Stress-Induced Protein Unfolding in Red Blood Cells and
Mesenchymal Stem Cells
Jonathon L. Meyer
Senior Comprehensive Paper
Department of Chemistry
The Catholic University of America
April 17, 2008
Protein Unfolding
Abstract
It has been hypothesized that induced protein conformation changes contribute to cellular
deformation. Knowing that structure maintenance is vital to protein function, studies were
conducted to reveal the effects of forced unfolding of protein tertiary and quaternary structures.
To obtain evidence that this process was effective, specific amino acid sites only present in the
unfolded conformation were labeled. In this paper, the folding-unfolding characteristics of
spectrin and non-muscle myosin II, found in red blood cells and Mesenchymal stem cells,
respectively, are reviewed. Quantitative mass spectrometry, sequential two-dye labeling, onedimensional
polyacrylamide
gel
electrophoresis,
densitometry,
Western
blots,
and
immunofluroescence labeling were among the methods used to identify the presence of unfolded
proteins in these cells. Within red blood cells, spectrin was found to unfold proportionally with
stress and temperature. Construct labeling and its kinetics also identified specific and exposed
cysteine residues. ATPase-dependent non-muscle myosin II in stem cells undergoes stress
required to activate other proteins. Inhibition of its ATPase activity proved to stop protein
expression and relax its coiled structure, shielding cysteine sites. Ultimately, the results provided
evidence that specific cytoskeletal proteins were stress-sensitive and prone to forced unfolding.
This led to the development of methods that identify sites where cell deformation can be
monitored.
2
Protein Unfolding
Introduction
The complete three-dimensional structure of a protein determines its unique function in
an organism and plays an essential role in preservation. When this structure collapses, or unfolds,
protein function is lost, leading to defects, including cell deformation.1 The stable conformation
of a protein provides a natural resistance to unfolding; however, when subjected to heat or other
forms of stress, non-covalent and certain covalent bonds break, leaving a dysfunctional strand or
conglomerate of amino acids. Identifying and understanding stress-sensitive protein unfolding
assists in locating the sites that promote cell deformation.
Protein Folding
Protein structure is considered and discussed at four different levels. The process by
which amino acid chains fold into functional proteins occurs through a series of mechanisms.2
The primary structure consists of a two-dimensional polypeptide strand composed of a specific
amino acid sequence. Three-dimensional arrangement begins with the secondary structure, and
several types exist in peptides and proteins. Hydrogen bonding between carboxyl and amino
groups within the polypeptide occur to form alpha helices, beta-pleated sheets, or loops. Alpha
helices are formed from a continuous region of the polypeptide chain. All their hydrogen bonds
are oriented in the same direction along the helical axis, giving a dipole moment that places a
slightly positive charge on the amino end and a partially negative charge on the carboxyl end. 3
This alignment naturally attracts ligands of opposite charges to corresponding ends of the
polypeptide chain. Beta-pleated sheets, on the other hand, consist of interacting strands from
several regions of a polypeptide chain that are built on top of one another. Chains in this
structure are either parallel or anti-parallel, indicating that all amino acids run in the same
direction (amino terminus to carboxyl terminus), or successive strands alternate directions. The
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Protein Unfolding
hydrogen bonding pattern between C=O---H—N groups in adjacent strands is irregular in the
parallel and regular in the anti-parallel arrangement.
