Laboratory Exercise
Myoglobin Structure and Function: A
Multiweek Biochemistry Laboratory
s
Projectw
Todd P. Silverstein*
Sarah R. Kirk
Scott C. Meyer
Karen L. McFarlane
Holman
From the †Department of Chemistry, Willamette University, Salem,
Oregon, 97301, From the
Abstract
We have developed a multiweek laboratory project in
which students isolate myoglobin and characterize its
structure, function, and redox state. The important laboratory techniques covered in this project include sizeexclusion chromatography, electrophoresis, spectrophotometric titration, and FTIR spectroscopy. Regarding protein structure, students work with computer modeling
and visualization of myoglobin and its homologues, after
which they spectroscopically characterize its thermal
denaturation. Students also study protein function (ligand
binding equilibrium) and are instructed on topics in data
analysis (calibration curves, nonlinear vs. linear regression). This upper division biochemistry laboratory project
is a challenging and rewarding one that not only exposes
students to a wide variety of important biochemical laboratory techniques but also ties those techniques together
to work with a single readily available and easily characC 2015 by The International
terized protein, myoglobin. V
Union of Biochemistry and Molecular Biology, 43(3):181–
188, 2015.
Keywords: inquiry-based teaching; biophysical methods; protein
structure function and folding; computational biology; active
learning; laboratory exercises
Introduction
The relationship between protein structure and function
has been a crucial aspect of biochemistry for the last 50
years or more. In the modern undergraduate biochemistry
course, discussion of the isolation, characterization, structure, and function of proteins takes up over one-fourth of
the typical one-semester course. Laboratory projects focusing on computer modeling of protein structure have been
increasingly common in the literature since about 2000
[1–7]. Almost all biochemistry laboratory texts feature projects in which students isolate a protein from a natural
source, purify it, and then assay its biochemical activity
[8–15]. In addition to these independent projects that last
223 laboratory periods and are centered on individual
w
s Additional Supporting Information may be found in the online
version of this article.
Scott C. Meyer’s current address is: Department of Chemistry,
Benedictine University, Lisle, IL, 60532
*Address for correspondence to: Department of Chemistry, Willamette
University, Salem, OR 97301. E-mail: tsilvers@willamette.edu
Received 25 July 2014; Accepted 2 November 2014
DOI 10.1002/bmb.20845
Published online 27 February 2015 in Wiley Online Library
(wileyonlinelibrary.com)
Biochemistry and Molecular Biology Education
enzymes, several semester-long biochemistry laboratories
devoted to a single protein or enzyme have been published
[8, 16–25]. Although the thematic unity of working on one
specific protein for the whole semester is attractive, not all
important experimental methods can be applied to a single
protein.
Therefore, instead of designing an entire course around
one protein, we decided to embed an extended protein
structure/function project within our semester-long upper
division laboratory course, Experimental Biochemistry I
(CHEM 346). We devote six 3-h laboratory periods in this
course to an extended project on myoglobin (Mb). The
remainder of the course includes a number of other multilab period projects that introduce our students to experimental methods such as HPLC, GCMS, phospholipid membrane dynamics, laser light scattering, spectrophotometric
and fluorimetric determination of enzyme activity, and
electrochemical biosensors.
The heme protein myoglobin is found in most muscle
tissue. Like hemoglobin (Hb), Mb contains a heme-bound
Fe(II) cation that can be oxidized to the Fe(III) form
(metMb). For many decades it was believed that the main
function of the Mb-heme-Fe(II) cofactor was to bind O2, as
well as CO, N2, nitrite, and azide ligands. Recent evidence
suggests that in fact, the major biological function of Mb
may be to catalyze the nitrite/nitric oxide interconversion
181
Biochemistry and
Molecular Biology Education
in muscle [26, 27]. Unlike Hb, which is tetrameric and
binds oxygen cooperatively with a Hill coefficient of n 5 2.8,
Mb is monomeric and binds oxygen noncooperatively
(n 5 1). Mb is easily extracted from muscle tissue and spectrophotometric characterization of the Fe-heme is facile, as
it absorbs strongly in the visible and UV ranges. In fact, the
visible absorption of heme-coordinated Fe(II) in Mb is
responsible for the reddish appearance of fresh (reduced)
meat, whereas the visible absorption of heme-coordinated
Fe(III) in metMb is consistent with the brownish color of
old (oxidized) meat [28, 29].
