This document summarizes research characterizing the mumps virus nucleocapsid-binding domain (NBD) protein via fluorescence spectroscopy and circular dichroism. Researchers created a variant of the mumps NBD protein called F366W by introducing a tryptophan mutation using site-directed mutagenesis. This allowed stability measurements using fluorescence spectroscopy. The mutated protein was expressed in E. coli and purified. Fluorescence spectroscopy and circular dichroism were used to monitor the protein's tertiary structure as it was subjected to thermal and chemical denaturation conditions. Results from these experiments provided insights into the stability and folding of the mumps NBD protein.

1. 1
Characterization of Mumps F366W NBD via Fluorescence
Spectroscopy and Circular Dichroism
Abstract:
The paramyxovirus viral polymerase is tethered to its template via weak, transient
interactions mediated by its nucleocapsid-binding domains (NBDs). The NBD domains from
different viruses vary between intrinsically unstructured (as in the mumps virus) and highly
stable (as in the measles virus). Nevertheless, the individual domains display significant
sequence and structural similarity, forming a very simple 3-helix bundle. We are using the NBD
domains from mumps and measles as a model system to investigate the basis for structural
specificity and its contribution to coupled folding and binding processes.
The mumps NBD has significant alpha-helical secondary structure in solution, but lacks
tertiary structure. Ongoing experiments in the lab are seeking to engineer the mumps NBD, and
we desired to devise a variant which would enable a more convenient experimental measure of
its tertiary structure. We substituted a tryptophan (W) for phenylalanine(F) using site-directed
mutagenesis to create the F366W variant of the mumps NBD, so that stability measurements
could be carried out using a fluorescence spectrophotometer. The wild-type mumps NBD may
be induced to fold using the osmolyte trimethylamine N-oxide (TMAO), and its helical structure
may be disrupted using urea or temperature. The F366W protein was subjected to thermal melts
and chemical titrations with urea and TMAO for comparison to the wild-type protein. The
fluorescence signal at 320 - 350 nm was monitored to judge the amount of tertiary structure that
was induced or denatured, and compared to circular dichroism measurements. Results indicate
that the tryptophan substitution had limited effect on the ensemble of conformations of the
protein in the absence of urea or TMAO and that the fluorescence from the tryptophan may be
used as an alternative measure to facilitate folding studies.

2. 2
Introduction:
The study of protein structure and function is a fundamental part of biochemical research.
It is known that the mumps nuclear binding domain (NBD) protein is in a molten globular state
in its native environment. This means that the protein is partially folded and unfolded in native
conditions.
Figure 1: Proposed folded/unfolded equilibrium
This figure shows the predicted folded/unfolded equilibrium arrangement of the mumps NBD protein in native
conditions. This figure was adapted from Dr. Andrew Hausrath's talk on virus model formulation.
This specific protein has been studied via circular dichroism (CD) and it is known to have alpha-
helical secondary structure. With this folding arrangement, there is a lack of inherent tertiary
structure. Lacking the stabilizing tertiary interactions the NBD protein will exist as a collection
of three isolated alpha helices (Kingston et. al.). The tertiary structure of this protein has not
been readily characterized. In order to better characterize the tertiary structure of this protein a
fluorescent probe was inserted into the hydrophobic core of the NBD protein.
The fluorescent probe was inserted into the mumps NBD via site directed mutagenesis
utilizing overlap extension polymerase chain reaction (PCR). PCR is based around three steps.
First, the denaturation of the template DNA strand into single strands. Next, the single strand of
template DNA is annealed to primers containing templates for new strand synthesis. Finally, the
primed strands will be extended into new double stranded DNA containing the desired mutation

