Probing Molecular Landscapes of Streptavidin–Biotin Unbinding Direct force measurements of the unbinding strength of single streptavidin/biotin pairs opened the way for examining the mol
Trang 1306 Chen and Moy
4 Repeat wash twice
5 After removing supernatant from last wash, resuspend beads in 0.01% BSA in PBS
6 Add 100μl of washed beads to neutravidin-coated plate Beads should adhere to
the dish almost immediately
III Applications
A Probing Molecular Landscapes of Streptavidin–Biotin Unbinding
Direct force measurements of the unbinding strength of single streptavidin/biotin pairs opened the way for examining the molecular determinants of ligand–receptor unbinding
(Merkel et al., 1999) As predicted by the Bell Model, the rupture force of the individual
streptavidin–biotin bonds increased with increasing loading rate (Bell, 1978; Evans and Ritchie, 1997) Figure 3 shows the dynamic response of streptavidin and a streptavidin mutant, W120F, in which the tryptophan residue at position 120 was replaced by a phenylalanine As shown, the mutation altered the force spectrum of the streptavidin– avidin interaction The analysis of these force spectra provides a direct approach for
probing the landscape of the streptavidin–biotin unbinding (Yuan et al., 2000).
The streptavidin–biotin force measurements were carried out as described in earlier sections with a streptavidin-functionalized tip and an agarose bead To ensure that single bonds were being measured, the frequency of adhesion events was reduced to 30% by restricting indentation depth and/or by adding excess free biotin By simple statistical analysis based on the Poisson distribution, one is thus ensured that 80% of the measured
events are due to the rupture of single-molecule pairs (Merkel et al., 1999) The loading
rate of the measurement is dependent on both the elasticity of the system and the speed
Fig 3 Loading rate dependence of the rupture force in the unbinding of the streptavidin–biotin ( ◦) and W120F-biotin ( •) The force measurements revealed two loading regimes in the unbinding of the complexes.
Both regimes in force spectra were fitted to the Bell model (Yuan et al., 2000).
Trang 214 Single-Molecule Force Measurements 307
at which the cantilever is retracted The loading rate was varied by pulling the molecules apart at different cantilever retraction rates The elasticity of the system was determined
by measuring the slope of the retract trace
B Probing Adhesion Receptors on Cells
We applied AFM toward measuring ligand–receptor unbinding on the surface of liv-ing and fixed cells to examine the effect of receptor crosslinkliv-ing on receptor/ligand unbinding strength (Chen and Moy, 2000) For these experiments, the cantilever tip was functionalized with biotinylated concanavalin A (C2272, Sigma) using the methods out-lined earlier Measurements were carried out at room temperature in glucose-free RPMI
supplemented with 0.01% BSA and 0.01 mM MnCl2 Glucose was eliminated from the culture medium to prevent potential competitive binding with the Con A-functionalized tip and Con A receptors on the cell MnCl2was a source of Mn2+, a necessary cofactor for Con A binding BSA was added not only to reduce nonspecific binding but also
to provide a permissive environment for adhesion of cultured NIH-3T3 fibroblast to the bottom of an uncoated plastic tissue culture dish Measurements were carried out on both unfixed and lightly fixed cells to determine if crosslinking of Con A receptors would have an effect on receptor unbinding strength A minimal applied force of 250 pN was used in these measurements and the scan speed was maintained at 1μm/s.
Compared to measurements on agarose beads, unfixed cells had much longer regions
of stretch before final separation between the tip and membrane (compare Fig 1A and 4A) Typical distances spanned 500 nm Thus, receptors seemed to be anchored to cell tethers Rupture force measurements revealed a stronger rupture force for chemically
Fig 4 Force versus extension curves acquired from Con A-functionalized AFM tips interacting with Con A receptors on the surface of NIH-3T3 cells that were (A) not fixed and (B) fixed with glutaraldehyde Histograms
of rupture force between Con A-functionalized AFM tips and Con A receptors on (C) untreated cells and (D) glutaraldehyde-fixed cells Arrows in (D) indicate quantized peaks at 80, 160, and 240 pN following fixation of cells in glutaraldehyde.
Trang 3308 Chen and Moy
fixed cells (173 ± 6.1 pN) compared to unfixed cells (86 ± 2.6 pN) (Figs 4C and 4D) Moreover, differences in cell compliance were readily apparent from the slope of the retract trace as the tip pulled on the surface of the cell Force histograms revealed multiple quantal peaks that were absent in the unfixed cell histograms (Fig 4D), suggesting that much of the increase in rupture force was due to a shift toward cooperative binding of cells In addition a shift in the first peak indicated that changes in loading rate resulting from changes in cell elasticity could also lend to the increase in rupture force following fixation
Acknowledgments
This work was supported by grants from the American Cancer Society and the NIH (1 R29 GM55611-01)
to VTM.