We describe here our six-period project, in which students isolate Mb from ground beef [29], spectrophotometrically characterize its oxidized and reduced forms [29],
probe its ligand binding equilibrium [30], perform molecular modeling on its structure and that of its homologues
[31], and use FTIR to characterize its secondary structure
in the native and denatured forms [32]. We have based our
extended Mb project on the four papers cited above
[29–32], adapting and expanding the projects beyond what
is described in the original source papers. Specifically, we
have added new methods of analysis (e.g., SDS-PAGE),
adapted to changes in molecular modeling websites, and
added extensive quantitative data analysis and curve-fitting
components.
Student Learning Objectives:
1. Proficiency in carrying out the following biochemical
techniques: centrifugation and protein isolation; column
chromatography and SDS-PAGE (proteins); and use of
protein structure databases
2. Strengthened understanding of the following biochemical
topics: protein structure modeling, characterization, and
denaturation; protein homology and evolution; and
protein-ligand binding equilibrium
3. Increased competence in biochemical data analysis,
including: x-y scatter plots and curve fitting (both linear
and nonlinear regression); and calibration curves
4. Increased confidence and mastery in using the following
instruments: UV-Vis and FTIR spectrophotometers; gel
electrophoresis; and in silico computer modeling of protein structure
5. Enhanced ability to: employ critical thinking and scientific reasoning; speak and write effectively about scientific concepts.
Teaching and Laboratory Procedures
This extended laboratory project centered around Mb
requires six 3-h laboratory periods, which include some inclass time for data analysis. Detailed instructions are provided in the Supporting Information. In laboratory Period 1,
students extract Mb from ground beef, reduce, and oxidize
portions of the sample, use a Sephadex column to “desalt”
the excess oxidizing agent, and use UV-Vis absorbance to
182
characterize the reduced and oxidized samples. In Period 2
students employ SDS-PAGE to characterize the extracted
samples. In Period 3, azide binding to metMb is examined
via spectrophotometric titration. In Periods 4 and 5, students use online databases, websites, and software to characterize the structure of Mb and a selected novel homolog.
In the final period, students employ FTIR spectroscopy to
probe protein secondary structure in both the native and
thermally denatured conformations.
Students work in pairs throughout this experiment.
Because use of the FTIR spectrophotometer takes up all
3 h of Period 6, we can only schedule use of the instrument if students are paired. During Periods 1 and 3, each
student pair has access to its own small Thermo-Electron
Genesys-10 UV-Vis spectrophotometer. In Period 2, all
pairs mount their gels and run from a single electrophoresis power supply. In Periods 4 and 5, paired students sit
adjacent to each other, but each works on his/her own
desktop computer in our computer room. We have run
this project six times, every spring since 2009. Course
enrollment has varied from 7 to 20 students, averaging
10212.
Period 1, Part IA: Myoglobin Extraction,
Separation, and Spectral Characterization
To begin the project, students extract Mb from ground beef
in a single 3-h laboratory period, following a modification
of the protocol of Bylkas and Andersson [29]. In this part of
the project, students work from the original literature
source; their laboratory manual contains screenshots of
selected paragraphs taken directly from the Bylkas and
Andersson paper (see Supporting Information). A portion of
the extracted Mb is fully reduced from Fe(III) to Fe(II) with
dithionite (turning deep red) and a separate portion is fully
oxidized to Fe(III) with ferricyanide (turning brown). Following oxidation, the metMb is separated from excess ferricyanide using a Sephadex G-25 size-exclusion gel chromatography “desalting” column. The three forms of Mb
(original extract, reduced, and oxidized) can then be characterized spectrophotometrically. Reduced Mb features O2
bound to the Fe(II) center in the heme. It has prominent
peaks in the visible range at about 540 and 580 nm, and its
Soret band is at 417 nm with e417 5 128,000 M21 cm21
[29]. The oxidized metMb has H2O bound to the Fe(III)heme; its visible peaks appear at 505 and 620 nm, and its
Soret band is at 409 nm with e409 5 179,000 M21 cm21
[29]. Given the prominent changes in the wavelength and
molar absorptivities of the Soret band absorbance peaks, it
is straightforward to spectrophotometrically determine the
concentrations of Mb and metMb, and monitor changes in
Fe(II)/(III) redox chemistry and ligand binding.