3. 3
(Delidow et. al.). Overlap extension allows complementary oligo primers and PCR to generate
DNA strands that will have ends that overlap (Ho et. al.). Combining these overlapping
segments and annealing them allows the 3' overlap of each strand to become the primer for 3'
extension of the complimentary strand (Ho et. al.). Overlap extension PCR is an extremely
accurate way to introduce mutations in the genetic code since approximately only 1 in 4000
nucleotides suffers a random substitution. In this case, the mumps DNA was mutated adding a
tryptophan into the DNA sequence instead of the native phenylalanine residue. The addition of
the tryptophan residue allowed the use of fluorescence spectroscopy to monitor changes in
protein folding and unfolding.
Before monitoring the protein via fluorescence spectroscopy the mutation had to be
confirmed. Confirmation of the mutation was done via sequencing. Sequencing was completed
in a different laboratory and the results were returned via email. (Figure 1 illustrates sequencing
data showing the PCR had successfully added the desired mutation.) Since the desired mutation
was successful, the protein had to be expressed in cell cultures before being purified and studied
via fluorescence spectroscopy and CD.
Protein expression was done via Ligation-Independent Cloning (LIC) into petHSUL
vectors. LIC cloning of PCR products allows the attachment of the small ubiquitin related
modifier (SUMO) and a histidine (his) tag. "... LIC adds flanking sequence extensions to the
target gene that are longer than those used in TA-cloning, but shorter than those used in
recombinogenic methods" (Weeks et. al.). This allows LIC to be used whenever the sequence
requirements in the flanking areas are met. The SUMO tag was used to allow quick purification
of the final LIC product. SUMO, when fused to a target protein, is known to increase solubility
of the targeted protein (Weeks et. al.). The mutated DNA strands were inserted into vectors and

4. 4
transformed into cell colonies. The petHSUL vectors were transformed into DH5α Gold cell
line.
Bacterial transformation is the process by which bacterial cells (i.e. e. coli) take up naked
DNA molecules. Transformation is a useful technique because if the newly inserted DNA is
recognized by the host cell's DNA polymerase the bacterial cell will continue to replicate the
foreign DNA along with the DNA that is already part of the bacteria (Bickle et. al.). In order to
be able to transform DNA into cells, the cells have to be competent. Competent cells are cells
which were treated with a solution of calcium chloride early in cell growth. This allows the
chloride ions to enter the cells with water swelling the cell (Bickle et. al). This swelling is
necessary for the uptake of DNA during bacterial transformations.
Once the desired protein mutation had successfully been introduced into a cell line, cell
cultures could be created. These cells cultures were used to grow 4 L of bacteria. These 4 L of
bacteria were used to grow e. coli containing the NBD protein with the mutation. Once the 4 L
of bacteria had grown to the optimal optical density they were spun down via centrifugation.
The pellet was kept and sonicated. Sonication destroyed the cell membrane exposing all of the
cell contents. After sonication the bacteria was ran over a nickel column that isolated the protein
via a SUMO-His tag that was already attached to the NBD. The SUMO-His tag stuck to the
column allowing the left over contents of the cell to flow through the column. This left behind
the mutated NBD protein on the nickel column. After nickel column purification the protein was
dialyzed to exchange buffers. The purified NBD protein could then be studied via fluorescence
spectroscopy once presence of the mutated protein was confirmed via an SDS-PAGE gel.
Fluorescence spectroscopy is a technique that utilizes a spectrophotometer. Fluorescence
spectrophotometers are designed to quantify fluorophores. A fluorophore is a molecule that

5. 5
absorbs photons at a specific wavelength when exposed to light. A common biological
fluorophore is tryptophan (W). Tryptophan fluoresces because of its aromatic structure.
Tryptophan fluoresces at roughly 320 - 350 nanometers (nm). The intensity at which tryptophan
fluoresces can be used to monitor the change in protein folding or unfolding. When a molecule
is denatured, it is exposed to a more polar solvent. When this happens, the fluorescence of
tryptophan shifts to a higher wavelength. This is known as a red shift. Red shifts can be used to
judge protein denaturation. Since the NBD protein was mutated to include a fluorophore in the
hydrophobic core, this allowed for investigation of properties associated with tertiary structure.
Figure 2: Desired Mumps NBD Mutation
This figure highlights the desired F366W mutation in the mumps NBD. The mutation takes place in the
hydrophobic core.
Monitoring the protein via fluorescence spectroscopy allowed the conditions to be changed
around the purified mumps NBD protein. The conditions were changed thermally and chemically
to see how the protein reacted. Thermally, the temperature was increased from 4 C to 80 C. The

