tracking unfolding and refolding reactions of single proteins using atomic force microscopy methods

Methods xxx (2013) xxx–xxx

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Methods

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Tracking unfolding and refolding reactions of single proteinsusing atomic force microscopy methods

Paul J. Bujalowski a, Andres F. Oberhauser a,b,c,⇑a Department of Biochemistry and Molecular Biology, University of Texas Medical Branch at Galveston, TX 77555, United Statesb Department of Neuroscience and Cell Biology, University of Texas Medical Branch at Galveston, TX 77555, United Statesc Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch at Galveston, TX 77555, United States

a r t i c l e i n f o a b s t r a c t

Article history:Available online xxxx

Keywords:Single-moleculeBiophysicsAtomic force microscopyProtein foldingUnfoldingOsmolytesChaperones

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⇑ Corresponding author at: Department of NeuUniversity of Texas Medical Branch, Galveston, TX 77

E-mail address: [email protected] (A.F. Oberhau

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During the last two decades single-molecule manipulation techniques such as atomic force microscopy(AFM) has risen to prominence through their unique capacity to provide fundamental information onthe structure and function of biomolecules. Here we describe the use of single-molecule AFM to trackprotein unfolding and refolding pathways, enzymatic catalysis and the effects of osmolytes and chaper-ones on protein stability and folding. We will outline the principles of operation for two different AFMpulling techniques: length clamp and force-clamp and discuss prominent applications. We provide pro-tocols for the construction of polyproteins which are amenable for AFM experiments, the preparation ofdifferent coverslips, choice and calibration of AFM cantilevers. We also discuss the selection criteria forAFM recordings, the calibration of AFM cantilevers, protein sample preparations and analysis of theobtained data.

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1. Introduction

Since its invention in 1986 [1], the AFM has evolved from beinga resolution imaging tool to a versatile technique used to manipu-late and detect single molecules [2–9] as well as to measure theinteraction forces between single proteins [10–12]. Single-mole-cule techniques harness several different disciplines of science(biology, physics, chemistry, material science and computer sci-ence) allowing better analysis and understanding of protein func-tion with unprecedented resolution. Here we will discuss thestudy of the unfolding and refolding pathways using single mole-cule force spectroscopy (SMFS) techniques. This technique wasdeveloped during the 1990s and allows doing experiments at thesingle-molecule level under physiological relevant conditions.One of the main advantages of this technique is that it allowstracking the structural dynamics during protein folding reactionsand can capture transient folding intermediates and misfoldedstates that cannot be identified by bulk studies. Another advantageof single-molecule AFM is the relatively easy sample preparation.One of the most important applications of SMFS has been donein the field of protein folding/unfolding [2–5,7,8,13–29]. SMFShas been also used to investigate enzyme catalysis as well as theeffects of osmolytes and molecular chaperones on protein folding

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ki, A.F. Oberhauser, Methods (2

[30–33]. In this paper, we describe SMFS operating principles,practical implementation and we will discuss a few noteworthyexamples.

2. Basic principles of single-molecule force spectroscopymethods

2.1. SMFS instrumentation

The AFM is composed of two main parts: a XYZ stage scanner(sometimes separated into Z and XY stages) and an optical head(Fig. 1A). A small cantilever plays the role of microscopic force sen-sor, a thin and flexible piece of silicon (about 200 lm in length and10 lm in thickness) that ends with a small pyramid-shaped stylus.A laser beam is shined on the back of the cantilever in order totrack the cantilever bending when it contacts the sample (Fig. 1).The force is measured using Hooke’s law, F = kcDx, where Dx repre-sents the cantilever deflection, kc spring constant (stiffness of thecantilever). The optical signal bouncing off the end of the cantileveris then amplified in such a way that a small bending of the canti-lever, of only a few nanometers, results in a large change in thephotovoltage of the detector. The SMFS method can measure forcesin range of 1 to more than 1000 piconewtons and changes in thelength of proteins with nanometer and millisecond resolution[3,4,24,26,29,34]. The most common SMFS modes are the length-clamp (Fig. 2A), which yields a force–extension curve and theforce-clamp, which gives an extension–time curve (Fig. 2C).

