize the area.

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In order to conduct this experiment, PCR mutagenesis was used to introduce mutations in

the Ftz F1 ligand binding domain. These mutations were sequenced and then transformed into

bacteria. Afterwards, the bacteria was lysed and purified for protein samples. A thermal shift

assay was then used to determine the melting point of each protein sample as well as the melting

points of each protein while being bound to the LxxLL peptide. The results showed that the

protein folding curves were normally shaped similar to the wild type + peptide sample graph.

This supported our first hypothesis and indicated that the mutations were binding to the peptide

and were more stable when doing so. The results also showed that the melting points of GK +

peptide and GY + peptide were significantly higher than the wild type + peptide melting point,

thus exhibiting a higher stability. The LF + peptide and LV + peptide samples displayed a

significantly lower melting than the wild type + peptide sample, thus exhibiting a lower stability.

Our first and third hypotheses were supported. However, our second hypothesis was only

partially supported. We had predicted that LF + peptide samples should be more stable than the

wild type + peptide samples since the mutation had substituted a less massive amino acid for a

more massive one. Therefore the dynamic behavior should have been slowed down thus

stabilizing the pocket. However, this may not have occurred because the leucine to phenylalanine

mutation may have been too overwhelming thus resulting in a lower stability. Although the

findings in this project may imply that mutations slowing down the dynamic behavior of Ftz F1s

ligand binding pocket may be more stable and thus result in the upregulation the gene being

coded for and vice versa, further experimentation is needed to be able to conclude any

relationship between these stabilizations and how they relate to transcriptional activity.

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Impact

Nuclear receptors are a large family of transcription factors that are involved in important

physiological functions such as growth, development, and homeostasis. Apart from this, nuclear

receptors have recently been identified to play key roles in many pathological disorders, such as

cancer, diabetes, rheumatoid arthritis, asthma etc. Therefore, these transcriptional regulators have

come into great scientific interest within the past few decades due to their applications in drug

discovery and biomedical research. Most nuclear receptors, as discussed, are modulated by

binding to a corresponding ligand, while some nuclear receptors have no known ligands.

Understanding the way orphan receptors are activated and repressed through a ligand binding

domain or pocket may aid in the discovery of novel, highly effective and specific drugs for a

variety of human diseases(Ottow, 2008).

Synthetic ligands are used as a structure based drug design tool to manipulate the

functionality of nuclear receptors. Currently, 34 of the top 200 most prescribed drugs target a

nuclear receptor and account for over 30 billion dollars in pharmaceutical sales due to their

effectiveness and economic viability (Moore et al, 2006). This project attempts to investigate this

drug design in orphan receptors where its gene expression is controlled by its ligand binding

domain. The results of this project may imply that by decreasing movement through mutation in

highly dynamic ligand binding pockets, the stability of the receptor may increase thus up

regulating the corresponding gene being coded for. The opposite may be implicated for

increasing movement by mutation in highly dynamic ligand binding pockets. Because it is likely

that in vitro profiling will translate into a physiological outcome in vivo, the outcomes of this

project may be applied to the LRH1 ortholog in creating a drug to target the synthetic ligand

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binding domain for the treatment of liver, intestinal, ovarian and pancreatic cancer. Currently,

these cancer therapies cost approximately $30,000 per month. Engineering a drug to target LRH1

would decrease this cost by $20,000 (Glover, 2015). This project allows for a more thorough

understanding of how synthetic ligand binding pockets should be targeted for treatment and may

be applied into real world therapy.

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