Download Problem of Focus - Clarkson University

Developing a Biosensor from the Periplasmic Mercury Binding
Protein MerP
Honors Program Thesis Proposal
Marianna Worczak ‘06
Dr. Linda Luck
Department of Biology and Chemistry
Problem of Focus:
The thesis goal is to design a biosensor for mercury using the periplasmic bacterial
protein MerP. Current methods for sensing mercury in the environment often involve lab
analysis of samples and methods such as spectroscopy/spectrometry. Such methods can
be burdensome for fieldwork, expensive, or impractical for immediate mercury detection.
Since MerP binds very specifically to mercury it serves as an ideal target protein on a
biosensor.
Background Information:
Mer P is a periplasmic mercury binding protein present in some strains of bacteria that
display resistance to mercury (II) ions. A periplasmic protein is a protein is located in the
layer between the bacteria membrane and cell wall in the periplasmic layer. MerP is
7500 kd in weight, 72 amino acids in length, and with a tertiary structure with two major
alpha helices (Powolowski 1999). Mercury binds to the protein by the means of two
cysteine residues at positions 14 and 17(Powolowski 1999). Before binding to mercury
the cysteines must be in their reduced form, in the oxidized form they exist as a cystine,
which contains a disulfide bond between the two cysteines. (Quian 1998). When the
protein binds to the mercury the protein undergoes a conformational change which allows
the protein to be recognized by a transmembrane protein, MerT which transports the
mercury across the plasma membrane. The bacteria are thus able to bind mercury,
transport it through its membrane into its cytoplasm where it is detoxified. Mercury is
detoxified by reduction from Hg2+ to its elemental form Hg0 (Quian 1998).
Many studies have already been done to understand both the structure and dynamics of
mercury binding. In particular a NMR study by Quian et al (1998) produced threedimensional structural models. This model, as well as other models is located in the NIH
data base. The protein has two alpha helices, one beta sheet and a variety of areas that
undergo structural dynamics. The mercury binding site is located between the two
Cysteine residues at the end of one of the helices. Both the C and N termini are located
in proximity to one another and located at the opposite end of the molecule. Figure 1
depicts a RasMol ribbon model of the NMR structure derived by Quian et al.
Figure 1: Ribbon Structure of MerP protein with cysteine residues shown in red space filing design. The structure
depicts the two alpha helices and one beta sheet. The mercury binds between the two cysteine residues at the end of
both helices.
The problem to be addressed involves preparing a purified sample of MerP in order for
use in the production of a mercury biosensor. Biosensors are one of many new
applications for genetic engineering. Many bacterial proteins, such as MerP, can bind to
different environmental toxins. The interest in using such proteins for biosensors is
because proteins bind very specifically and efficiently to their substrates. Hence an ideal
biosensor should be able to bind even a very small concentration of the toxin in the
environment. Biosensors can be used to detect the amount of a toxic substance or even
be used to sequester and remove such toxins (Huang 2003). The binding constant for
MerP is 2.8 μM Hg-2 (Sahlman 1997) indicating that it would be ideal to use for a
biosensor.
Current research on biosensor development has been sparked by the relative insufficiency
of current spectrometric methods for measuring heavy metal concentrations in natural
environments. Currently, methods such as graphite furnace or flame atomic emissions
spectroscopy are available to measure very low concentrations of heavy metal in prepared
samples. However such techniques can be expensive when extended to widespread
contamination monitoring, and are difficult to use for in situ measurements (Malitesta
2005). Hence biosensors provide a cost effective, convenient method of monitoring
without jeopardizing sensitivity.
Initial studies by Powoloski et al (1999) on mercury resistant Eschercia Coli allowed for
the gene encoding the protein to be isolated. The protein was found to be located in the
transposon Tn 21 in E. Coli BL21(DE3). This section of the transposon was then cloned
into pCA plasmid vectors and used to create genetically engineered bacteria expressing
the MerP protein. The goal of research is to use this plasmid to grow bacteria and isolate
substantial quantities of the protein. Ideally the plasmid can be further manipulated to
develop a version of the MerP protein that can be bound to the gold surface of a quartz
crystal microbalance.
Proposed Research Steps:
The goal of proposed research is based on the hypothesis that a derivative of MerP can be
engineered to bind to a gold surface. To do so would involve using previously employed
methods to visualize and isolate the protein and then use the same methods to recover the
engineered protein. Our goal is to replace one of the amino acids far from the Hg binding
site for a cysteine residue. This would allow us to use a gold-sulfur bond to bind the
protein to the gold surface of the QCM at a place that does not interfere with Hg binding
or the conformational change taking place when Hg binds. If this can be achieved, then a
secondary goal will be to determine its potential as a biosensor for Hg detection in an
aqueous environment such as lakes, ponds and drinking water.
The first step in proposed research is to grow bacteria with the plasmid and express the
protein. This first step is called transformation where the plasmid is put into a special
strain of bacteria. The next step is induction of the plasmid to produce the protein. To
visualize the protein one must use a nondenaturing polyacrylamide gel. Since the protein
is only 7200kd in length, a gel of about 15% polyacrylamide may be necessary to
separate the protein. Since 12% polyacrylamide gels are traditionally used, a specialized
gel recipe will need to be developed. The protein also has a positive charge so a
specialized procedure can be utilized to run the gels in the opposite as normal SDS gels.