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Fig. 1. Schematic diagram of the AFM apparatus and associated control electronics. (A) The AFM consists of two main parts: the scanner (micromanipulator) and an opticalhead. The center point of the system is a small cantilever that functions as a microscopic spring. The cantilever is brought into contact with the sample and its bending isdetected by shining a laser on its back; the light bounces off and is captured by a split photodetector (split into two regions: ‘‘a’’ and ‘‘b’’ photo-signals). A tiny deformation of afew nanometers causes a large alteration in the photovoltage of the detector due to optical amplification of the signal. The photovoltage is then converted into a force signal.The AFM is very sensitive it can measure forces in the range of pico-newtons and distances of only few angstroms. (B) Two modes can be used to stretch single molecule:length-clamp or force-clamp modes. The standard length-clamp mode allows the control of the position (L) and measurement of the resulting force (F) which is calculatedfrom the laser deflection (a � b)/(a + b). The force-clamp mode measures force and then compare it with a set value thus generating an error signal that is fed to aproportional, integral and differential amplifier (PID) whose output is connected directly to the piezoelectric positioner. Reproduced with permission from [76].

2 P.J. Bujalowski, A.F. Oberhauser / Methods xxx (2013) xxx–xxx

2.2. Length-clamp mode

By moving the sample away from the AFM tip, a stretching forceis applied to the protein. The length-clamp (or constant velocity)mode records force–extension curves obtained by pulling a singleprotein in the z axis (Fig. 2B). The interpretation of force–extensioncurves is not always straightforward. The recorded force peaks canoriginate from many sources which include not only the unfoldingof protein domains but also detachment of other molecules fromany of the two anchoring points or disentanglement of molecules.This problem was solved by using native multi-domain proteins(such as titin, tenascin or spectrin) [4,35,36] or recombinant poly-proteins [37–40]. For multi-domain and polyproteins the recordedforce–extension curve resembles ‘‘sawtooth’’ pattern which repre-sents the sequential unfolding of individual domains. This period-icity allows unequivocal identification of single molecules. Thetypically forces required to unfold single proteins are in the rangeof 50–500 pN (at pulling speeds of about 1 lm/s) [2].

2.3. Force-clamp mode

The force-clamp mode controls the force applied to a proteinthrough a feedback mechanism that maintains the applied forceconstant and quickly corrects the distance between the coverslipand the AFM tip. The force feedback is based on a proportional,integral, and differential (PID) amplifier whose output is connecteddirectly to the piezoelectric positioner [41,42]. The time responseof the feedback and piezo is critical. The frequency response of cur-rent PID amplifiers and piezoelectric positioners are limited to 5–10 ms, which in most cases is adequate to study the unfoldingand refolding reactions. However, recent advances in piezos andPID amplifiers have pushed the limit in the sub-millisecond range(about 150 ls [24]). These new high-speed force clamp spectrom-eters allow the study of the recoil dynamics of single polypeptidechains under force [24]. The force-clamp mode allows the precise

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control of the end-to-end distance of the protein with nanometerresolution [13,41]. For example, when a constant stretching forceis applied to a multidomain protein (such as titin) or a polyprotein,the domains unfold stochastically in an all-or-none fashion leadingto a stepwise increase of the end-to-end length of the protein(Fig. 2B). Force-clamp SMFS techniques are currently being usedto tackle fundamental problems in biology such as protein folding[13,43–45] and chemical mechanisms in enzyme catalysis[23,30,46–49]. Force-clamp SMFS techniques allow the direct mea-surement of the mechanical stability of proteins (energy barriers)and the kinetics of unfolding and refolding pathways and the loca-tion of kinetic barriers [13,41,42,46,50–54].

