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AthenaES™ Projects
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Construction of Polyproteins for use in Atomic Force Microscopy Studies
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Client Mayo Clinic, Rochester, MN, Dr. Julio Fernandez,
Principal Investigator
This work was published in the Proceedings of the National
Academy of Sciences, USA in 1999. (See Carrion-Vazquez M.,
A.F. Oberhauser, S.B. Fowler, P.E. Marszalek, S.E. Broedel,
Jr. J. Clarke, and J.M. Fernandez. 1999. Mechanical and chemical
unfolding of a single protein: a comparison. Proc Natl. Acad.
Sci. USA. 96(7):3694-3699.)
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Engineering a polyprotein
The atomic force microscope (AFM) is a remarkably simple
instrument. Originally used for measuring surface contours of
individual molecules, it has provided important information about
the structure of proteins. In the force-measuring mode, AFM is
capable of measuring forces down to piconewtons and can resolve
force changes caused by the displacement of its probe by a fraction
of a nanometer1. Single molecule measurements are
routinely done (see Fisher et al. for a review2).
Since measurements can be made on many individual molecules in a
short period of time, a statistical evaluation of the measurements
can be performed. Perhaps the most powerful aspect of AFM is that
the measurements can be done in an aqueous environment, allowing for
the study of biological material under conditions that resemble those
in vivo. The folding and unfolding of the protein can be studied
in the presence of substrate, product, and co-factors. The temperature
can be controlled as can the buffer conditions, i.e., ionic strength,
pH, solvents, etc.
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Figure 1.
A) Schematics of our custom built atomic force microscope.
The AFM was constructed using a Digital Instruments (DI, Santa Barbara,
CA, USA) AFM detector head (AFM-689) mounted on top of a Physik
Instrumente (PI; Waldbronn, Germany) single axis piezoelectric
positioner. The PI positioner has a capacitative sensor resulting
in a Z axis resolution of 0.1 nm (P-732.ZC). The data acquisition
(Force) and the voltage control of the movement of the piezoelectric
positioner (Dzp) are done by means of a PC-mounted data
acquisition board (AT-MIO-16X; National Instruments) and controlled by
custom made LabView (NI) software. The spring constant, kc,
of each individual AFM tip (Si3N4 tip NPS, DI)
is calibrated in solution, before each experiment. Kc
varies between 30-120 mN/m. The force is measured by the deflection of
the cantilever and the extension can be calculated from the piezo
travel (Dzp).
B) Stretching and unfolding Ig-type domains with AFM. 1)
shows an unstretched polypeptide with 4 Ig domains adsorbed to an AFM
tip; 2) the stretching of the protein requires force and this is
monitored as a deflection of the cantilever; 3) the unfolding of a
domain increases the protein length, relaxing the cantilever back to
its resting position.
C) Idealized force-extension curve for the stretching of a
multi-domain polypeptide and the subsequent unfolding of a single
domain. The numbers correspond to the stages marked in B.
The AFM functions like a miniature phonograph. A sharp tip mounted
on a cantilever interacts with the sample, causing minute deflections
that can be calibrated as a force. The design of our AFM is shown
in Figure 1. An example of how we have applied AFM to the analysis
of a protein is described in detail below. In a typical experiment,
the protein sample is placed on a gold-coated cover slip that is
attached to a piezoelectric positioner. We engineered recombinant
proteins so that one end (the C-terminus) is anchored to the gold
substrate. Random segments of the protein (titin) are then picked
up by adsorption to the AFM tip and stretched for up to several
hundred nanometers (Dzp, Fig. 1B). When the protein is
stretched, it pulls on the cantilever causing small cantilever
deflections (Dzc) that can be converted into units of
force using the cantilever spring constant, kc (Fig. 1C).
As the protein is stretched the force increases in a non-linear
(non-Hookean) fashion; this is the energy required to decrease the
degrees of freedom of the polypeptide (an entropic-spring behavior
that is a common property of polymers). If the protein is further
stretched the probability for the unfolding of individual domains
becomes higher. When a domain unfolds, the AFM tip travels back to
its resting position and the force goes back to zero (Fig. 1B and 1C).
Construction of a polyprotein is necessary because the mechanical
properties of a single module could not be studied using current AFM
techniques. A single module is only 89 residues and will extend only
for a short distance. The length of a single domain falls into a region
where we always observe a large amount of non-specific interactions
between the AFM tip and the substrate (<30nm). In contrast, tandem
repeats of many modules extend well beyond the region of nonspecific
interactions and generate periodical patterns that amplify the features
of the individual modules and allow for a high signal-to-noise ratio.
One of the aims of our research has been to reduce the interference
from non-specific interactions.
Recombinant DNA techniques were used to construct direct tandem repeats
of a single Ig domain from I band titin (I27). Two methods were employed
to synthesize and express recombinant multimers of I27, with similar
results. One of them was a multiple step cloning technique modified
from a previously described method3 that makes use of four
restriction sequences (BamHI, BglII, SmaI and KpnI ) to build even
multiples of I27. This approach adds two new amino acids (Arg and Ser)
to the I27 sequence such that the repeated monomer becomes [I27-RS]
n. Using this method we have constructed a protein composed
of eight tandem repeats (I27RS8 , Fig. 2).
The second method was based on single step cloning of high order multimers
into a custom-made expression vector4,5. This method relies
on a single type of restriction site, AvaI, to build linear multimers of
the I27 monomer. The AvaI sequences add two new amino acids (Leu and Gly)
to the I27 sequence such that the repeated monomer becomes [I27-LG]
n. Using this method we constructed a protein composed of
eleven tandem repeats (I27LG11, Fig. 2D). Expanding
on this theme, we have since constructed polyproteins for the titin
domains I28 through I34 including an alternating I27-I28 construct. Each
was expressed in E. coli, purified by Ni-affinity chromatography, and
subjected to AFM analysis.
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Figure 2.Construction of poly-I27 proteins.
A) Agarose gel stained with ethidium bromide showing the size
of the I27-RS multiples (right lane).
B) Coomassie blue staining of the purified I27RS
8 protein (~90 kDa) separated by SDS-PAGE.
C, D) Summary of the sequence of the I27RS
8 and I27LG11 constructs.
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References
| 1. |
Czajkowsky, D. M. and Shao, Z. 1998. Submolecular resolution of single macromolecules with atomic force microscopy. FEBS Lett. 430:51-54. |
| 2. |
Fisher, T. E., Oberhauser, A. F., Carrion-Vazques, M., Marszalek, P. E., and Fernandez, J. M. 1999. The study of protein mechanics with the atomic force microscope. TIBS 24:379-384. |
| 3. |
Kempe, T. Kent, S.B., Chow, F., Peterson, S.M., Sundquiest, W.I., L?Latien, J.J., Harbrecht, D., Plunket, D. and DeLorbe, W.J. 1985. Gene 39:239-245. |
| 4. |
Hartley JL, and Gregori TJ (1981). Cloning multiple copies of a DNA segment. Gene 13, 347-353. |
| 5. |
Graham, G.J. and Maio, J.J. 1992. BioTechniques 13:780-789. |
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