References
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Trang 6CHAPTER 15
Forced Unfolding of Single Proteins
S M Altmann and P.-F Lenne
European Molecular Biology Laboratory Cell Biology and Biophysics Programme Meyerhofstrasse 1
69117 Heidelberg, Germany
I Introduction
A General Scheme
II The Biological System
A Spectrin Proteins
B Protein Engineering
C Preparation of Samples
D Results III Forced Unfolding
A Atomic Force Microscopy: Force Spectroscopy Mode
B Force Curves
C Force-Clamp Experiments
D Refolding
IV Analysis
A Sorting Data
B Multiple Pickup
C Fitting Procedures
V Models
A Questioning Unfolding Pathways
B A Single-Parameter Model for Forced Unfolding Using Three States
C Monte Carlo Simulations
VI Conclusion and Prospects
A Biological Implications VII Appendices
A The Double-Sensor-Stabilized AFM
B Data Acquisition and Evaluation Techniques
C Calibration
D Monte Carlo Simulations References
METHODS IN CELL BIOLOGY, VOL 68
Copyright 2002, Elsevier Science (USA) All rights reserved. 311
Trang 7312 Altmann and Lenne
I Introduction
The present chapter is focused on forced unfolding of proteins by atomic force microscopy (AFM) Protein folding remains one of the most fascinating mechanisms
of biology Different approaches can be used to understand this complex mechanism During the last years, exciting advances have been made trough new detailed
experimen-tal and theoretical studies [for a review, see Brockwell et al (2000)] Forced unfolding
is among the new experimental techniques promising new insights into the energy land-scapes of protein folding processes
AFM provides experimenters with the means to manipulate single molecules under physiological conditions This powerful new tool can produce the forces necessary either
to rupture ligand–receptor bonds (Florin et al., 1994) or to stretch DNA More recently, the AFM has been applied to unfold proteins (Rief et al., 1997) For a review, see Fisher
et al (1999).
The unfolding of proteins by applying a force to single proteins attached between a surface and an AFM tip complements more classical techniques using either temperature
or chemicals as denaturants This approach provides single-molecule information that has not been available previously It is of particular interest for proteins that are under mechanical stress in living cells as, for example, the muscle protein titin or the cytoskele-ton protein spectrin In this chapter we will present the method of forced unfolding and illustrate its use to probe the mechanical properties of a single-spectrin domain
A General Scheme
The general scheme for forced unfolding of protein resembles a “fishing” experience Proteins attached on a surface are picked up with the silicon–nitride tip of a flexible cantilever (Fig 1) The probability of fishing one or more molecules depends not only
Fig 1 Experimental scheme for protein fishing.
Trang 815 Forced Unfolding of Single Proteins 313
on the density of proteins on the surface but also on the interactions between the tip and the protein (See Section II,B) Once a protein is picked up, it can be stretched to more than 10 times its folded length (depending on its folded structure) reaching almost its total contour length
The extension of the elastic, already unfolded part of the protein produces a restor-ing force that bends the cantilever This bendrestor-ing, and therefore the force, can be mea-sured with the high precision of the AFM With proper sample preparation and well-adapted instrumental techniques, single-molecule unfolding processes generate a signature, i.e., a force–distance profile, which can be clearly distinguished from back-ground noise and other events not related to unfolding processes (see Sections III,B and IV.A)
II The Biological System
A Spectrin Proteins
Spectrin is a member of a large family of actin-binding proteins These are able to crosslink actin filaments into loose networks or tight bundles This property makes the members of the spectrin family scaffolds for both cytoplasmic and membrane assem-blies Forming a two-dimensional network in red blood cells, spectrin molecules are
assumed to provide the cell with special elastic features (Elgsaeter et al., 1986) This
ability of the spectrin molecule to contract and expand has been attributed to the mod-ular structure made of repeats, initially identified by Speicher and Marchesi (1984) Moreover, this ability seems to be a key element for structures, also containing spec-trin, that are regularly subjected to mechanical stresses in cellular complexes ranging from muscle Z bands to stereocilia Recent experiments have also demonstrated that spectrin acts as a protein accumulator that traps and stabilizes proteins at specific points
on cell membranes (Hammarlund et al., 2000; Moorthy et al., 2000; Dubreuil et al.,
2000)
The basic constituent of spectrin chains is the repeat which typically has 106 amino acids and is made of three antiparallelα-helices separated by two loops, folded into a left-handed coiled-coil (Fig 2) (Pascual et al., 1997; Djinovic-Carugo et al., 1999; Grum
et al., 1999) Grum et al (1999) proposed a model for the flexibility of spectrin, based
on structural data
It is rather difficult to deduce the mechanical response of the molecule under stress from structural data, since the energy landscape of proteins is unknown Mechanical properties of proteins must be measured directly, because they depend on a particular pathway along a preferred direction through the energy landscape This can be done by AFM
Rief et al (1999) studied the natural α-spectrin chain by AFM, which is composed of
homologous but not identical domains When the chain is stretched, it is not possible to know which domains unfold first Hence, the study of engineered constructs consisting of identical domains provides more insight into the mechanical stability and the unfolding
features of the spectrin repeat (Lenne et al., 2000).