2.4. Preparation of surfaces – the choice of coverslips

In SMFS experiments, the protein of interest is immobilized on asubstrate and then by physisorption (i.e., nonspecific adsorption)the protein is attached to the tip of cantilever therefore beingcaught between coverslip and cantilever. For immobilization pur-poses, the protein constructs are commonly engineered with ter-minal cysteine-tags and then adsorbed onto a gold-coatedcoverslip; the interaction of gold with thiol groups is quite strongand ruptures at around 1 nN [55]. If the protein of interest has sol-vent exposed cysteine residues, then terminal histidine tags can beused for immobilization on nickel–nitriloacetic acid (Ni–NTA)functionalized coverslips [56,57]. A potential disadvantage is thatthe unbinding forces between His-tag and Ni–NTA are around50 pN and thus are lower than typical unfolding forces of Ig-likedomains (>100 pN) but compatible with spectrin, ankyrin or C2 do-mains (they unfold at forces <50 pN).

2.4.1. Glass coverslipsGlass coverslips (12 or 15 mm diameter, 1 oz, Ted Pella, Inc.)

should be cleaned by thoroughly spraying them with 70% ethanol,rinse them with MilliQ water and then sonicate them for 30 min in



Fig. 2. Protein unfolding events captured with length- and force-clamp AFM. (A) Example of single protein unfolding events observed using AFM in length-clamp mode (A)and a force-clamp mode (C). (A) In the length-clamp mode each force peak corresponds to the unfolding of individual domains. The recorded force–extension curve resemblesa ‘‘sawtooth’’ pattern. Each force peak corresponds to the unfolding of a single protein domain. The spacing between force-peaks is related to the length of the polypeptidechain upon domain unfolding. In the case of the titin I27 domain the measured increase in contour length is about 28 nm which correspond to the unraveling of about 75amino acids that were packed into core of the domain [34]. The last peak shows the detachment of the protein from the cantilever. The unfolding peaks in the sawtoothpattern vary randomly in amplitude with an average value of �200 pN which is a result of stochastic nature of the unfolding process of single domains. (B) Cartoon diagramshowing the different steps during the mechanical unfolding of a domain in a polyprotein. The AFM tip picks up a single protein (1) and starts pulling on it (2). When sufficientforce is applied (around 200 pN) the domains begin to unfold (3) until it is fully unfolded and reaches its maximum extension length (4). (C) In the force-clamp mode, theapplied force is constant. The polyprotein unfolding resembles ‘‘staircase’’ pattern. Here each step of the extension–time curve corresponds to individual unfolding events andshows increases in the length of the protein. (D) Frequency histogram of step sizes for the titin polyprotein (n = 99). The main peak is centered at 22.4 nm. Three minor peakshave average step size of about 48, 67, and 89 nm. The data were obtained at constant force of 147 pN. Reproduced with permission from [76].

P.J. Bujalowski, A.F. Oberhauser / Methods xxx (2013) xxx–xxx 3

10% (v/v) Hellmanex solution. The coverslips are then washed withwater and sonicated for 30 min in MilliQ water. Before use, cover-slips should be dried using a stream of nitrogen gas.

2.4.2. Gold-coated glass coverslipsGold-coated coverslips are prepared by depositing a layer of

chromium/nickel (1 nm) and then a 50 nm layer of high-puritygold (99.9%; Goodfellow), under vacuum, at a pressure of 1–2 � 10�6 mbar using a vacuum evaporator (the chromium coatingis necessary to achieve strong bonding between gold and glass)[58,59].

2.4.3. Nickel–NTA-coated glass coverslipsGlass coverslips (20–40 round glass coverslips, 15 mm diame-

ter, 1 oz, Ted Pella, Inc.) are placed in a plastic container containing20 N KOH for 13 h. The KOH solution is discarded; the coverslipsare washed with water and transferred into an aqueous solutioncontaining 0.02% (v/v) acetic acid and 2% (v/v) 3-mercaptopropyl-trimethoxysilane. They are placed in a 90 �C oven for about 1 h.The coverslips are washed with water and dried. The SH silanegroups are reduced with 100 mM dithiothreitol (DTT). The cover-slips are rinsed with water and reacted with 20 mg/ml N-[5-(3-maleimidopropylamido)-1-carboxypentyl]iminodiaacetic acid(Dojindo Molecular Technologies, Inc.) in 10 mM 3-(N-morpho-lino)propanesulfonic acid-KOH (pH 7.0) for 30 min. The coverslipsare rinsed again with water. Finally the coverslip’s surface is


activated by a reaction with 10 mM NiCl2 for 10 min, rinsed withwater and stored at room temperature in a glass desiccator [60].