Period 2: SDS-PAGE
In a subsequent 3-h laboratory period students use SDSPAGE to separate and visualize the protein components of
both the original Mb extract, and the desalted metMb
Myoglobin Structure and Function
FIG 1
SDS-PAGE gel of Mb solutions (representative student results). Lanes 1 and 8: Novex protein standards; Lanes 2 and 9: PP1 protein standards; the
three smallest standards are 10, 15, and 20 kDa;
Lane 3: Mb (horse skeletal muscle, Sigma M-0630);
Lane 4: supernatant of ground beef extraction;
Lane 5: supernatant 1 dithionite; Lanes 6, 7, and
10: 10, 20, and 15 mL (respectively) of supernatant 1 ferricyanide, Sephadex G25-desalted. In
Lanes 327 and 10, MW(Mb) 5 16.2 6 1.8 kDa; in
Lanes 427 and 10, the Mb band accounts for
20 6 3% of the protein in each lane.
column eluent. Here students are introduced to electrophoresis, including loading and running the gels, quantifying
TM
the bands (using GelDoc ), and calculating molecular
weights and protein composition of each mixture (Fig. 1).
Students determine the molecular weight of Mb in their
mixtures and compare it to the literature value (17.6 kDa
for Sigma M-0630); they also determine the percentage of
Mb in each mixture (20 6 3%), showing that the desalting
Sephadex G-25 column merely serves to remove the excess
ferricyanide, but does not appreciably separate any of the
proteins in the mixtures.
Period 3, Part IB: Myoglobin Function: Ligand
Binding
Students perform a spectrophotometric titration to monitor
the binding of azide to Fe(III) in metMb and characterize
the azide/metMb binding equilibrium. This modification of
the experiment described by Marcoline and Elgren [30]
also takes a single 3-h laboratory period. By following
changes in the 540 (or 580) nm peak, students can calculate r, the ratio of bound ligand to total protein, and [L]free,
the equilibrium concentration of free (unbound) azide
ligand. Fitting the r vs. [L]free plot to the equation for hyperbolic saturation [Eq. (1)] allows a best fit determination of
the protein-ligand dissociation equilibrium constant, Kd,
and the number of bound ligand molecules per protein, n.
r5
n
11Kd =½Lfree
5
n½Lfree
½Lfree 1Kd
(1)
Typically, a linearized form of the results, such as a
Scatchard plot or a double-reciprocal plot, would be used
to determine n and Kd. However, it has been pointed out
Silverstein et al
FIG 2
MetMb/azide spectrophotometric titration (representative student results). Total initial [Mb] 5 16.1
mM; changes in metMb absorbance at 543 nm
were followed upon addition of azide aliquots
amounting to final [N3-] 5 2 – 948 mM. (a) r vs.
[N3-]free fit to the equation for hyperbolic saturation
gives Kd 5 35.6 6 2.0 mM, n 5 0.973 6 0.013, and
R2 5 0.997. (b) The double-reciprocal plot, 1/r vs. 1/
[N32]free gives intercept 5 1.9 6 0.8, slope 5 21 6 3
mM21, and R2 5 0.83 for all 12 points (solid line);
this yields calculated values of n 5 0.52 6 0.22 and
Kd 5 11 6 5 mM. Omitting the two lowest concentration points (1.1 and 3.9 mM free azide) gives intercept 5 1.075 6 0.023, slope 5 32.4 6 0.5 mM21, and
R2 5 0.998 (dashed line); this gives calculated values of n 5 0.930 6 0.020 and Kd 5 30.1 6 0.8 mM. A
Scatchard plot of these data (r/[N32]free vs. r) gives
similar large error in the two lowest concentration
points. [Color figure can be viewed in the online
issue, which is available at wileyonlinelibrary.com.]
many times that linearizing data often magnifies error in
the derived parameters [33–35]. Students are therefore
instructed to analyze their results using both linear regression of linearized data and nonlinear regression of the
hyperbolic curve, and discuss which is more reliable. Students find that n does indeed equal one for azide binding to
metMb, and Kd is in the 10–100 mM range (see Fig. 2), in
agreement with the literature (4–80 mM) [36]. Figure 2b
clearly shows how error is magnified in the doublereciprocal plot for the two lowest concentration points.