Other secondary structures, such as loops, are loose and mobile, but are still well defined,
recognizable, and play a major role in protein function. Loops are generally caused by reversals
of the polypeptide chain due to hydrogen bonding between carboxyl and amino groups.3 When
these elements of proteins lie on the surface, they usually interact with surrounding molecules;
antigen binding sites, for example, reside on loops.4 Certain secondary structures are unique to a
protein, such as the helical structure of collagen. This extracellular protein contains three helical
chains comprised mostly of glycine and proline-hydroxyproline residues. Rather than the
presence of hydrogen bonds to maintain the structure, steric repulsion of the pyrrolidine rings
found in proline and hydroxyproline stabilize the helical shape. The three strands then wind
around each other through hydrogen bonding, creating a superhelix.3
Interaction between amino acids located at distant points in the primary structure that
may be spatially near each other is described within the tertiary structure. This involves the
folding of the alpha helices or beta-pleated sheets to create a compact conglomeration of
polypeptides into globules. This process places hydrophobic groups on the inside of the fold and
polar groups on the outside interacting with the environment. Myoglobin, for example, is folded
into eight α helices, with its interior composed almost entirely of nonpolar residues, such as
leucine, methionine, and valine.3 Hydrogen bonding within this nonpolar center becomes very
strong. In addition, when loops occur at active sites, in the tertiary structure, they have a special
role in catalysis as they carry key residues for binding and catalysis. A quaternary structure exists
for many proteins that function in multi-subunit form. Each subunit consists of a separate folded
polypeptide chain that joins together to complete protein conformation.3
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Protein Unfolding
Protein Unfolding
Certain proteins, such as cytoskeletal proteins, are designed to withstand external forces
to preserve their structure. Yet, natural processes, such as cell adhesion, produce small amounts
of force that potentially cause conformational changes in proteins that result in their unfolding.5
The precise mechanism by which this occurs is unknown and cannot be ascertained by thermal
or structural tests. However, under laboratory conditions, successful protein denaturation
simulations have been executed displaying the destruction of hydrogen bonds and salt bridges.6
While tests conducted at the cell level have shown no direct evidence that this unfolding occurs
due to external forces, direct, single-molecule measurements that analyze the mechanical
properties of individual proteins have shown unfolding through reversible extension in
cytoskeletal, motor, and matrix adhesion proteins, whereby the folded protein structure breaks
apart and unwinds to the secondary structure or primary peptide chain.1 In order to identify areas
that are force-sensitive, specific amino acids visible only in denatured proteins must be labeled
under live conditions. In a number of studies, a shotgun approach was carried out where the
method involved labeling cysteine molecules found in cell proteins that were subjected to
physiological stress.
Cysteine
The amino acid cysteine is a moderately hydrophobic molecule, similar in structure to
serine, with a sulfhydryl group replacing the hydroxyl group, as displayed in Figure 1, and a pK a
of 8.3. The presence of the sulfhydryl group makes the molecule more acidic, giving it greater
reactivity and allowing it to form disulfide bonds with other cysteine molecules. This latter
component plays a role in the stabilization of proteins.3
5
Protein Unfolding
Figure 1. Structure of the sulfhydryl amino acid, cysteine.3
When found within proteins, cysteine is often hidden by the tertiary or quaternary
structure, preventing it from being visualized by fluorescence imaging. However, previous
denaturation studies have successfully labeled the thiol moiety of the molecule in purified
proteins, one of them being the protein spectrin, found in red blood cells.1 Other experiments
have shown the catalyzed break down of disulfide bonds when single proteins experienced
forced unfolding. These results suggest that forced conformation changes in proteins can expose
hidden cysteines, particularly in cytoskeletal proteins.
An in situ “Cys shotgun” approach was carried out using sequential 2-dye-labeling of
specific cytoskeletal proteins in human red blood cells and Mesenchymal stem cells.
Fluorophores of different colors were utilized to identify then isolate the exposed cysteine
domains in the unfolded proteins. Red blood cells lend themselves to a more direct and basic
approach for forced unfolding under fluid stress, therefore these experiments were executed first,
followed by the Mesenchymal stem cells, which show denaturation under cell-generated stress.1
I. Red Blood Cells and Spectrin
Human red blood cells (RBC), or erythrocytes, have the capability to effectively deform
under the stress of blood flow, particularly when passing through small capillaries in tissue. The
cytoskeleton membrane of RBCs allows for this deformation to occur, and once stress levels
6
Protein Unfolding
decrease, the cell regains its normal shape.8 The mechanical strength of the RBC membrane is
provided by a hexagonal network composed of 6 spectrin dimers that extend from the central
actin protofilament, ankyrin, and protein 4.3 (Figure 2). The latter two proteins form a
suspension complex that act as the site for anchoring the actin-spectrin network to the lipid
bilayer. The protein spectrin is comprised of α and β chains which create a cross-link with the
actin protofilament. These chains differ in length, the α-chain having twenty-one domains and
the β-chain having seventeen, running anti-parallel to each other. Each domain contains
approximately 106 amino acids that together form three anti-parallel alpha helices. These helices,
in turn, fold into a left coil, with the α and β domains connected through helical linkages.