2.5. AFM cantilevers – choice and calibration

There are two main physical parameters of AFM cantilevers thatare important for SMFS experiments: the spring constant, kc, andthe resonant frequency, fc [24,58,61]. The spring constant affectsthe force sensitivity and the resonant frequency limits the re-sponse time of the measurements. The ideal cantilevers for SMFSmeasurements are those with small kc (<10 pN/nm, for high forcesensitivity) and high fc (>1 kHz, for high response time). For exam-ple, Bruker MLCT cantilevers have a kc and fc of about 20 pN/nmand 1 kHz (measured in water) and Olympus Biolever BL-RC150VBcantilevers have a kc and fc of about 40 pN/nm and 10 kHz, respec-tively. The BL-RC150VB cantilevers are much smaller than theMLCT (60 lm vs 300 lm in length) and hence have a much lowerviscous drag coefficient making them ideal for experiments requir-ing a very fast response time and low force noise [24].

The AFM cantilever spring constant can differ significantly fromthe value given by the manufacturer; therefore, it has to be cali-brated before each experiment [28]. The most commonly usedmethod for determining the spring constant of a cantilever is thethermal method, which models the cantilever as a damped simpleharmonic oscillator fluctuating in response to thermal noise[11,62,63]. The mechanical properties of the cantilever are relatedto the frequency and amplitude of these oscillations. Hence, its




spring constant can be calculated using the so-called equipartitiontheorem: hDx2i = kbT/k, where hDx2i represents the mean-squaredisplacement noise, kb is the Boltzman constant and T is tempera-ture. This calculation has a typical error of ±20% [11,64]. It must benoted also that the position of the spot on the back of the cantileverhas an influence on the determination of its spring constant andthat a method has been developed to correct this effect [65]. Analternative method to the thermal noise one is based on the shiftin the resonance frequency of the cantilever after the addition ofa small mass. This method is more accurate but it requires special-ized equipment [66].

2.6. Construction of polyproteins for SMFS experiments

In order to construct polyproteins, several recombinant DNAtechniques and cloning methods have been developed [37–40].The polyproteins are expressed in Escherichia coli strains and thenpurified by affinity chromatography.

2.6.1. Cloning and expression strategiesDNA fragments preparation: design proper primers to amplify

the target protein DNA fragments and introduce different restric-tion enzyme sequences on both ends of each target DNA fragmentusing PCR. Then, the target fragments are purified, introduced intoan expression vector and the proteins are then expressed usingstandard protocols [37–40,67].

2.6.2. Polyprotein purificationThe E. coli expressing the polyproteins are dissolved in lysis buf-

fer containing protease inhibitors and are lyzed by sonication or byusing a French press. Ni–NTA resins are normally used to purifypolyproteins. Before use, the resins should be equilibrated withbuffer. Then, the supernatant of cell lysate is mixed with the resin.The binding process takes around 30–60 min at 4 �C with gentleagitation to keep the resin completely mixed with lysate. The resinis settled by gravity and the supernatant is collected. Then, the re-sin is rinsed with wash buffer (containing 25 mM imidazole) sev-eral times before adding the elution buffer (containing �250 mMimidazole). The proteins are eluted by adding the elution bufferand stored at 4 �C. The proteins are then identified and analyzedby sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS–PAGE) [60].

2.7. Protein sample preparation for SMFS experiments

In a typical experiment, a small aliquot of the purified polypro-teins (�1–10 ll, 10–100 lg/ml) are allowed to adsorb onto gold- orNi–NTA-coated glass coverslips for about 5–10 min and thenrinsed with buffer (usually with phosphate buffered saline (PBS))to remove unbound proteins. The AFM tip is brought into contactwith the coverslip for several seconds to allow a polyprotein to at-tach to the tip. Then the tip is retracted; stretching a single poly-protein should result in a characteristic sawtooth unfoldingpattern. The probability of picking up a protein is characteristicallyvery low (about 5%) because the concentration of proteins has to bekept low enough to ensure pulling of single molecules. Addition-ally, the contact between AFM tip and protein occurs at randomlocations, thus most of the AFM protein traces do not show theunfolding of all the domains.