Even omitting these points gives errors, relative to the
183
Biochemistry and
Molecular Biology Education
FIG 3
Mb structure comparison. (A) solved structure of sperm whale Mb, pdb file 1VXA; (B) Swiss model predicted structure
of Mb from unicorn icefish (Channichthys rhinoceratus), UniProt file E5G628. Color key for amino acid side chain types
at the protein surface: white 5 hydrophobic; red 5 negative; blue5 positive; pink 5 polar neutral; yellow 5 aromatic;
green 5 proline. Note the heme binding cavity in the center of both structures; most of the surface directly above this
cavity is identical in both (A) and (B). Besides these and other similarities, six key differences between the two forms of
Mb are marked above.
values derived from nonlinear regression, of 215% and
24.5% in Kd and n, respectively; the Scatchard plot is a little better, with errors of 29% and 21.7%, respectively.
pare the 3D structure of original protein to that of its novel
homolog.
Periods 4 and 5
Part II: Myoglobin Three-Dimensional Structure:
Native vs. Denatured
Myoglobin Structure on the Web
In 1998, Leon et al. published a biochemistry laboratory
project in which students used internet websites and freeware to model protein structure [31].1 Leon et al. describe
a project in which students select a protein whose structure
has already been solved and deposited in the Protein Data
Bank (PDB). They then carry out a series of four operations
on the internet: (a) use search programs to find a “novel
homolog,” i.e., a homologous protein whose structure has
NOT yet been solved; (b) submit the amino acid sequence
of the novel homolog to secondary structure prediction programs and compare secondary structures of the original
protein and its novel homolog; (c) submit the amino acid
sequence of the novel homolog to Swiss Model to obtain a
predicted three-dimensional structure; and finally (d) com-
1
In order to increase the diversity of protein structures examined, we have half of the students start with myoglobin, a predominantly alpha-helical protein, and the other half start with
the predominantly beta-sheet protein, lysozyme.
184
It takes students about 4.5 h to finish operations (a–d) listed
above. We have our students use the final 1.5 h in laboratory
period 5 to prepare figures for an oral presentation in which
they carefully characterize the major differences and similarities between the original protein and its novel homolog.
All of the websites described by Leon et al. have
changed dramatically in the nearly 2 decades since the
original publication. Some things have gotten easier. For
example, in 1998 one had to visit three separate websites
to obtain a protein’s structure coordinates (PDB), structure,
and function information (UniProt/SwissProt), and perform
homology searching (Blitz). Currently all of this information
can be obtained from the PDB website and links therein.
On the other hand, other aspects of the project have gotten
more difficult. The protein visualization program recommended by Leon et al., Rasmac, is woefully outdated; in
addition, a useful protein structure comparison program
that we discovered around 2000, Protein Explorer, has not
been supported since about 2006. However, an even better
freeware program, PyMOL, has since come into wide use
[2, 37238]. This program not only allows homologous proteins to be aligned and visualized, but it also has many different viewing modes that can be implemented. We encourage students to explore the capabilities of PyMOL on their
Myoglobin Structure and Function
own, but we require that they use at least three different
views to compare their two proteins: van der Waals surface; electrostatic surface; and polarity/hydrophobicity (Fig.
3). The former two views are built into PyMOL, and the latter is provided by the “color_by_restype” script written by
researchers at Queens University in Kingston, Ontario [39].
Structure of Native and Unfolded Myoglobin
Characterized by FTIR
In the sixth and final 3-h laboratory period of our Mb
structure and function project, students use FTIR to
observe spectroscopic differences in native, folded proteins that are predominantly alpha-helical (e.g., Mb) and
those that are predominantly beta-sheet (e.g., lysozyme).
Students can determine the actual percent helix vs. sheet
by accessing structural information at the Protein Data
Bank (pdb) website, and compare this to the FTIR spectroscopic results that they get for each protein. They then
incubate these proteins at elevated temperatures and use
changes in FTIR peaks to characterize the thermal denaturation process (Fig. 4a).
This final sub-project is based on the experiment
described by Olchowitz et al. [32]. We have made two major
modifications in order to dramatically improve data analysis.
First, we found that in order to get the expected sigmoidal
changes in FTIR peaks with changes in temperature, careful
baseline subtraction, and normalization is required. In Fig.
4a the spectra are baselined at 1700 cm21 and normalized
at the a-helix amide I peak, 1651 cm21. We describe these
processes in detail in our Supporting Information. Second,
we encourage our students to analyze their results in order
to obtain a denaturation temperature, Tden (also called TU or
Tmelt in the literature); this is the temperature at which the
equilibrium between native/folded protein and denatured/
unfolded protein features a 50/50 mixture.