Cysteine is present in both chains, twenty in α-spectrin and fifteen in β-spectrin.
Ankyrin
Protein 4.3
Figure 2. Schematic layout of a RBC membrane showing the spectrin-actin
interaction.9
7
Protein Unfolding
Through examination of homology models and high-resolution crystal structures some
cysteine molecules are shown to be shielded by the tertiary coil of the protein. 1 This layout
makes spectrin ideal for examining its denaturated state under stressed conditions (Figure 3).
Under standard conditions, the amino acid is shielded by the coiled tertiary structure; however,
forced extension reveals the thiol group of the molecule, allowing it to react with a labeling dye.1
Figure 3. A spectrin molecular dynamics simulation displaying the presence of
Cys1167 in the β-chain (bottom view).1
Cysteine labeling in Spectrin
The most accurate technique to label cysteine domains is through thiol-sensitive
fluorophores. A water soluble, naphthalene fluorophore, 5-({[(2-iodoacetyl)amino]ethyl}amino)naphthalene-1-sulphonic acid (IAEDANS), acts as a ligand and binds to exposed sulfhydryl
groups, in this case, cysteine.10 N-(4,4-Difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-sindacene-2-yl) iodoacetamide (BODIPY-IA), is another Cys-reactive fluorphore that was used
as an alkylating agent for the sheared RBCs.
The experimental procedure to identify unfolded membrane proteins in human red blood
cells began with reversible lysis of cell samples, removing the hemoglobin and adding the
IAEDANS dye to bind to exposed cysteines. Any non-labeled samples were alkylated with
iodoacetamide (IAM). The samples were then split, one fraction experienced stress and the other
fraction remained static to use as a control. The proteins within the samples were separated
8
Protein Unfolding
through one-dimensional SDS polyacrylamide gel electrophoresis (PAGE). Results of this
procedure, as shown in Figure 4, contained high fluorescence with both chains of spectrin,
suggesting that cysteines were exposed due to denaturation. The gel also isolated spectrin as the
only cytoskeletal protein in RBCs that showed significant fluorescence, indicating that other
proteins were not as reactive to fluid stress.1
Figure 4. 1-D SDS PAGE results of sheared cell samples displaying the
separation of various membrane proteins present in RBCs.1
Following electrophoresis, the spectrin bands displaying IAEDANS fluorescence
underwent liquid chromatography coupled tandem mass spectrometry (LC-MS/MS) in order to
detect specific cysteine sites. Table 1 displays a list of confirmed dye-labeled sites in both the α
and β chains. Most of the labeled sites occur on the surface of the protein, where cysteine is
easily visualized, making internal cysteine molecules harder to label until denaturation and IAM
labeling has occurred. To compensate for this problem, chromatograms of specific sites in shear
and static samples were compared to further quantify fluorescence. One particular site, Cys1203 in
α12 as shown in Figure 5, displayed a greater ratio between IAEDANS and IAM labeling in the
shear sample than in the static sample, indicating more cysteines were exposed. Further
9
Protein Unfolding
Table 1. Identified Cysteine sites in IAEDANS labeled spectrin (A=α-chain, B=βchain).1
A
B
“Y” indicates detection in either a sheared or static sample, while “ND” implies a
non-detected site. The ratios of shear samples to static samples are, at times equal,
but enough evidence indicates greater fluorescence with sheared cells.