6. 6
protein was then cooled from 80 C to 10 C since the water was not able to be cooled back to 4 C.
Chemically, the protein was subjected to increases of Urea ranging from 0 M to 8 M in various
increments. The protein was also titrated with an additive trimethylamine N-oxide (TMAo)
under the same conditions. TMAo is known to promote folding of molten globular proteins.
"The physical basis of this effect is proposed to be the unfavorable interactions between TMAo
and the polypeptide backbone, which preferentially raises the free energy of partially unfolded
states, shifting the equilibrium toward the folded form of the protein" (Kingston et. al.).
Comparing the fluorescence data with TMAo and without TMAo allowed for protein stability
conclusions to be made. Tertiary structure formation could be inferred from the protein stability
under different conditions.
Circular dichroism is a technique which measures the differences in absorption of left-
handed polarized light versus right-handed polarized light. This technique is very useful because
in the far-UV spectrum (from 190 – 260 nm) the chromophore is the peptide bond in the protein.
This means the amount of secondary structure present in the protein in various environments
could be easily detected. The CD apparatus is also useful when measuring secondary structure
when conditions around the protein in question are changed either thermally or chemically.
Materials and Methods:
Creating Mutated Genetic Code via PCR:
The required materials for PCR are: mutated upper and lower primers (ordered from a
supplier), a template DNA strand, dNTP, Taq Polymerase, and 10x PCR buffer. The machine
utilized was an Eppendorf Mastercycler Gradient. First, combine 220.5 μL molecular biology
grade water to 30 μL 10x PCR buffer, 30 μL plasmid template, 6 μL of the upper and lower

7. 7
primer, 6 μL dNTP, and 1.5 μL of Taq polymerase. This solution was then separated out into six
individual solutions containing 50 μL each into 0.2 mL PCR tubes. These tubes were then
spaced across the desired temperature gradient. The Eppendorf Mastercycler Gradient was then
programmed to run 60 seconds at 94 C, 30 seconds at 94 C, 30 seconds at 65 C, and 30 seconds
at 72 C. This program was repeated 40 times. After the 40th time, the temperature was held at
72C for 10 minutes. After that the temperature was held at 4 C. The PCR product had to be
isolated via a 1% agarose DNA gel.
Isolating PCR Product via 1% Agarose DNA Gel:
To isolate the PCR product a DNA gel was used. DNA gels have lower melting points
making them ideal to isolate products to be sequenced. To make the gel 0.4 g of low melt
agarose was combined with 36 mL of de-ionized water and 4 mL 10X TAE buffer in a 50 mL
beaker. This solution was then microwaved for 30 seconds or until the solution appeared clear.
The mixture was left alone for one minute then poured into a gel well with the comb inserted.
The gel was allowed to cool for five minutes before the PCR product was loaded into it. While
the gel was cooling the running buffer and samples were prepared. The running buffer was 25
mL 10X TAE buffer with 225 mL de-ionized water prepared in a large graduated cylinder. The
samples were prepared by adding 10 uL of the DNA sample from PCR with 2 μL 6X loading
dye. The loading dye consisted of a mixture of 6X DNA dye with gel green. The gel green was
added so the gel would appear under ultraviolet light. Once the gel had cooled the samples were
loaded into the wells, and the running buffer was added. The gel was run at 80 volts until the
DNA sample had moved about 3/4 the way down the gel. To check if the mutation was

8. 8
successful, the PCR products had to be excised out of the gel and purified. Gel purification was
done with a QIA Miniprep kit using the supplied spin protocol, buffers, and a micro centrifuge.
Ligation-Independent Cloning into petHSUL Vector:
LIC started with a t4 DNA polymerase treatment of the solution to be inserted. This was
done by mixing 29.5 μL of the gel-purified PCR product in water to 10 μL 5X T4 DNA
polymerase buffer, 0.5 μL 100 mM dGTP, 0.5 μL 100X BSA, 8.5 μL DNAse free water, and 1
μL T4 DNA polymerase in a 1.5 mL eppendorf tube. This was incubated for 30 minutes at 25 C.
After the 25 C incubation, the solution was incubated at 75 C for 20 minutes to deactivate the T4
polymerase. The insert and vector were mixed. This was done by taking three tubes containing
one of the prepared LIC ready petHSUL vectors. One spare tube was also used. The tubes were
labeled A - D. In tube A 1 μL of vector was added to 5 μL of the insert. Tube B was 1 μL of the
vector to 1 μL insert. Tube C was 1 μL vector and 0 μL insert. Tube C was the vector only
control. Tube D was 0 μL vector, and 5 μL insert. Tube D was the insert only control. The last
tube E was empty and this was the selection control. After the tubes were prepared, the vector
was annealed and inserted. This was done by incubating tubes A - D at 75C for precisely 2
minutes. After incubation the tubes were allowed to cool to room temperature for 5 minutes. To
each tube 10 mM EDTA was added to equal volume of insert and vector making the EDTA 5
mM. 50 μL of thawed DH5α cells were added to each tube and gently mixed. These cells were
then incubated for 20 minutes on ice. At this point, the vectors could be transformed.