2.8. Analysis of traces

The elasticity of the unfolded polypeptide is commonly ana-lyzed using the worm-like chain (WLC) model of polymer elasticity[4,61,68].


FðxÞ ¼ kbTp

14

1� xLc

� ��2

� 14þ x

Lc

" #

The WLC equation predicts the entropic restoring force (F) gen-erated upon extension (x) of a protein in terms of its contour length(Lc) and persistence length (p) which characterizes the orienta-tional correlation of segments in the chain. This analytical approx-imation is used to fit the WLC model to experimental force–extension curves [68,69]; to include stiffness of the chain, a modi-fied WLC chain with ‘‘elastic modulus’’ parameter can be used [70].Using AFM and optical tweezers, several groups have determinedthe persistence length of single unfolded polypeptide chains torange between 0.3 and 1.0 nm [4,39,45,68,71–75]. It is significantthat this length is approximately equivalent to the distance of0.4 nm between alpha carbon atoms in a polypeptide chain.

The AFM recordings are selected using the following criteria: (i)the trace should have clean initial force–extension curve afterretraction from the surface (i.e., little or no unspecific interactions);(ii) force–extension curves of single polyproteins should havedetachment forces higher than 300 pN (most domains unfold atforces in the range of 50–200 pN) to be sure that the protein iscompletely extended and unfolded. Typically about 1 in 500–1000 of force–extension traces fulfill these criteria. The initial con-tour length of the folded protein (Lc) and the contour length incre-ments (DLc) are estimated by fits of the WLC model to the force–extension curves that lead to each force peak; the zero length pointis defined as the point where the AFM cantilever tip contacts thecoverslip. These parameters are estimated from hundreds offorce–extension recordings and used to construct unfolding forceand increase in contour length histograms.

3. Tracking protein unfolding reactions

When weak interactions responsible for the mechanical resis-tance of a protein fold are broken due to the external applied forcethe protein unfolds. In the force–extension traces, the distance be-tween the two consecutive peaks represents increase of the con-tour length which is characteristic for the each protein fold. Thusit becomes a ‘‘molecular fingerprint’’ because that allows theunmistakable identification of the multi-domain protein. The firststudies of protein unfolding reactions using AFM were done usingthe sarcomeric striated muscle protein titin [4] and the extracellu-lar matrix protein tenascin [35]. These showed characteristic saw-tooth patterns with regular spaced force peaks. In these unfoldingSMFS experiments, the recorded unfolding peaks correspond to theunfolding of single-protein domains (Fig. 2A and B).

The force-clamp experiments generate length–time traces at afixed force (Fig. 2C). Each step in the length–time trace representsan unfolding event. The height represents the change in the con-tour length after unfolding of protein at applied force (Fig. 2D).The dwell time between steps is related to the mechanical stabilityof the protein [76]. Additionally, the force-clamp mode allowsobtaining the kinetic parameters of the unfolding reaction suchas unfolding probabilities, unfolding dwell-time distributions,unfolding and folding trajectories or ensemble of transition states[13,41–43,51,53,54].

SMFS has been used to track the mechanoenzymatics of giantprotein kinases [77–80]. The titin kinase domain is involved instrain sensing that occurs during muscle activity. The mechanicalproperties of the kinase domain and flanking Ig/Fn domains ofthe Caenorhabditis elegans titin-like proteins twitchin and TTN-1were studied using SMFS [80]. AFM pulling experiments showedthat kinase domains have mechanical resistance and they unfoldin a biphasic, stepwise fashion at forces �50 and 80 pN. The firststep probably represents movement of the autoinhibitory domain




from the catalytic pocket. Unfolding of the kinase domain requiresapplication of additional force. The obtained results strongly sug-gest that titin kinase function as a force sensor. The conformationalchanges during strain-induced activation of human titin kinase do-main have been also studied using SMFS [81]. The process is regu-lated by two autoinhibition mechanisms. In the first one, ATPbinding site is blocked by C-terminal regulatory tail and the secondone includes tyrosine autoinhibition of the catalytic base by tyro-sine-170. The pulling experiments indicated that mechanical forceis able to activate titin kinase due to its capacity to release the ac-tive site for ATP binding by unfolding of the autoinhibitory domain.The forces required for activation of ATP binding are lower than theforces of unfolding the surrounding titin domains.