10
Protein Unfolding
Figure 5. Chromatogram profile of α-spectrin Cys1203 in a shear and static cell
sample. Note that the overall intensities of the static peaks are significantly higher
than the shear peaks.
shear/static distinction occurred by treating spectrin with 2-nitro-5-thiocyanobenzoic acid
(NTCB) and performing a Western blot. This reagent cleaves peptide bonds near reduced,
un-labeled cysteines, therefore cleavage patterns should differ between static (unmodified) and
stressed cells (modified). Results supported distinct cleavage patterns both quantitatively and
qualitatively, conveying the presence of altered, or exposed, cysteine domains.
Spectrin Construct Labeling
Many shear-sensitive cysteine sites within the chains were completely buried and
shielded by the tertiary structure; however, forced unfolding broke thiol bonds, extending the
structure and causing the dissociation of the spectrin tetramer, exposing cysteines. Cys-β1167, in
sample β-R8-9, is one such example where exposure occurred and in order to further study its
binding sensitivity, a recombinant multidomain molecule providing a high concentration of the
β-R8-9 tetramer was constructed and named sample β-R5-9.1 This construct was analyzed for
temperature dependence. Fixed labeling at different temperatures was introduced followed by
fluorescence imaging and mass spectral analysis. The results in Figure 6 indicated that only
temperatures above 25°C caused unfolding in this protein. This observation correlates with the
results of other tests conducted at this temperature, suggesting that a transition midpoint
temperature was reached. Sample β-R5-9, for example, has been studied by atomic force
11
Protein Unfolding
microscopy (AFM) and circular dichroism, which indicated that above 25°C, the helical structure
of spectrin deteriorates and mechanical stability of the membrane is lost.1
Figure 6. Results for β-R5-9 construct determining temperature dependence based
upon the percentage of folded proteins present. Melting temperatures are based on
circular dichroism.1
A previously studied site, Cysα1203, was also studied using the recombinant, sample αR12. Unlike sample β-R5-9, this recombinant only contains partial cysteine shielding. The
construct underwent a similar test where the fraction of unfolding was studied under ureadenaturing conditions versus native conditions. Results in Figure 7 showed the immense increase
in fluorescence for those samples that were denatured, while those that were not, displayed
lowered levels of fluorescence. This observation directly correlates to the steric protection
hypothesized to exist for partially exposed cysteine sites. In other words, under native conditions,
some cysteine are naturally shielded from reacting with certain compounds, however, when a
protein is unfolded, this barrier is removed, enabling cysteine domains to bond freely.
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Protein Unfolding
Figure 7. Results for the α-R12 construct, measuring the rate of fluorescence and
its temperature dependence on protein unfolding.1
Spectrin Kinetics Labeling
An additional experimental technique that supplements identification of buried cysteine
sites is determining the rate at which samples are labeled by the IAEDANS and BODIPY-IA
fluorophores. This method furthers distinguishes between sheared and static samples. It was
proven that cysteine sites on the surface of spectrin were labeled to saturation within 30-60
minutes, following a first-order rate dependence (Figure 8).1 The higher fluorescence values on
Figure 8. Kinetics labeling comparing shear versus static spectrin samples with
IAEDANS dye.1
the shear samples were consistent with previous results, however, comparison of the first-order
rate constants showed that static fluorescence developed faster than shear fluorescence (k = 0.13
13
Protein Unfolding
min-1 for static versus k = 0.04 min-1 for shear). This was due to the locations of cysteine sites for
each sample; only surface-exposed domains were labeled in the static samples, while the
unshielded domains were additionally labeled in the shear samples as the protein unfolded. This
implied that unfolding was a slow process and therefore resulted in a lower labeling rate.
Following these results, the sequential two-dye labeling process was administered. The
principle of this method involved the application of a dye to label cysteines in the static proteins
and then adding another dye following stress application. Static samples were treated with
IAEDANS to saturation. At this point, shear stress was induced to these cells and the BODIPYIA green dye was injected. Spectrin labeling using densitometry was found to increase further,
proportional to time and stress. For example, at stress levels of 0.8 Pa and 1.7 Pa the shear/static
labeling ratio increased from about 5 to 6.5, respectively, after an hour (Figure 9).