9. 9
Bacterial Transformation:
Bacterial transformations were done in the afternoon so that the cells would not
overgrow. Tubes of competent cells were taken from the -80 C freezer and gently thawed by
putting the tubes on ice. 50 μL of the thawed cells were added to a 1.5 mL sterile microtube. To
this tube, 1 μL of the DNA solution was added and gently mixed by swishing around the pipette
tip. The mixture was allowed to sit for 20 minutes so the DNA could adhere to the cells. The
cells were then heat shocked by placing them in the 42 C heat block for exactly thirty seconds.
The cells were then placed on ice for precisely two minutes. 700 μL LB broth was added to the
tube of cells and placed in the incubator at 37 C for an hour while shaking. After incubation, the
cells were plated on LB plates containing the appropriate antibiotic. The antibiotic used for this
procedure was ampicillin, so the plates used were LB/amp plates. Two plates had 300 μL of
cells added to them. Another plate had 30 μL of cells added to it. The plates were incubated
overnight at 37 C.
Growing 4 L of e. coli:
First,label test tubes (i.e. 1 - 4). To each tube add 5 mL LB broth and 5 μL of AMP. A
single colony from the bacterial transformation was picked with a sterile stick and swished into
solution. These cultures were allowed to grow overnight at 37 C while being shaken. 4 L of e.
coli was made by taking 2 mL of overnight culture and added to a fresh 1 L flask of LB. This
was repeated three more times. To each 1 L flask, 0.1 g of AMP was added. The flasks were
allowed to grow at 37 C while being shaken until the optical density reached 0.5.

10. 10
Isolation of Desired NBD Protein:
4 L of bacteria was evenly split into 1 L centrifuge tubes. These tubes were centrifuged
for 45 minutes at 18,000 RPM. The supernatant was discarded and the pellet was kept. The
pellet was added to a stainless steel lysis beaker. 100 mL of the lysis buffer was added and
stirred on ice for the remainder of the sonication. The lysis buffer was 50 mM sodium phosphate
200 mM sodium chloride pH 7. To this cell/lysis buffer solution 10 mg hen egg white lysozyme
was added and stirred for an additional 15 minutes. Soncation was done with a Fisher Scientific
550 sonic dismembrator. Once sonication was complete the products were centrifuged. The
supernatant was kept and the pellet was discarded. The supernatant was run over a nickel
column. A 5 mL NTA column was washed with 100 mL of the same lysis buffer. The
supernatant was poured slowly to not disturb the column matrix. Once the supernatant was
loaded, the column was washed with lysis buffer twice. While being washed 4 mL fractions
were collected. These fractions were referred to as the flowthrough. The column was then
washed with elution buffer while 4 mL fractions were again collected. The elution buffer was 50
mM sodium phosphate 200 mM Imidazole pH 7. Once these fractions had been collected an
SDS-PAGE gel was taken to confirm presence of protein after purification. The fractions that
contained protein were pooled and dialyzed overnight in the cold room in 10 mL of 20 mM
sodium phosphate, 200 mM sodium chloride pH 8.
Analyzing mumps F366W NDB on fluorometer:
The fluorometer used for each experiment was a Varian Cary Eclipse fluorescence
spectrophotometer. The purified NBD protein could be analyzed on the fluorometer because of
the inserted tryptophan. The first experiment ran was a temperature gradient. The excitation

11. 11
wavelength was set at 279.06 nm. The scan went from 300 nm - 450 nm. A single cell peltier
accessory was used control temperature of the cuvette in the fluorometer. 200 μL F366W NBD
was added to 800 μL 20 mM sodium phosphate, 200 mM sodium chloride pH 8 buffer. The
starting temperature was set at 4 C. Then the temperature was increased to 5 C. From 5 C the
temperature was increased in 5 C increments until 80 C was reached. Five minutes were allowed
for the NBD/buffer to reach equilibrium after each temperature change. The next experiment
was cooling the NBD/buffer solution from 80 C to 10 C in 5 C increments again allowing five
minutes between trials for equilibration. The cooling and heating experiments were then repeated
with the addition of 2.5 M TMAo solution in the buffer. The buffer used with TMAo was 20
mM sodlium phosphate, 200 mM sodium chloride, 2.5 M TMAo, pH 8. Data was collected and
analyzed with MatLab®.
The next set of experiments run on the fluorometer was the chemical titrations of the
NBD protein with urea and TMAo. The concentration of mutant NBD protein was 67 μM, and
during the chemical titrations the concentration of mutant NBD was 6.7 μM. Titrating the NBD
with urea was done by preparing twelve numbered 1.5 mL eppendorf tubes. Each tube had 30
μL NBD protein added to it. To each tube the appropriate volume of urea was added. The
concentrations of urea ranged from 0 - 8 M. For each trial the excitation wavelength was 279.06.
The scan started at 300 nm and went to 450 nm. The slits were set at 5 mm. The PMTs were set
at 600 V. Between each trial the cuvette was washed with a cuvette washing apparatus hooked
up to a vacuum pump. The wash was always water followed by ethanol followed by acetone.
The TMAo titration was carried out in a similar manner. There were twelve numbered 1.5 mL
eppendorf tubes. Each tube received 30 μL of protein. To each tube the appropriate volume of
TMAo was added. The concentrations of TMAo ranged from 0 - 2.3 M. The flurometer set up