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Fig 2 Structure of the spectrin repeat: a left-handed antiparallel triple-helical coiled-coil (Protein Data Bank ID 1AJ3).
B Protein Engineering
Protein engineering is required to
1 Fix the protein to both a surface and the tip
2 Construct polyproteins to amplify the features of unfolding of one domain These can be handled much more easily than single-domain proteins
3 Construct mutants for a detailed study of the relation between structure and me-chanical stability
The application of AFM for protein unfolding has so far been restricted to a small group
of proteins Most of published works were focused on natural or engineered proteins, organized in linear arrays of globular domains This is the case for natural spectrin, titin, and fibronectin To our knowledge, only two works dealt with nonmodular proteins,
namely, the bacteriorhodopsin (Oesterhelt et al., 2000) and the HPI protomer (Muller
et al., 1999).
Nonmodular proteins are difficult to handle in the AFM, as it is not easy to attach the proteins at one end on a surface and at the other to the tip without affecting their structure
Trang 1015 Forced Unfolding of Single Proteins 315
With modular proteins, even if the protein is not so ideally attached, the elements of the chain, which are not directly attached to a solid surface, span the gap between the surface and the tip and can contain one or few domains that lend themselves to forced unfolding
In this case the force traces will exhibit a sawtooth-like pattern that gives a fingerprint
of the modular protein
The changes introduced upon adsorption of proteins to a surface are still poorly un-derstood This process can be at least partially controlled by engineering specific ends The thiol group of cysteine allows proteins to be attached specifically onto a gold-coated surface The spectrin clones were therefore fused with a COOH Cys2 tag for immobi-lization purposes A cysteine residue could be as well engineered at the other (properly oriented) terminus of the protein to enable specific attachment of the gold-coated AFM
tip to the cysteine (Oesterhelt et al., 2000) But the same trick cannot be used for the
surface and the tip at the same time
Oesterhelt et al (2000) used two-dimensional (2D) crystals of proteins that fix the
orientation the proteins The specific interactions of the supporting lipid layer results
in the ordering of the proteins In the case of bacteriorhodopsin, the proteins form 2D crystals spontaneously In the 2D lattice, the protein has a well-defined direction and only
a part of the protein is accessible to the tip But it is a quite particular case of a protein that forms large 2D crystal domains easily Protein engineering also allows constructing
modular proteins from ones the are not naturally modular Yang et al (2000) used an
original strategy to polymerize lysosyme proteins by solid-state synthesis
A system that would guarantee uniform orientation of the molecules is still needed
To preserve the native states of the protein, few or no specific interactions are required
A good candidate is the N-nitrilo-triacetic acid (NTA)/His tag system, which is widely
used in molecular biology to isolate and purify histidine–tagged fusion proteins Here the
histidine tag acts as a high-affinity recognition site for the NTA chelator Schmitt et al.
(2000) have shown that the binding forces between histidine–peptide and NTA chelator are in the 50-pN range The strength of such bonds is too small to prevent detachment
of the molecule before the total unfolding of a protein
C Preparation of Samples
One can use different surfaces to attach proteins Glass and mica are suitable to
non-specifically adsorb proteins [compare Norde et al (1986)] It is preferable though to use
specific interactions to immobilize the protein and to forbid the detachment of it during stretching We used engineered proteins (see Section II,C) with a cysteine residue at one end, which can form a specific bond with the gold-coated surfaces The proteins were suspended in PBS or another suitable buffer at a concentration ranging from
10 to 100 μg/ml To prepare the working buffers, it is recommendable to use
ultra-pure water rather than double-distilled water This guarantees that the salt concentra-tions, which are very important for the adsorption characteristics, are well defined A small drop (20–50μl) of the protein solution is deposited on the surface Proteins were
absorbed during 10 min, and samples were washed with PBS (10 times with 100μl)
afterwards