Figure 9. Comparison of shear/static labeling ratios over stress using two-dye
sequential labeling.1
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Protein Unfolding
Temperature dependence was also observed for kinetics labeling of spectrin. In samples
that were dyed with IAEDANS for sixty minutes, fluorescence did not increase until after 25°C,
rising steadily until thermal disruption was induced at 40°C. Figure 10 shows this dependence,
potentially suggesting that cysteine sites only react with the fluorophore at a specific temperature
range. However, this dependence was tested with another RBC protein, actin, the protofilament
to spectrin known to also carry cysteine sites. Using both shear and static cells, the same
procedure was carried out and results indicated that even at temperatures above 25°C, no change
Figure 10. Fluorescence versus temperature dependences of spectrin and actin.1
in fluorescence was observed. It was not until thermal disruption of the cells was induced that a
dramatic increase in fluorescence occurred in the actin sample. Since the change was not
observed below 40°C, the denaturation of actin was shown to be independent of temperature.
Ultimately, this examination proved the specificity of IAEDANS to spectrin unfolding and also
displayed the kinetic dependence of labeling on temperature.
15
Protein Unfolding
II. Mesenchymal Stem Cells and NMM II
The second type of cell displaying forced protein conformation changes is the MSC.
These are adult stem cells found in bone marrow that often transform into fat, muscle, skin, and
nerve cells, in addition to cartilage, bone, ligaments, and tendons.11 During differentiation in
which the stem cell spawns its specificity, these cells generate traction forces on their own matrix
that supersede the fluid stresses on red blood cells, accumulating to around 1000 Pascals. 1 This
stress was hypothesized to reveal hidden cysteine residues. A major protein essential for this
process is non-muscle myosin II (NMM II), depicted in Figure 11. Structurally similar to muscle
myosin, this molecule contains two heavy chains and four light chains and exists in two distinct
isomers, myosin IIA and myosin IIB.12 Functions of the protein include cytoplasmic
contractility, cytokinesis, and membrane trafficking. Many of these processes are carried out
through phosphorylation, where the release of a phosphate from NMM II bonds with another
protein, such as actin, and activates it, allowing the function to be executed. Though the isomers
are closely identical in structure, they are distributed in different areas of a cell, suggesting that
functional variations exist too. For example, it has been hypothesized that myosin IIB plays a
role in exocytosis, a process that involves vesicle secretion from the cell membrane.14
16
Protein Unfolding
Figure 11. Depiction of the protein, Myosin II. The head is comprised of the four
light chains and part of the two heavy chains. The tail consists of the remainder of
the heavy chains.13
Blebbistatin inhibits NMM II expression
In order to relieve stress from stem cells and suppress NMM II activity, an inhibitor must
be introduced. One study called for the drug blebbistatin, a potent uncompetitive inhibitor that
was known to specifically target skeletal muscle and non-muscle myosin II.15 Named after its
ability to prevent bulges in the cell membrane when undergoing apoptosis, this compound works
by inhibiting ATPase activity (Figure 12), an essential process that activates other proteins, such
as actin, which is responsible for cell motility and cytokinesis. The drug binds to the ADP-Pi
product complex in myosin, changing its open conformation to a closed-state and preventing the
release of the phosphate which normally bonds to actin.15 Ultimately, this inhibition softens and
relaxes the cell, as shown in Figure 13 using immunofluorescence labeling; it also prevents
shielded cysteine exposure and stops differentiation from occurring.