12. 12
was the same as the urea titrations. Again, in between each run, the cuvette was washed
following the same procedure. Data was analyzed with Microsoft Excel.
Analyzing mumps F366W NBD via CD:
The CD used for all of the CD experiments was an OLIS DSM-20 CD. Prior to running
the samples on the instrument the protein had to be concentrated. Concentrating the protein was
done via a Centricon centrifugal protein concentration kit. The protein was concentrated
approximately three-fold. There were two batches of samples prepared to run on the CD. One
sample batch consisted of 30 μL concentrated F366W NBD to 270 μL 20 mM sodium
phosphate, 200 mM sodium chloride pH 8 buffer. The other batch of samples was 30 μL
concentrated F366W NBD to 270 μL 20 mM sodlium phosphate, 200 mM sodium chloride, 2.5
M TMAo, pH 8. These samples were kept on ice until they were run on the CD. The CD took
two types of measurements. Before any samples were measured on the CD a scan of the buffer
was taken first. This buffer scan was subtracted from the average values obtained from the
actual experiment before plotting the data. The first type of measurement was a scan looking for
changes in alpha helical structure with and without TMAo. This scan went from 260 nm to 205
nm with an integration time of 12 seconds. There were a total of five scans taken. The average
values of the scan are reported in the results section. The next measurement taken by the CD
was a thermal melt of the protein with and without TMAo. The melt went from 5 C to 80 C in
two degree increments. The protein was allowed to equilibrate for two minutes between each
temperature change. The reading was taken at 222 nm. The data was averaged and plotted via
Microsoft Excel.

13. 13
Results:
Mutagenesis via PCR
Figure 3: Sequencing data highlighting successful F366W mutation
Figure 3 shows the sequencing data. This data was generated by sending a PCR product to
another laboratory to be sequenced. The red box highlights the successful F366W mutation.
Protein Purification
Figure 4: SDS-PAGE gel illustrating successful purification of mumps F366W NBD

14. 14
This figure is a picture of the SDS-PAGE gel showing that the mutated mumps NBD protein was
successfully purified. The lanes are as follows. Lane 1: Standard BSA protein ladder, lane 2:
post-sonicate mix, lane 3: column wash, lane 4: supernatant post sonication/centrifugation, lane
5: column flow-through, lane 6: first 4 mL fraction, lane 7: second 4 mL fraction, lane 8: third 4
mL fraction, lane 9: fourth 4 mL fraction, lane 10: fifth 4 mL fraction. This figure also shows
that the protein was most concentrated in lanes 7 and 8. After purification the fractions that
contained protein were pooled.
Fluorescence Melts and Titrations
Figure 5: Raw excitation and emission spectra of mumps F366W NBD
This figure shows raw fluorescence data showing excitation (red) and emission (blue) of mumps
F366W NBD during a thermal melt. The temperature range was from 10 C to 60 C in ten degree
increments, then a jump from 60 C to 80 C. This jump was intended to see if there was a large
decrease in signal. It is clear that the fluorescence decreases with increasing temperature.

15. 15
Figure 6: Maximum fluorescence intensity plotted against temperature for mumps F366W NBD
This plot is the excitation and emission maximums from figure four plotted together against
temperature. This shows a clear decrease in signal intensity as temperature increased.
Figure 7: Excitation and emission spectra of mumps F366W mumps NBD in 2.5 M TMAo
Mumps NBD Fluorescence with 2.5 M TMAo
Fluorescence(a.u.)