The denaturation equilibrium, in which the folded,
native protein (N) denatures, and unfolds (U) can be characterized by an equilibrium constant (Kden 5 KU 5 [U]eq/
[N]eq, and by an enthalpy and entropy for the process, DH U
and DS U. Given a change in some FTIR spectroscopic
parameter y as the protein unfolds, the dependence of the
denaturation equilibrium on T, TU, and DH U, is shown in
Eq. (2) (see Supporting Information, Appendix 4):
y5
yloT 1yhiT e
11e
DH 1
U 1
R ðTU 2T Þ
DH 1
U 1
R ðT U 2 T Þ
Compound
Danger
Hazard rating
NPFA hazard 5
Eye damage; skin
2, GHS eye
irritation Harmful
if swallowed, inhaled,
damage 5 1
or absorbed through skin
Sodium azide Fatal if swallowed, or
GHS dermal
absorbed through skin
toxicity 5 1,
GHS oral
toxicity 5 2,
GHS aquatic
toxicity 5 1
Potassium
Harmful to aquatic life
GHS aquatic
ferricyanide
toxicity 5 3
Sodium
dithionite
(bisulfite)
Common Pitfalls
Project
Section
Part IB:
Part IIA
Part IIB
Problem
At lowest azide
concentrations,
calculated
concentration
of free azide is
negative.
Amino acid
sequence
differences
between UniProt
vs. PDB sites.
Swiss Model fails
Protein aggregates
at high T
Determining the
effect of T on
protein structure:
(2)
Using nonlinear regression to fit the data in Fig. 4 gives
best fit values for both TU and DH U. Students can then
compare their results to literature values for Mb unfolding
in aqueous buffer: TU 5 81–83 C, DH U 5 100–130 kcal/mol
[40242]. Student results suggest (Fig. 4b) that in D2O, Mb
unfolds at a lower temperature (65–70 C) than it does in
aqueous buffer, but with a DH U that is the same or higher
(100–300 kcal/mol).
Silverstein et al
Hazards
Solution
Set these free azide
concentrations
to the minimum
value, zero.
Find and omit
the N-terminal
signal sequence.
Select a different
template
For T 85 C, incubate
for only a few minutes.
Have students consider as
many spectral changes
as seems productive. We
plotted the relative
absorbances of
three different peaks
in Fig. 4b, but
the shift in kmax of
the amide I peak
can also be
informative [32].
Student Reports
For Part I (Mb extraction/characterization and azide binding), students write informal lab reports in which they
address questions focused on data analysis to hone their
critical thinking skills. They use Beer’s Law to calculate Mb
concentrations, and discuss how heme-iron oxidation alters
185
Biochemistry and
Molecular Biology Education
FIG 4
FTIR characterization of Mb thermal denaturation (representative student results). (a) Relative absorbance (Ak/A1651) of
60 mg/mL Mb in D2O, at eleven different temperatures, 22–87 C. Note the appearance, above 64 C, of shoulders at 1618
and 1680 cm21, and an aggregated protein peak at 1570 cm21. (b) The three wavelengths in the spectra in (A) that
change dramatically with T are plotted: R shoulder, A1618/A1651 (black circles); L shoulder, A1680/A1651 (blue diamonds);
and aggregated protein peak, A1570/A1651 (blue diamonds). Fitting the R shoulder points using Eq. (2) gives
DH U 5 290 6 70 kcal/mol and TU 5 65.79 6 0.15 C; fitting the L shoulder and aggregated protein points gives
DH U 5 120 6 50 kcal/mol and TU 5 68.3 6 1.2 C. [Color figure can be viewed in the online issue, which is available at
wileyonlinelibrary.com.]
UV-Vis absorbance. Students use SDS-PAGE standard protein mobilities to prepare a calibration curve to determine
molecular weights; they compare their Mb molecular
weight to the literature value, and also establish the fraction of Mb in their samples. For Part IB, students use both
linear and nonlinear regression to determine Kd and n for
azide binding to metMb.
For Part II (protein structure modeling and thermal
denaturation) students prepare an oral presentation in
which they carefully characterize the major differences and
similarities between Mb and their chosen novel homolog.
They use FTIR amide I peaks to distinguish between proteins that are primarily a-helix versus b-sheet, and to follow temperature-driven changes in secondary structure.
Finally, students use nonlinear regression of their FTIR
peak versus T data to determine DH U and TU and compare
to literature values.