Actin-activated
Basal
17
Protein Unfolding
Figure 12. Depiction of the effects of increasing blebbistatin concentration in
NMM II samples on the activation of actin.15
Figure 13. Immunoflourescence images of Mesenchymal stem cells under
stressed (top) and relaxed (bottom) conditions. NMM II is believed to be
responsible for the tension.1
The same Cys-shotgun method used for RBCs was carried out for these cells, using an
alternative fluorophore, called monobromobimane (mBBr).1 This dye covalently bonds to the
free thiol groups found in reduced cysteine molecules. The formed linkage creates a relatively
high fluorescence, labeling exposed cysteine that are unshielded under the cells natural stress
pattern.17 Following cysteine labeling, 1D SDS-PAGE and densitometry were carried out with
18
Protein Unfolding
LC-MS/MS analysis of any prominently labeled bands. Figure 14 displays the highest level of
fluorescence in NMM II samples, with a labeling ratio favoring the tensed (uninhibited) state.
This observation suggests NMM II contains the greatest number of shielded cysteine residues,
having the largest tensed-to-relaxed ratio. Other prominent proteins in MSCs show mBBr
labeling as well. Filamin, an actin-binding protein, displays a similar ratio as NMM II, with
greater fluorescence in the tensed state. Vimentin, a mesenchymal intermediate filament protein,
and actin, however, both indicate that cysteines were more exposed in the relaxed (inhibited)
state, counteracting the former prototype. Ultimately, tests prove that while blebbistatin directly
inhibits NMM II, this suppression affects the activation of other proteins too and either promotes
or prevents cysteine exposure.
Figure 14. SDS-PAGE and densitometry results for MSCs showing the ratio of
fluorescence between standard and inhibited samples.
Differential Labeling of Cysteine sites in MSC proteins
It was hypothesized that injecting blebbistatin into MSCs would unorganize NMM II,
preventing ATP hydrolysis from occurring and keeping hidden many cysteine sites.
Immunofluroescence imaging of MSCs supported this statement, showing that myosin IIA
exhibited greater labeling with mBBr in the tensed (untreated) state of the cell, versus its induced
19
Protein Unfolding
relaxed state (Figure 15). A differentially labeled site, Cys90, identified through mass
spectrometry, particularly confirms this paradigm. Homology models of NMM II revealed that
Cys90 is located between the head and tail domains of the protein, burying the amino acid behind
other residues. Under normal conditions, stress activates ATPase which, once hydrolyzing ATP,
exposes the Cys90 site. When adding blebbistatin to the protein, relaxation of the MSC prevents
ATP hydrolysis, keeping this site buried.1
Figure 15. Immunofluorescence imaging of MSCs at both the tensed and relaxed
states.1
Another differential labeling site in MSCs was Cys327, the only cysteine site found in the
protein vimentin. As mentioned previously, vimentin was shown to have greater cysteine
labeling under the inhibited state, therefore residues within this protein should become exposed
under MSC relaxation. The Cys327 site is located in the central coil where coiled-coil
polymerization occurs. Typically, the protein monomer continually coils around neighboring
proteins to form a polymer chain. However, under the influence of blebbistatin, vimentin has
been shown to depolymerize. This non-polymerized conformation reveals the Cys327 site,
suggesting that the polymerized forms shield the site from being exposed. Further evidence is
shown in Figure 16, where Western blots and immunofluorescence imaging display greater
20
Protein Unfolding
signaling in the monomer form versus the polymer form. While no correlation can be made on
the direct inhibition of vimentin by blebbistatin, observations confirm that the inhibitor promotes
the uncoiling of the vimentin polymer chain, thus revealing Cys327.