16. 16
This plot shows the excitation (red) and emission (blue) of mumps F366W NBD during a
thermal melt with the addition of 2.5 M TMAo. This plot also follows the same thermal melt
procedure as figure 4. The peak at approximately 420 nm is a fluorescent impurity in the stock
TMAo solution. This fluorescent impurity did not affect results in any way.
Figure 8: Comparison of max fluorescence intensity with and without TMAo
This figure shows a comparison of max fluorescence intensity with and without TMAo. It is
clear there is an increase in intensity at each data point. This figure illustrates an increase of
signal intensity with and without the addition of TMAo
Peak Fluorescence Comparison with and without TMAo
Fluorescence(a.u.)
+ TMAo
- TMAo

17. 17
Figure 9: Melt of mumps F366W NBD in the presence of 2.5M TMAo
This plot shows the emission of mumps F336W NBD recorded at 339 nm. The F366W NBD
was in 2.5 M TMAo during this thermal melt. The temperature range for this melt was from 4 C
to 5 C, then 5 C to 80 C in five degree increments.
Figure 10: Normalized melt comparison with and without 2.5 M TMAo

18. 18
This plot is a normalized comparison of a thermal melts with and without TMAo. This plot
followed the same temperature range as in figure 8. The plot was created by plotting the
normalized maximum fluorescence intensity against temperature in Kelvin. This plot shows a
clear shift in intensity when the mumps F366W NBD was in the presence of 2.5 M TMAo.
Figure 11: Chemical titration of mumps F366W NBD from 0 - 7.2 M Urea
Figure 10 shows a plot of a mumps F366W NBD titrated with urea. The urea titration ranged
from 0 M urea to 7.2 M urea. The plot was generated using maximum fluorescence and plotting
the corresponding intensities against the concentration of urea used. This plot shows a slight
decrease in intensity from 0.4 M urea to 1.6 M urea.
0
20
40
60
80
100
120
140
160
0 1 2 3 4 5 6 7 8
Fluorescence(a.u.)
[Urea] (M)
Urea Titration Measuring Max
Fluorescence Intensity

19. 19
Figure 12: Chemical titration of mumps F366W NBD from 0 - 2.3 M TMAo
This figure shows a plot of mumps F366W NBD titrated with TMAo. The concentration range
of TMAo ranged from 0 M to 2.3 M. Again, fluorescence intensity was recorded at 339 nm and
plotted against the corresponding concentration of TMAo used. This plot shows a slight increase
in intensity from 0 M TMAo to 0.63 M TMAo.
Figure 13: Chemical titration of mumps F366W NBD from 0 - 3.2 M Urea
0
50
100
150
200
250
300
0 0.5 1 1.5 2 2.5
Fluoresence(a.u.)
[TMAo] (M)
TMAo Titration at 4C Measuring Max
Fluorescence Intensity
0
20
40
60
80
100
120
0 0.5 1 1.5 2 2.5 3 3.5
Fluorescence(a.u.)
[Urea] (M)
Fluorescence vs. [Urea] at 342 nm

20. 20
Figure 13 is a plot of another mumps F366W NBD urea titration. The concentration range for
this plot ranged from 0 M urea to 3.2 M urea. This was an attempt to recreate the slight decrease
in intensity seen in figure 10. The data appears to have a downward trend but it is inconclusive.
Figure 14: Chemical titration of F366W mumps NBD from 0 - 1.26 M TMAo
Figure 14 is a plot created by titrating mumps F366W NBD with TMAo. The concentration
ranges from 0 M TMAo to 1.26 M TMAo. This was an effort to obtain data illustrating the
increase in fluorescence seen in figure 11. There seems to be a slight increase in intensity in this
plot as well.
0
50
100
150
200
250
0 0.2 0.4 0.6 0.8 1 1.2 1.4
Fluoresence(a.u.)
[Urea] (M)
Fluorescence vs. [TMAo] at 338nm

21. 21
CD and CD Melts
Figure 15: CD spectra of mumps F366W NBD
This figure shows the CD spectra of the mumps F366W NBD. There is evidence of alpha helical
structure between 223 and 217 nm.
Figure 16: CD comparison with and without TMAo
-400
-200
0
200
400
600
800
200 210 220 230 240 250 260
MolarEllipticity(degcm2/dmol)
Wavelength (nm)
F336W CD Spectra
-800
-600
-400
-200
0
200
400
600
800
200 220 240 260 280
MolarEllipticity(degcm2/dmol)
Wavelength (nm)
F66W CD Comparison
-TMAo
+TMAo

