Andrew Reed - march 2016

Thesis Introduction (rough draft)

Urea Denaturation of the protein TIAM 1 PDZ QM+C4

Motivation/significance
To understand the nature of living things we need to understand the cellular and
molecular level, to watch and observe the basis of life itself. The cell has a sense of mystery to it
that we humans still do not know how to explain its action in complete and full detail. For
example cells may start to wander and replicate without direction or meaning even when
signaled to stop and disperse. This specific cell phenomena (one of many) known as cancer is yet
to be completely understood by todays scientific community. A possible answer to this process
is the TIAM 1 protein which is a structure in human cells that deals with communication and
metastasis. To understand the function of this transmembrane protein the characteristics of its
structure must be understood. In this study changes caused by the chemical Urea will be
observed. According to the National Cancer Institute (Figure 1), in 2016 an estimated 1,685,210
new cases of cancer will be diagnosed, of which 595,690 will be fatal cases. The National Cancer
Institute states, The most common cancers in 2016 are projected to be breast cancer, lung and
bronchus cancer, prostate cancer, colon and rectum cancer, bladder cancer, melanoma of the skin,
non-Hodgkin lymphoma, thyroid cancer, kidney and renal pelvis cancer, leukemia, endometrial
cancer, and pancreatic cancer. In 2010 the amount of expenditures for cancer care totaled in
around 125 billion dollars and is expected to rise around 150 billion dollars in 2020 (National
Cancer Institute).

Figure 1: American Cancer society statistical figure for 2015 (Cancer

statistics 2015).

Background information
The protein of interest in this study is formally known as TIAM 1 PDZ QM + C4 also
known as T-cell lymphoma invasion and metastasis inducing protein. This protein is a
transmembrane protein which shows a relation between its expression in human tissue and the
development of tumors and cancer. In studies performed by Bin Chen and colleagues, it was
found using tissue micro array and other forms of analysis that there is a positive correlation
between the over expression of Tiam 1 protein and the increase in metastasizing tumor cells in
cancer cases, specifically in this study cases of hepatocellular carcinoma (Bin Chen 2011). In the
research performed by Xingjun Guo and colleagues the balance between Tiam 1 and Rac1 was in
question. They found that the progression of pancreatic cancer is partially controlled by the
balance between Tiam 1, Rac 1, and Rhoa (Guo 2013). To better understand this relation between
the Tiam 1 protein and tumors/ cancer the purpose of this research is to better understand the
structure of the PDZ domain of the protein. This domain is specific to mediating protein to
protein interaction.
Tiam 1 PDZ QM+C4 is the specific name of the sample used in this research. It is
a mutant domain of the T cell lymphoma protein ranging from 80 90 amino acids. This is a
small fraction of the entire protein as shown by Figure 2 and Figure 3. Of the entire macro
structure 4 amino acids have been engineered and differ from the wild type protein which is what
the QM in the sample name stands for; Quad mutant.
2

Figure 2: Figure shows the ribbon structure of the Protein Tiam 1 PDZ, PDZ
domain is color coded pink. (madej 2014)

Figure 3: Figure shows the ribbon structure of the PDZ domain detached
from the Tiam 1 protein (madej 2014).

The four engineered amino acids were selected by Dr. Fuentes and his research group at the
University of Iowa, who stated the reasoning for this was that NMR chemical shift perturbation
experiments indicated that the Tiam1 PDZ/Syndecan1 and PDZ/Caspr4 complexes where
different structurally and the four amino acids selected four change were mostly likely the cause.
The PDZ domain is a short hand name that stands for the first letter of three proteins that share
the domain. P stands for post synaptic density protein PSD 95, D stands for Drosophila disc
large tumor suppressor DLG), Z stands for Zonula occludens 1 protein . The C4 stands
for the binding partner specific to the PDZ domain; Caspr 4, which is also joined by another
binding partner Syndecan 1, which is absent in this research. Caspr 4 is of the NCP family of cell
recognition molecules that is expressed in the ervous system. According to Dr. Fuentes and his
colleagues at the University of Iowa, Tiam 1 is a Rac specific guanine nucleotide exchange
factor. It is related in the mechanisms oncogenicity, tumor invasion, and metastasis. It activates
the Rho-like GTP-ase Rac. Tiam 1 protein is known to contain several protein to protein
interaction domains one of which is PDZ, allowing Tiam 1 to help hold together and organize
signaling complexes at cellular membranes. They also play a key role in anchoring receptor
proteins in the membrane to cytoskeletal components. The TIAM 1 protein modulates Rho-like
proteins and connects extracellular signals to cytoskeletal activities, indirectly effecting the
process of cell adhesion and migration (shepherd 2010). The Tiam 1 to Rac pathway is the
indirect connection Tiam 1 has to effecting the cytoskeletal structure of the cell, and thus the
connection Tiam 1 has to generation of cancer cells. Tiam 1 is a GDP dissociation stimulator
protein that catalyzes the process of exchange from GDP (guanine di-phosphate) to GTP
(guanine tri-phosphate). This process then activates Rho-like GTPases which have been
implicated in the regulation of the actin cytoskeleton which controls the morphology, adhesion,
and motility of cells (michiels 1995).
In this research the application of the substance Urea will be used as a chemical means to
denature the PDZ domain of the Tiam 1 protein. Urea is ( C H 4 N 2 O ) a compound formed in
the liver from ammonia produced by the deamination of amino acids that is used in a white
odorless pellet/crystal (National Center for Biotechnology Information). It causes the
intermolecular forces of the PDZ structure to change and deform such as di-sulfide bonds,

hydrogen bonding, and Van Der Waals forces. This denaturation with basic effects is shown in
Figure 4.
Figure 4