186
Student Feedback/Assessment
We assessed this project (and the entire Experimental Biochemistry course sequence) in two ways: with a pre/post
laboratory skills exam, and with a survey of student
alumni. Of the 33 questions on the exam, 8 covered concepts related specifically to this protein structure laboratory project (e.g., protein separation, structure characterization, ligand binding). On these eight questions, student
performance improved from 53% correct before the start of
the course, to 85% correct at the end (and 91% after the
second semester of the course sequence). In addition to
these exam results, as instructors of the course, we
observed consistently an increase in both skills and confidence level as students moved through the course.
We carried out a survey of our alumni to assess the
impact of this laboratory sequence on their perceived knowledge of techniques, biochemical topics, and instrumentation.
Myoglobin Structure and Function
We had 18 people respond out of a total 23 accessible Biochemistry Track alumni over 4 years. From the survey
results, it was clear that this laboratory was successful in
meeting our intended goals. Students felt confident in their
understanding of all the major biochemical topics covered, as
well as in their ability to analyze biochemical data. One student wrote the following about what s/he got out of the course:
“I have a solid foundation for lab technique and good lab
practices upon which to build, and have an ability to learn
new protocols quickly to become both proficient and efficient
with them.” Interestingly, while students rated highly their
knowledge of all of the instruments that they employed in the
course, they expressed the greatest confidence in their ability
to use the instruments that they were repeatedly exposed to.
In particular they rated the course highly for helping them to
develop critical thinking skills and scientific reasoning (4.6 on
a Likert scale of 1 to 5) and to speak about scientific concepts
(4.5). Students rated satisfaction with their ability to write scientifically even more highly (4.8). In short, the results of our
pre/post laboratory skills exam and our alumni survey allow
us to conclude that this project did indeed achieve the learning objectives that we aimed for (see list above).
Further Extensions
We decided to focus our protein structure/function project on
myoglobin because it is readily obtained (from ground beef
and in purified form), easily characterized (by spectrophotometry), and has been used in a number of published biochemistry laboratory projects. There are undoubtedly many other
proteins that fit this bill and could be substituted for Mb.
We have on occasion included another interesting laboratory project dealing with protein denaturation. As published [43], this project uses various concentrations of guanidine hydrochloride to denature chymotrypsin. Fluorescence
spectroscopy is then used to probe both the structure of the
protein as well as its enzymatic activity; the unique aspect of
this particular laboratory project is that students simultaneously assay changes in both protein structure and function
as a result of denaturation. Although we have not adapted
this technique to Mb, we believe that it would not be difficult. In fact, due to the intrinsic absorbance of the Fe(II/III)
center in Mb, UV-Vis spectrophotometry could probably be
used in place of fluorimetry. In order to probe the effect of
denaturation on protein structure, one would simply follow
changes in the UV-Vis (or fluorescence) spectrum with guanidine concentration. To probe the effect on protein function, one would select a few representative guanidine concentrations and perform azide binding titrations at each
one. Presumably, as denaturation increases, the Kd for azide
binding should also increase (weaker binding).
Summary
We have developed an extended project for an upper division biochemistry laboratory course that uses Mb as a cen-
Silverstein et al
tral theme to investigate several important experimental
techniques, as well as the relationship between the structure and function of a protein. Over six 3-h laboratory periods, the students isolate Mb from muscle tissue, spectroscopically characterize the oxidation state of its heme
moiety and its ligand binding, analyze the protein’s structure via molecular modeling programs, and track its thermal denaturation with FTIR. We have expanded and combined previously described experiments [29232], and also
updated procedures to reflect changes in the availability of
structure analysis software like PyMOL [2, 37, 38]. This
project has the advantage of fulfilling two sometimes disparate goals in laboratory curriculum development: It
introduces students to a wide variety of biochemical techniques, while at the same time organizing the project around
a central, biologically relevant theme.
Acknowledgements
The authors thank Willamette University for its extensive
long-term support of laboratory curriculum development,
and our colleague Dr. Alison Fisher for her help with this
manuscript. This material is based upon work supported
by the National Science Foundation under Grant No. DUE1044737. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the
authors and do not necessarily reflect the views of the
National Science Foundation.
References
[1] Bain, G. A., Yi, J., Beikmohamadi, M., Herman, T. M., and Patrick, M. A.
(2006) Using physical models of biomolecular structures to teach concepts of biochemical structure and structure depiction in the introductory chemistry laboratory. J. Chem. Educ. 83, 1322–1324.
[2] Stockman, B. J., Asheld, J. S., Burburan, P. J., Galesic, A., Nawlo, Z.,
and Sikorski, K. F. (2014) Design and characterization of a Zn21-binding
four-helix bundle protein in the biophysical chemistry laboratory. J.