22. 22
This plot is comparing the CD data with and without TMAo of the mumps F366W NBD. There
is a fair amount of noise with the TMAo sample which resulted in the inconsistent data. This
noise is attributed to the presence of free TMAo in the sample which disrupts some of the chiral
light that would otherwise hit the protein.
Figure 17: Melt of mumps F366W NBD
This plot shows a melt of the mumps F366W NBD. Each data point was measured at 222 nm to
observe the loss of alpha helical structure. From the plot it is apparent there is a loss of signal for
part of the melt.
-900
-800
-700
-600
-500
-400
-300
-200
-100
0
-5 5 15 25 35 45 55 65 75 85
MolarEllipticity(degcm2/dmol)
Temperature (C)
F366W Melt

23. 23
Figure 18: Melt comparison with and without TMAo
This plot shows the melt data for mumps F366W NBD with and without TMAo. In both cases
data was gathered at 222 nm to monitor loss of alpha helical structure. There was a loss of signal
in both cases.
-1200
-1000
-800
-600
-400
-200
0
0 20 40 60 80 100
MolarEllipticity(degcm2/dmol)
Temperature (C)
F66W Melt Comparison
-TMAo
+TMAo

24. 24
Discussion:
The overall goal of this experiment was to introduce a fluorescent probe into the core of
the mumps NBD and compare secondary structure formation to tertiary structure formation via
fluorescence spectroscopy and CD. Figure 3 shows the sequencing data showing the successful
of the native phenylalanine to tryptophan at the 366 position. This mutation was coined F366W.
Figure 4 shows the presence of the purified F366W NBD. This purified protein was used when
running various experiments. There are two bands in lanes 7-10. This was thought to be
attributed to the protein existing as partially folded and unfolded in solution.
Fluorescence:
Figure 5 was the first evidence that the fluorescent probe was successfully introduced
into the purified NBD. This is because there is fluorescent signal, without the fluorophore there
would not have been any signal present on the plot. This plot also shows what little tertiary
structure is present without TMAo can be removed by adding heat to the protein. The
fluorescence signal decreases as heat is increased indicating a loss of tertiary structure. Figure 7
shows the addition of TMAo during the same melt. There is a peak at 420 nm. This peak is a
fluorescent impurity in the stock TMAo solution. This peak did not affect results in any way
since the tryptophan fluorescence was recorded at 320-350 nm. Figure 8 is a comparison of
mumps F366W NBD peak fluorescence with and without TMAo. There is a clear increase in
fluorescence signal when TMAo was present in solution with the mumps NBD. This increase in
signal is attributed to more tertiary structure formation when TMAo is present in solution. The
increase in tertiary structure corresponds to the increase of signal during the thermal melt when
TMAo was present in solution. There was an increase in signal because the tertiary structure

25. 25
formation increased the overall stability of the protein. Figure 10 shows another thermal melt
comparison with and without TMAo. This data was normalized so it could be analyzed on the
same plot. This experiment was an effort to fit a standard sigmoid denaturation curve to the data.
The protein did not exhibit standard two-stage denaturation. It did not follow a standard two-
stage denaturation because the protein exists as partially folded and unfolded when in its native
conditions. The red line shows the protein in native conditions and the curve does not look
sigmoid. There is a decrease in signal so what little tertiary structure existed was removed via
temperature. When TMAo is present in solution with the protein, the protein is thought to
proceed to the folded configuration. The blue line illustrates the same melt but the protein was in
the presence of TMAo. There is a shift in signal and the curve appears more sigmoid. It is
thought that TMAo forces the protein to fold and take on tertiary structure because TMAo
creates unfavorable interactions between the TMAo and the polypeptide backbone of the protein
(Kingston et. al.). This shift in fluorescence intensity is consistent with the formation of more
tertiary structure when TMAo is present.
Figure 11 shows a chemical titration of mumps F366W NBD with urea. The
concentration of urea was increased from 0 - 7.2 M. There is a loss of signal from 0.4 M to 4 M
urea. This loss of signal corresponds to a loss of tertiary structure. This data was expected since
urea is known to denature protein. The signal increases steadily after the urea concentration rose
above 4 M. This phenomenon has not been explained. Figure 12 shows a chemical titration of
the mumps F366W NBD with TMAo. The concentrations ranged from 0 - 2.3 M. There is a
clear increase in signal intensity from 0 to 0.63 M. After 0.63 M the signal is roughly the same.
This data shows that TMAo does induce more tertiary structure formation since the signal
increased when the TMAo concentration was increased in solution. Figure 13 was an attempt to