Specialized dye is also used in this procedure.
We will also design the mutation primers to genetically engineer for the new cysteine
residue for immobilizing the protein to the QCM. The molecular modeling program
RasMol will be used to visualize the protein and selecting a site far from the mercury
binding site and away from any conformational important area. The ideal residue to alter
must also be on the outside of the protein and not folded internally. Once an ideal
location for a mutation has been isolated, the DNA sequence must be examined and a
primer designed. The DNA sequence is readily available in the NIH sequence database.
Primer design can be achieved by mixture of manual and computer based examination of
the sequence. The Sequence Translator of JustBio will be used to align amino acid
residues and codons to facilitate the process of primer design. IDT is the company to buy
the primers and check to see if they are compatible for this experiment.
Once the primer for the mutation is designed and purchased, it will be used to induce the
mutation in the plasmid. Again the new plasmid will be inserted into the bacteria by
transformation by the same protocol as used to grow the original plasmid. Once the
plasmid has been successfully taken up by the bacteria and expressed, a polyacrylamide
gel will be also used to visualize the protein.
Successful detection of the engineered protein would then require purification. The exact
protocol for isolating the protein in pure sample will require a fair deal of
experimentation. We will use the osmotic shock procedure since the protein will migrate
to the periplasmic space after it is produced. Column chromatography will not be
necessary for the purification since a 100% pure protein is not required for the QCM
experiments. In addition columns are difficult since the protein does not contain any
tryptophan and monitoring the protein as it comes off the column will not be feasible by
UV-Visible spectroscopy.
The major goal of thesis work is to 1) work out an gel system to visualize the protein 2)
design primers for new cysteine mutant 3) do the PCR experiments for the mutation 4)
work out an isolation procedure for the mutant protein. If these goals are successfully
met, work will be carried further to immobilize the protein to the gold surface of the
QCM at the cysteine residue that has been newly inserted. Test can then be run to assess
the ability of the biosensor in the detection of mercury. However, this may be beyond the
scope of the thesis and be continued by another person in the lab.
Preliminary Work: Methodology and Results:
The plasmid pCA was obtained from Justin Polowski of Concordia University.
Competent E.Coli cells (strain BL21DE3) were transformed to uptake the plasmid using
well published protocols. In short the cells were incubated with plasmid for 15 minutes
then heat shocked at 42 C. The cells were then heat shocked to close the pores that were
opened making them competent. The cells were then grown with an incubation period of
1 hour at 37C. Since the plasmid also contains an ampicillin antibiotic resistance gene,
the cells were then plated onto LB/ampicillin plates overnight. Only colonies containing
and expressing the plasmid were able to display ampicillin resistance and grow on the
plates. The transformation process was successful as several well-shaped and large
colonies developed. The following photographs depict plates after the transformation
process was completed:
Figure 2: Left LB/Ampicillin plates with cultures of transformed bacteria containing pCA plasmid. Right another
culture with the same cells.
The transformed cells were then cultured in LB broth contain amphicillin at a
concentration of 50 μg/ml. Two of such cultures were prepared. Cultures were grown at
37°C overnight and were measured to ensure an absorbance of 660nm to assure a
turbidity of 0.80. Induction was then carried out by the addition of IPTG to allow the
expression of the protein. After induction was complete, a 15% polyacrylamide gel will
be run to determine if the protein is indeed present or not. The stacking gel consisted of
1.5M Tricine, 15% concentration of Arylamide/Bisacrylamide, Amonium persufate, and
TEMED and had an overall pH of 7.0. The separating gel will be prepared using a 4%
acrylamide/bisacryamide, 0.5M Tris/0.25 M Tricine at pH 7.4, ammonium persulfate and
TEMED.
Examination of NMR structures deduced by Quian et al (1998) from the NIH database
with RasMol was carried out to determine where the best location for an additional
cysteine. The purpose for adding a cysteine is to allow for adhesion to a gold template of
a biosensor hence the model was examined for a location far from the mercury binding
site, and extending outward in the proteins tertiary structure. Form these criteria it
hypothesized that Threoine (Thr) 50 would be an ideal location. Figure 4 illustrates both
a backbone structure of the protein to detail the relative location of Thr 50 to the mercury
binding site (Cys 14 and Cys 17). The figure also depicts a space filling model of the
protein illustrating that Thr 50 is oriented on the outside of the protein’s tertiary structure
and would serve as an ideal location for binding to a template to form a biosensor.
Figure 4 illustrating location of Threonine 50 and cysteine residues. At left a backbone structure depicts location of
Thr 50 far from Cys 14 and Cys 17 residues (mercury binding site), indicating an ideal loci to induce mutation. At
right, a space filling model depicts both the cysteine residues (red at top) and Thr 50 (at bottom). The threonine is
located on the outside of the protein further validating it as an ideal loci for mutation induction.