Chem. Educ. 91, 451–454.
[3] Schneider, T. L. and Linton, B. R. (2008) Introduction to protein structure
through genetic diseases. J. Chem. Educ. 85, 662–665.
[4] Lowery, M. S. and Plesniak, L. A. (2003) Some like it cold: A computerbased laboratory introduction to sequence and tertiary structure comparison of cold-adapted lactate dehydrogenases using bioinformatics
tools. J. Chem. Educ. 80, 1300–1302.
[5] Taylor, A. T. S. and Feller, S. E. (2002) Structural studies of phycobiliproteins from Spirulina: Combining spectroscopy, thermodynamics,
and molecular modeling in an undergraduate biochemistry experiment.
J. Chem. Educ. 79, 1467–1470.
[6] Ship, N. J. and Zamble, D. B. (2005) Analyzing the 3D structure of
human carbonic anhydrase II and its mutants using Deep View and the
Protein Data Bank. J. Chem. Educ. 82, 1805–1808.
[7] Sumter, T. (2009) Using JBC in the classroom. ASBMB Today. 10, 18.
[8] Farrell, S. O. and Ranallo, R. T. (2000) Experiments in Biochemistry, a
Hands-on Approach: A manual for the Undergraduate Laboratory,
Harcourt-Brace & Co., Orlando, FL, pp. 95–110; 133–142; 148–156; 169–
178; 189–206; 218–242; 253–260.
[9] Switzer, R. L. and Garrity, L. F. (1999) Experimental Biochemistry, WH
Freeman & Co., New York, pp. 135–162; 205–252.
187
Biochemistry and
Molecular Biology Education
[10] Waldmann, H. and Janning, P. (2004) Chemical Biology, A Practical
Course. Wiley-VCH, Weinheim, Germany, pp. 137–144.
[11] Boyer, R. (2000) Modern Experimental Biochemistry, 3rd ed., BenjaminCummings, San Francisco, CA, pp. 257–302, 357–370.
[12] Dryer, R. L. and Lata, G. F. (1989) Experimental Biochemistry, Oxford
Univ. Press, New York, pp. 375–441.
[13] Plummer, D. T. (1987) An Introduction to Practical Biochemistry, 3rd
ed., McGraw-Hill Book Co., London, pp. 160–162, 236–249.
[14] Stenesh, J. (1984) Experimental Biochemistry, Allyn & Bacon, Inc., Boston, pp. 55–77; 97–110, 135–236.
[15] Zeidan, H. M. and Dashek, W. V. (1996) Experimental Approaches in
Biochemistry and Molecular Biology, Wm. C. Brown Publishers, Dubuque, IA, pp. 29–78.
[16] Dryer, R. L. and Lata, G. F. (1989) Experimental Biochemistry, Oxford
Univ. Press, New York, pp. 375–408.
[17] Kirk, S. R., Silverstein, T. P., Holman, K. M., and Taylor, B. L. H. (2008)
Probing changes in the conformation of tRNAPhe: An integrated biochemistry laboratory course. J. Chem. Educ. 85, 666–673.
[18] Kirk, S. R., Silverstein, T. P., Holman, K. M., and Taylor, B. L. H. (2008)
UV thermal melting curves of tRNA in the presence of ligands. J Chem.
Educ. 85, 674–675.
[19] Kirk, S. R., Silverstein, T. P., Holman, K. M., and Taylor, B. L. H. (2008)
Metal-catalyzed cleavage of tRNAPhe, J. Chem. Educ. 85, 676–677.
[20] Kirk, S. R., Silverstein, T. P., Holman, K. M., and Taylor, B. L. H. (2008)
Fluorescence spectroscopy of tRNAPhe Y base in the presence of Mg
21 and small molecule ligands. J. Chem. Educ. 85, 678–679.
[21] Markwell, J. (1993) Focusing on a single enzyme. J. Chem. Educ. 70,
1018–1019.
[22] Craig, P. A. (1999) A project-oriented biochemistry laboratory course, J.
Chem. Educ. 76, 1130–1135.
[23] Wolfson, A. J., Hall, M. L., and Branham, T. R. (1996) An integrated biochemistry laboratory, including molecular modeling. J. Chem. Educ. 73,
1026–1029.
[24] Vincent, J. B. and Woski, S. A. (2005) Cytochrome c: A biochemistry
laboratory course. J. Chem. Educ. 82, 1211–1214.