During the last decade, SMFS has been applied to investigate themechanisms of enzyme catalysis [46,47,80,82]. In these studies,the force-dependence of an enzymatic reaction was measured byapplying a mechanical force on the substrate of the reaction. Anexcellent example here is the investigation of reduction of disulfidebond by thioredoxins enzymes [30,31,46,48]. First, the polyproteincomposed of I27 modules with engineered disulfide bonds be-tween residues 32 and 75 was constructed. Then, the AFM force-clamp mode was applied to track the unfolding of domains up todisulfide bonds. In presence of thioredoxins, disulfide bonds are re-duced allowing the unfolding of region of the domain that previ-ously remained inaccessible. Studies of both E. coli and humanthioredoxins showed that the reaction rate decrease as a functionof the applied force. Interestingly, different force dependence pat-terns were observed once applied force exceeds 200 pN. Addition-ally, the reduction rate accelerated with increasing force for E. colithioredoxin, whereas it became force-independent when humanthioredoxin was present. These studies were expanded to differentthioredoxins allowing the identification of two evolutionary linesof organisms: one for bacteria and the second for archaea andeukaryotes [83].

Fig. 3. Capturing single protein refolding reactions using SMFS. (A) Cartoondiagram showing the equilibrium between folded (i) and unfolded states (ii) of apolyprotein controlled by an applied mechanical force. (B) Refolding kineticsprobed with a double-pulse protocol (inset). The force/extension traces of an I278

polyprotein obtained by consecutively stretching (sawtooth pattern; trace i),relaxing (trace ii) and re-stretching after 10 s relaxation time (iii). All of thedomains unfolded in the first pull and spontaneously refolded during the relaxedperiod. Single molecule experiments show the stochastic nature of the processwhich is exhibited by small difference between unfolding forces in following pulls.Reproduced with permission from [44].

4. Tracking protein refolding reactions

The SMFS has also been used to study protein refolding reac-tions [23,24,84,85]. In these experiments, the protein is firststretched and then is allowed to refold by bringing back the AFMtip close to the coverslip. Folding can be observed by stretchingthe protein again under high force which results in protein unfold-ing (Fig. 3).

Refolding studies have been performed using a variety of pro-teins such as titin, tenascin, fibronectin, filamin, calmodulin, anky-rin and ubiquitin [4,13,35,86–88]. For example, analysis of therefolding kinetics of the titin-like protein projectin using SMFSshows that this protein functions according to a folding-based-spring mechanism [89]. Projectin is a member of the titin proteinsuperfamily that is found in invertebrate muscles and is responsi-ble for the high passive stiffness of insect indirect flight muscles,which are crucial to perform oscillatory work during flight. Therefolding kinetics of projectin was studied using a double pulseprotocol in which the time interval is changed (Fig. 4A); the datashows that projectin domains require only milliseconds to refold-ing (Fig. 4B). Remarkably, this protein can be subjected to hun-dreds of stretching–relaxation cycles with no signs of rundownor fatigue indicating a unique mechanical robustness (Fig. 4C).The length-clamp mode was used to investigate the refolding ofprojectin domains under force (Fig. 5). During the experiment, asingle projectin was first unfolded to 70 pN and then the forcewas dropped to 5 pN to give it time to refold to its native state.As the figure shows, before the protein reached its fully collapsedstate, there was a dramatic increase in the noise level with largefluctuations length which may reflect the transient formation of


secondary structures or intermediate folded states [89]. Then thestretching of the protein was repeated by stepping back the forceto 70 pN. The experiments clearly indicated that projectin domainscan refold under relatively high forces suggesting a robust refold-ing mechanism that may operate over a large range of sarcomerelengths.