Figure 16. Depiction of both tense and relaxed states of vimentin, the latter
showing cysteine exposure. Analysis of immunofluorescence imaging and
Western blots also suggest that monomer vimentin has higher fluorescence. The
image on the right depicts a vimentin dimer.1, 18
Conclusion
While many mechanical properties of proteins are still unknown, several correlations
have been made about their folding and unfolding tendencies that affect their ability to function
in cells. It was hypothesized that stress causes cell deformation via forced protein unfolding, so a
model was designed that examined specific molecules to prove protein dissociation. The amino
acid, cysteine, which often exists within the tertiary and quaternary structures of proteins, was an
appropriate molecule to tag in order to identify dissociated proteins since many of its sites are
shielded. In RBCs, for example, the protein spectrin was shown to unfold under various levels of
stress, exposing certain normally shielded cysteine sites. Using a variety of techniques, including
two-dye sequential labeling, 1D SDS-PAGE, densitometry, LC-MS/MS, and Western blots, it
was proven that spectrin unfolds proportionally to temperature and stress levels. Construct
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Protein Unfolding
labeling of known sites, such as Cysα1203 and Cysβ1167 further assisted in understanding the
parameters of spectrin unfolding.
Another type of cell, MSC, which experiences internal forces, was also analyzed. The
protein, NMM II, was known to cause a natural stress to the cell and reveal shielded cysteine
sites; however, injection of blebbistatin disorganizes the protein and causes inhibition of its
expression, preventing these sites from being exposed. Utilizing the same tests, cysteine residues
located throughout the NMM II structure were found to be more prevalent in the tensed form
versus the induced relaxed form. This pattern correlates with RBC results and further supports
the effects of forced protein unfolding (i.e. cysteine exposure).
Ultimately, these tests provided a means to prove stress-induced protein unfolding while
also offering specific information about certain proteins. While no direct correlation has been
made between the unfolding of these proteins and the deformation of their cells, it can be
inferred that deliberately suppressing protein expression results in a changed protein
conformation, which leads to cell deformation.
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Protein Unfolding
References
(1) Johnson, C.P.; Tang, Hsin-Yao; Carag, C.; Speicher, D.W.; Discher, D.E. Science 2007, 317,
663-666.
(2) Rief, M.; Gautel, M.; Oesterhelt, F.; Fernandez, Julio M.; Gaub, Hermann E. Science 1997,
276, 1109-1111.
(3) Berg, J.; Tymoczko, J.L.; Stryer, L. Biochemistry. 2007. 6th ed. New York: W.H. Freeman
and Company, 31, 40-49.
(4) Branden, C.; Tooze, J. Introduction to Protein Structure. New York: Garland
Publishing, Inc, 1991, 12-19.
(5) Beningo, K.A.; Dembo, M.; Kaverina, I.; Small, J.V.; Wang, Y.L. Cell Biology. 2001, 153,
881.
(6) Rader, A.J.; Hespenheide, B.M.; Kuhn, L.A.; Thorpe, M.F. PNAS. 2002, 99, 3540-3545.
(7) The Biology Project. 2003, Department of Biochemistry and Molecular Biophysics,
University of Arizona, <<http://biology.arizona.edu>> January 10, 2008.
(8) Vera, C; Skelton, R.; Sung, A. ABME. 2005, 33, 1387-1404.
(9) Stryer, L. Biochemistry. 1995. 4th ed. New York: W.H. Freeman and Company, 285.
(10) Thiol Reactive Probes Excited with Ultraviolet Light. 2006, Invitrogen Corp.
<<http://probes.introgen.com>> January 13, 2008.
(11) Kadereit, S. ISSCR. 2005, <<http://isscr.org>> January 13, 2008.
23
Protein Unfolding
(12) Bao, J.; Jana, S.S.; Adelstein, R.S. J. Biol. Chem. 2005, 280, 19594-19599.
(13) Blamire, J. BIO dot EDU. 2003, <<http://www.brooklyn.cuny.edu>> March 19, 2008.
(14) Togo, T.; Steinhardt, R.A. MBC. 2004, 15, 688.
(15) Kovács, M.; Tóth, J.; Hetényi, C.; Málnási-Csizmadia, A.; Sellers, J.R. J. Biol. Chem. 2004,
279, 35557–35563.
(16) Straight, A.F. et al. Science. 2003, 299, 1743–1747.
(17) Swem, L.R. et. al. EMBO J. 2003, 22, 4699–4708.
(18) Strelkov, S.V. et. al. EMBO J. 2002, 21, 1255-1266.
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