[25] Saderholm, M. and Reynolds, A. (2011) Jmol-enhanced biochemistry
research projects. J. Chem. Educ. 88, 1074–1078.
[26] Cossins, A. and Berenbrink, M. (2008) Myoglobin’s new clothes. Nature.
454, 416–417.
[27] Hendgen-Cotta, U. B., Merx, M. W., Shiva, S., Schmitz, J., Becher, S.,
Klare, J. P., Steinhoff, H. J., Goedecke, A., Schrader, J., Gladwin, M. T.,
Kelm, M., and Rassaf, T. (2008) Nitrite reductase activity of myoglobin
regulates respiration and cellular viability in myocardial ischemiareperfusion injury. Proc. Natl. Acad. Sci. USA 105, 10256–10261.
[28] Linenberger, K., Lowery-Bretz, S., Crowder, M. W., McCarrick, R.,
Lorigan, G. A., and Tierney, D. L. (2011) What is the true color of fresh
meat? A biophysical undergraduate laboratory experiment investigating
188
[29]
[30]
[31]
[32]
[33]
[34]
[35]
[36]
[37]
[38]
[39]
[40]
[41]
[42]
[43]
the effects of ligand binding on myoglobin using optical, EPR, and NMR
spectroscopy. J. Chem. Educ. 88, 223–225.
Bylkas, S. A. and Andersson, L. A. (1997) Microburger biochemistry:
Extraction and spectral characterization of myoglobin from hamburger.
J. Chem. Educ. 74, 426–429.
Marcoline, A. T. and Elgren, T. E. (1998) A thermodynamic study of
azide binding to myoglobin. J. Chem. Educ. 75, 1622–1623.
Leon, D., Uridil, S., and Miranda, J. (1998) Structural analysis and modeling of proteins on the web: An investigation for biochemistry undergraduates. J. Chem. Educ. 75, 731–734.
Olchowitz, J. C., Coles, D. R., Kain, L. E., and MacDonald, G. (2002)
Using infrared spectroscopy to investigate protein structure. J. Chem.
Educ. 79, 369–371.
Martin, R. B. (1997) Disadvantages of double reciprocal plots. J. Chem.
Educ. 74, 1238–1240.
Silverstein, T. P. (2008) Quantitative determination of DNA-ligand binding: Improved data analysis by nonlinear regression. J. Chem. Educ. 85,
1192–1193.
Silverstein, T. P. (2011) Nonlinear and linear regression applied to concentration vs. time kinetic data from Pinhas’s sanitizer evaporation project. J. Chem. Educ. 88, 1589–1590.
Stone, J. R., Sands, R. H., Dunham, W. R., and Marletta, M. A. (1996)
Spectral and ligand-binding properties of an unusual hemoprotein,
the ferric form of soluble guanylate cyclase. Biochem. 35, 3258–
3262.
Bethel, C. M. and Lieberman, R. L. (2014) Protein structure and function:
An interdisciplinary multimedia-based guided-inquiry education module
for the high school science classroom, J. Chem. Educ. 91, 52–55.
Yeturu, K. and Chandra, N. (2011) PocketAlign: Novel algorithm for
aligning binding sites in protein structures. J. Chem. Inf. Model. 51,
1725–1736.
My PyMOL Script Repository: Available at: http://pldserver1.biochem.
queensu.ca/rlc/work/pymol/(checked Jan. 2015).
Mehl, A. F., Crawford, M. A., and Zhang, L. (2009) Determination of
myoglobin stability by circular dichroism spectroscopy: Classic and
modern data analysis. J. Chem. Educ. 86, 600–602.
Moriyama, Y. and Takeda, K. (2010) Critical temperature of secondary
structural change of myoglobin in thermal denaturation up to 130 C
and effect of sodium dodcyl sulfate on the change. J. Phys. Chem. B
114, 2430–2434.
Shih, P., Holland, D. R., and Kirsch, J. F. (1995) Thermal stability determinants of chicken egg-white lysozyme core mutants: Hydrophobicity,
packing volume, and conserved buried water molecules, Protein Sci. 4,
2050–2062.
Silverstein, T. P. and Blomberg, L. E. (1992) Probing denaturation by
monitoring residual enzyme activity and intrinsic fluorescence: An
undergraduate biochemistry experiment. J. Chem. Educ. 69, 852–855.
Myoglobin Structure and Function