26. 26
recreate the drop in fluorescence signal between 0.4 – 4 M seen in figure 11. There is a
downward trend to the data but the data is inconclusive. Figure 14 was an attempt to recreate the
upward trend between 0 – 0.63 M seen in figure 12. There is an upward trend to the data. It was
thought that most of the tertiary structure formation was completed when at least 1.26 M TMAo
was present in solution with the NBD.
CD:
Figure 15 shows CD data of the mumps F366W NBD. There was the presence of alpha
helix shown by the dip in signal at 223 and 217 nm. This is an important figure because it shows
that the presence of the tryptophan in the hydrophobic core did not affect the formation of alpha
helical secondary structure formation. Figure 16 shows the comparison of the CD data with and
without TMAo. The TMAo spectra shows more alpha helical signal denoted by the larger drop
in signal at 220 nm. There was a fair amount of noise during the TMAo trials. Since the protein
was thought to be folded by 1.4 M TMAo, a future experiment would be to run the same
experiment again with 1.4 M TMAo present in solution to get clearer data. Figure 17 shows the
CD spectra of a thermal melt of mumps F366W NBD measured at 222 nm. The signal was
measured at 222 nm to observe the alpha helical structure of the protein. As temperature
increased there was a decrease in signal to 30 C. After 30 C the signal increased. This
phenomenon has not been explained. Figure 18 shows a melt comparison of the mumps F366W
NBD with and without TMAo. The melt with TMAo had a greater loss of signal. This is
consistent because the TMAo should have been creating tertiary structure which would stabilize
the secondary structure formation. Again, both plots show an increase in signal as the
temperature continues to increase. Further investigations into these phenomena are needed.

27. 27
Next Steps:
Since the F366W mutation was a success and it was confirmed via CD that the mutation
did not affect secondary structure, this mutation can be used in a number of ways. This mutation
can be utilized to characterize further mutants of the protein via fluorescence since it is a quick
and inexpensive way to get high quality data. Random mutants could be created using PCR with
the DNA template containing the F366W mutation and then characterized on the fluorometer.
Along this same line of thought the development of a high-throughput screening method could
be created. Utilizing a plate reader attachment for the fluorometer a 96 well plate could be used.
The 96 well-plate would allow for multiple measurements taken in a small amount of time. This
method could be used in tandem when screening multiple mutants at once. The mutants with
high signal would have more tertiary structure formation than those without tertiary structure
formation.
Another interesting experiment that could be done is to take a CD spectrum of the
aromatic region. If this method could be developed it would shed insight into the environment
around the tryptophan in the hydrophobic core. This data could be compared to that of the CD
taken from 260 - 205 nm to see how the molten state is coupled with the folded state. This
would require many hours in method development since getting CD data in the aromatic region
is notoriously difficult.
The last thing that needs attention is the answer to why the signal increases in both the
fluorescence and CD. The signal was thought to decrease when the protein was titrated with
higher concentrations of urea. Instead, the signal increased after the concentration was above 4
M urea. This would mean that somehow urea is encouraging tertiary structure formation. This is
probably not the case, so some investigation as to why this happens would be useful and

28. 28
informative. When the protein was melted and observed via CD the same thing happened. After
roughly 30 C the signal increased indicating more secondary structure formation. This is
counterintuitive since one would think the protein would continue to denature at higher
temperature as illustrated by fluorescence data. It would be interesting to know why the tertiary
structure loses signal at high temperatures but the secondary structure gains signal.

29. 29
References:
 Bickle, T. et al. (1982) in Nucleases eds Linn, S.M. and Roberts, R.G. (CSH, NY) p. 95-
100.
 Delidow, Beverly, et al. "Polymerase Chain Reaction." Methods in Molecular Biology.
15. (1993): 1-29. Web. 26 Mar. 2013. <http://link.springer.com/protocol/10.1385/0-
89603-244-2:1>.
 Heckman , Karin L. "Gene Splicing and Mutagenesis by PCR-driven Overlap Extension."
Nature Protocols. 2.4 (2007): 924-932. Print.
 Ho, Steffan, et al. "Site-Directed Mutagenesis by Overlap Extension Using the
Polymerase Chain Reaction." Gene. 77. (1989): 51-59. Print.
 Kingston, Richard, et al. "Structure of the nucleocapsid-binding domain from the mumps
virus polymerase; An example of protein folding induced by crystallization." Journal of
Molecular Biology. 394.4 (2008): 719-731. Web. 26 Mar. 2013.
<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2693374/>.
 Weeks, Stephen, et al. "Ligation Independent Cloning Vectors for Expression of SUMO
Fusions." Protein Expression and Purification. 53. (2007): 40-50. Print