Force-clamp SMFS techniques have been used to investigate therefolding pathways of single ubiquitin proteins [13]. In theseexperiments, a ubiquitin polyprotein was stretched at a high force,and then force was released allowing observation of the foldingprocess. The recorded folding trajectories were divided into dis-tinct stages. The first stage is very fast and most likely representsthe elastic recoil of the unfolded polypeptide which is caused bythe change in the pulling force. The second stage is characterizedby a long-lasting relaxation plateau and an increase in length fluc-tuations; whereas the third stage is marked by an abrupt decreasein the end-to-end length, reflecting the collapse to the nativefolded state. The results clearly showed that folding speed of theprotein depends on applied force and contour length of the un-folded protein. This study supports the notion that protein foldingunder force is characterized by a continuous collapse rather thanby a discrete all-or-none process.



Fig. 4. Unfolding and refolding kinetics of single titin-like proteins. (A) Refolding of projectin domains depending on relaxation time (100 ms, 1 s, 10 s). The refoldingprobability increases with increasing the time interval between stretching pulses. After 10 s, relaxation almost all domains refolded into their native state. (B) Fraction ofrefolded domains as a function of the time delay between stretching pulses. The solid lines are a two-exponential fit of the data to the function Nrefolded/Ntotal = A1(1 � e�t�b1) + A2 (1 � e�Dt�b2). Considering the heterogeneous nature of the Ig-like domains, we attribute the biphasic folding kinetics to heterogeneity in foldingkinetics among the different domains of projectin. (C) A series of force–extension and relaxation cycles collected from a single projectin molecule. The delay time betweeneach refolding cycle was about 20 s. In this example, the protein was unfolded and refolded a total of 52 times. Reproduced with permission from [89].


Recently, SMFS techniques were used to investigate unfoldingand refolding pathways of the transmembrane protein KpOmpA[15]. This protein mediates bacterial adhesion and in course of itsfunction, it is subjected to mechanical stress. SMFS experimentsshowed a stepwise unfolding of the protein where some of the b-strands unfolded in cooperative manner. The KpOmpA protein re-folded back into the membrane after the tensile load was released.The results suggest that KpOmpA may use a stepwise unfoldingmechanism to reduce the mechanical stress applied to membrane.These data suggest that cells may employ both unfolding andrefolding pathways of transmembrane proteins to perform differ-ent cellular tasks.

5. Computer simulations of mechanical unfolding and foldingreactions

SMFS experiments can resolve the forces required to unfold aprotein with single amino acid resolution [90,91] but they do notprovide detailed structural information. Further atomic-leveldescription of unfolding pathways is often obtained by comple-menting the experimental SMFS studies with steered moleculardynamics (SMD) simulations [92–94]. The magnitude of the forcesobserved in the SMD simulations does not directly correspond to


those measured with AFM. This is partially because the pullingspeeds are several orders of magnitude different. However, thesimulations are qualitatively consistent with the SMFS experimen-tal data. SMD simulations have been extensively used to examinethe mechanical unfolding of a wide variety of proteins [92,93,95–98]. The synergy between experimental data and computer simu-lations has significantly enhanced our understanding of proteinstructure and dynamics.

Recent AFM force-clamp spectroscopy experiments confirmedimportant computational predictions regarding the energy profilesof protein folding [43]. Statistical theories of protein folding predictthat proteins fold through a number of small conformationalensembles along a rugged funnel-like energy landscape surface[99,100]. Single protein force-clamp spectroscopy experiments di-rectly confirmed the existence of an ensemble of collapsed statesthat were able to convert to folded states, demonstrating the vastdiversity of the collapsed conformations [43].

6. Probing osmolytes and molecular chaperones using SMFS

SMFS has been used to analyze the effect of osmolytes andmolecular chaperones on protein stability and folding. Osmolytes