Since the location for mutation was identified, it was then possible to examine the DNA
coding sequence and choose an appropriate primer. Threonine 50 was determined to
have sequence of 5’ACT 3’ Coding sequences for Cys are either TGT or TGC. If the
sequence TGT is used a potential primer to for use may be: 5’GGC CTT GGT GTC
GTG AAA ACA GAC GAC GGC CTC CTC GAA-3’. The primer choice must still be
verified. If it is indeed a solid choice, it may be purchased from a biotechnology
company then used to induce the mutation.
Future Steps:
Aside from work already detailed above, there are several steps which still need to be
accomplished. First the bacterial cells need to be grown into a permanent culture so that
future experiments and protein extractions can be performed. Next, the protein alone
must be purified. Beyond detecting of the protein steps must be carried out to isolate and
purify the protein and to test reproducibility of techniques derived.
The second stage of experimentation will begin with inducing a mutation in the MerP
gene and growing cells possessing this trait. As detailed above, a primer with an altered
gene at residue 50 will be used to produce protein with an added cysteine residue. The
procedure for isolating the mutated protein as well as growing cells expressing the
protein will follow the same protocol as with the original transformation. Once the
protein has been isolated and purified, work on building a sensor from it can begin.
The final goal of the research involves attaching mutated protein to the gold template so
that it can serve as a biosensor. If the mutation is successful, the cysteine 50 should bind
directly to the gold surface and anchor the protein to a template. The plasmid used in the
experiment produces the protein in its oxidized (disulfide) form so Cys 14 and 17 will not
be available to bind the protein to the gold. This is beneficial as it ensures that the
mercury-binding site is intact. The final step will reduce the disulfide bonds of the gold
bound protein to dithiols, and use such to bind to mercury.
Timeline:
Date
March 2005
April 2005
May-Aug 2005
Sept-Oct.2005
November 2005
December 2005-Jan 2006
Jan 2006-Feb 2006
March 2006
April 2006
Goal
Isolate protein, develop primer
Induce mutation, attempt to recover protein
Writing Introduction and portions of
Materials and Methods already completed
Purify recovered protein, attach to gold
plate, continuing to write materials and
methods
Potentially Perform test on Mercury
Binding, continuing to write materials and
methods
Working on writing results, discussion
Writing and finalizing rough draft of
written section
Prepare PowerPoint presentation, submit
rough draft for revision
Present, and submit final paper
References:
Berglund, Anders et al. 2005. The equilibrium unfolding of MerP characterized by
multivariate analysis of 2D NMR data. Journal of Magnetic Resonance. 172:2430
Brorsson, Ann-Christin et al. 2004. The “Tow-stateFolder’ MerP Forms Partially
Unfolded Structures that Show Temperature Dependent Hydrogen Exchange.
Journal of Molecular Biology. 340: 333-344.
Huang, Chieh-Chen et al. 2003. Polypeptides for heavy-metal biosorption: capacity and
specificity of two heterogeneous MerP proteins. Enzyme and Microbial
Technology. 33:379-385.
Malitesta, C. and M.R. Guascito. 2005. Heavy metal determination by biosensors based
on enzyme immobilization by elecropolymerisation. Biosensors and
Bioelectronics. 20: 1643-1647.
Narita, Masaru et al. 2003. Diversity of mercury resistance determinants amoung Bacillus
strains isolated from sediment of Minamata Bay. BEMS microbiology Letters.
223:73-82.
Powolowski, Justin and Lena Sahlman. 1999. Reactivity of the Two Essential Cyseine
Residues of the Periplasmic Mercuric Ion-binding Protein MerP. The Journal of
Biological Chemistry.
Quian, Hong et al. 1998. NMR Solution Structure of the Oxidized Form of MerP, a
Mercuric Ion Binding Protein Involved in Bacterial Mercuric Ion Resistance.
Biochemistry 37:9316-9322.
Ravel, Jacques et al. 2000. Cloning and Sequence Analysis of the Mercury Resistance
Operon of Streptomyces sp. Strain CHR28 Reveals a Novel Putative Second
Regulatory Gene. Journal of Baceriology. 182: 2345-2349.
Sahlman, Lena et al. 1997. A mercuric Ion Uptake Role for the Integral Inner Membrane
Protein, MerC, Involved in Bacterial Mercuric Ion Resistance. The Journal of
Biological Chemistry. 272:29518-29526.
Serre, Laurence et al. 2004. Crystal Structure of the Oxidized Form of the periplasmic
Mercury-binding Protein MerP from Ralstonia metallidurans CH34. Journal of
Molecular Biology 339: 161- 171.
Wimmer, Reinhard et al. 1999. NMR Structure and Metal Interations of the CopZ
Chopper Chaperone. The journal of Biological Chemistry. 274: 22597-22603.
Yamahita, Shozo et al. 2001. Possible Physiological Roles of Proteolytic Products of
Actin in Neutrophils of Patients with Behcet’s Disease. Biol. Pharm. Bull. 24:
733-737.
Yeo, Chew Chieng et al. 1998. Tn5563, a transposon encoding putative mercuric ion
transport proteins located on plasmid pRA2 of Pseudomonas alcaligenes. FEMS
Microbiology Letters. 165: 253-260.