Figure 4: image showing the introduction of urea (red dot) to a native

natured protein in ribbon structure and how this affects the protein structure
and results in the denaturation of that protein in to a non-native form (Tao
2002).
Through this denaturation effect of urea the amino acid structure of the PDZ domain can be
analyzed and observed so as to better understand the structure of the PDZ domain. The severity
and order of which specific amino acids migrate from native structure regions will be observed
as the domain goes from natured to completely denatured. This will be done through the use of
nuclear magnetic resonance spectroscopy (NMR), which allows for the observation of the
denaturation of the domain. Specifically Hetero Nuclear Single- Quantum Correlation (HSQC)
NMR experiment will be utilized so as to produce a two dimensional spectra that will give a
picture of the structure of the PDZ domain. This technique involves the overlapping of two one
dimensional NMR spectra. One axis for proton
research

15

and the other for a heteronucleus, in this

dimensional NMR spectra producing a 2 dimensional spectra that gives the real

time location of each amino acid. When done in consecutive experiments movement can be
observed.

Previous research
Research done in this field of study includes the thermodynamic denaturation of proteins
done by Bruce E. Bowler. In this journal insight is given as to the stability of the residual
structure of proteins obtained through NMR. Data showed that the role of hydrophobic clusters
in stabilizing denatured state residual structures is great and that electrostatic interactions are
important in modulating the stability of the denatured state. Thermodynamic methods are used to
provide insight in to the role of excluded volume, chain stiffness, and loop persistence in the
action their conformational preferences of highly disordered proteins. New insights into protein
folding and novel methods to manipulate protein stability are emerging from this work (Bowler
2007). Many forms of research are being done in the scientific community that incorporate the
technique of Nuclear Magnetic Resonance spectroscopy for means of analysis including the
before stated research by Bruce E. Bowler. Research done by Michela Candotti and colleagues
also includes this technique as well as an interest set towards the action of urea denaturation of
ubiquitin and how its coiled natured state compares to its denatured state caused by native
activities within a cell. There were multiple sessions where the sample protein was unfolded by
different means and in different circumstances. This was analyzed using NMR to give
information on the nuances in the proteins unfolding action in correspondence to the changing
conditions. This research group was able to define the unfolded state ensemble, understand the
energetics stabilizing unfolded structures in urea, describe the differential nature of the
interactions of the fully unfolded proteins with urea and water, and characterize the early stages
of protein refolding when chemically denatured proteins are transferred to native conditions.
These results allow for information to be gained on the forces that act upon a protein in its
natural circumstances as well as a protein denatured in non-natural circumstances that have the
presence of urea. However these results are specific to the protein Ubiquitin they still give a
point of reference as to how might the Tiam 1 protein might interact with urea. In the results it is
stated that 8M urea is the point at which the protein becomes completely denatured (Michela
Candotti 2013).

Goals/research objectives

The basis of this research set upon the structure of the Tiam 1 PDZ domain entails the
following goals to gain information in the area of structure and integrity of the PDZ domain. The
specific goals include first the analysis of samples and the identification of the PDZ domain
through HSQC NMR spectroscopy. Once this is obtained then we must successfully map the
amino acids in the sample and compare to known amino acid positions. The urea concentration
range of denaturation then needs to be determined through a stepwise urea addition to efficiently
obtain a range for future research and experiments and the elongation of sample life. This will
allow for optimal structure change observations. Tracking, recording, and analysis of change in
the PDZ domain structure must be done (order and magnitude of change in amino acids) in
relation to urea concentration change in the sample. Once analysis is done comparison and
sharing of the results is needed, as well as possible new areas of research pertaining to the Tiam
1 protein and what effects the new found information of the PDZ domain may have on new
research.

References
Cancer Statistics. (n.d.). Retrieved March 05, 2016, from http://www.cancer.gov/aboutcancer/what-is-cancer/statistics
American Cancer Society | Information and Resources for Cancer: Breast, Colon, Lung, Prostate,
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overexpressed in most malignancies, is a novel tumor biomarker. Molecular Medicine
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The Tiam1 PDZ Domain Couples to Syndecan1 and Promotes CellMatrix Adhesion.
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Biological Sciences - Biophysics and Computational Biology:

Michela Candotti, Santiago Esteban-Martn, Xavier Salvatella, and Modesto Orozco
Toward an atomistic description of the urea-denatured state of proteins
PNAS 2013 110 (15) 5933-5938; published ahead of print March 27, 2013,
doi:10.1073/pnas.1216589110
Guo, X., Wang, M., Jiang, J., Xie, C., Peng, F., Li, X., . . . Qin, R. (2013). Balanced Tiam1-Rac1
and RhoA Drives Proliferation and Invasion of Pancreatic Cancer Cells. Molecular
Cancer Research, 11(3), 230-239. doi:10.1158/1541-7786.mcr-12-0632