Fig. 5. Collapse of unfolded protein domains under force. (A) Cartoon representation of a projectin single molecule held under different force values. The cantilever is broughtinto a contact with a polyprotein (1), and then force is applied on the molecule (2). Protein domains start unfolding (3) until all of them are unfolded and protein reaches itsmaximum length (4). Then the force is released (5) until domains will refold to their native states. The cantilever was drawn at an angle for illustration purposes only. (B) and(C) In this experiment, the protein was unfolded and extended at high force 70 pN. The observed steps correspond to the unfolding of several domains. Then the force wasdropped to about 5 pN (marked by arrow); after 6 s the force was increased to 70 pN, and after another 15 s it was lowered to 40 pN and finally to 0 pN. The applied force isshown in (C). Reproduced with permission from [89]. (D) Schematic representation of the folding energy landscape under a stretching force (pathway in cyan) and no appliedforce (pathway in black). The diagram represents mapping of a possible conformations of a protein and their corresponding levels of Gibbs free energy. The protein’s foldedstate corresponds to its free energy minimum.


such as trehalose, sorbitol or trimethylamine N-oxide (TMAO) canact as ‘‘chemical chaperones’’ and are potent stabilizers for manyproteins and capable of reversing protein misfolding and or aggre-gation [101–103]. Several neurodegenerative diseases such as Alz-heimer’s, Huntington’s and Parkinson’s have been attributed toproblems associated with protein misfolding and aggregation.There is considerable interest in developing new effective thera-pies based on small molecules for the treatment of these devastat-ing pathologies. Promising results on the modulation of proteinaggregation by naturally occurring osmolytes have been recentlyreported [101–106]. Hence, the use of osmolytes as chemical chap-erones to stabilize proteins/peptides that misfold and aggregate inneurodegenerative diseases is an attractive concept for drugdevelopment.

Recent studies have shown that the mechanical properties ofproteins can be affected by presence of osmolytes [44,107,108].Increasing concentration of urea which is a well-known denaturingosmolyte results in a remarkable decrease in the mechanical stabil-ity of PKD (polycystic kidney disease) domains [32] (Fig. 6A). Addi-tion of protective osmolytes such as sorbitol and trimethylamineN-oxide effectively counteract the effect of urea (Fig. 6B). Therefolding rate of PKD domains was found to decrease significantlyin presence of urea but it is restored to near normal rates when sor-bitol or trimethylamine N-oxide were added.

SMFS has been used to study the effects of molecular chaper-ones on the folding of the motor domain of myosin [33]. Molecularchaperones assist in the folding of proteins [109]. Myosins are ac-tin-dependent motor proteins that convert chemical energy fromATP hydrolysis into mechanical work. They play essential roles ina wide variety of cellular motility processes, ranging from musclecontraction to cleavage furrow ingression during cytokinesis. TypeII myosin heavy chains have a molecular weight of �225 kDa andconsist of an amino-terminal globular motor (or head) domain


and carboxyl-terminal rod domain. The motor domain harborsthe sites for actin-binding and enzymatic activity. In contrast tothe rod domain, the motor domain folds in a chaperone-dependentfashion [110–114]. Expression of myosin motor domains in bacte-ria results in misfolding. Molecular chaperones appear necessaryfor de novo folding and structural maintenance of the myosin head.The interaction between myosin and its chaperone UNC-45 wereanalyzed using SMFS techniques by coupling titin I27 domains tothe myosin motor domains [115]. By chemically coupling a titinI27 polyprotein to the motor domain of myosin, a ‘‘molecular re-porter’’ was introduced providing a specific attachment point anda well characterized mechanical fingerprint in the AFM measure-ments. This approach enabled the study of the folding pathwayof the motor domain and directly tracks the effects of the chaper-one UNC-45. Single molecules of derivatized full length myosin(Fig. 6C) or the motor domain were subjected to repeated cyclesof mechanical stretching, separated by a waiting time of 10 s atzero force. The I27 unfolding pattern is apparent in the initialunfolding trace (1), but absent in the second (2) and third (3)traces, indicating that the unfolded motor domain interferes withrefolding of the I27 domains. The characteristic low-force plateaustemming from the rod (between dashed lines) is visible in alltraces in Fig. 6C, indicating the rod folds as an independent entityand is not affected by misfolding of the motor domain. In the ab-sence of UNC-45, the I27 saw-tooth pattern is observed only inthe initial unfolding cycle, indicating that the misfolded motor do-main interferes with the refolding of the I27 domains. In the pres-ence of 1 lM UNC-45, full recovery of folded I27 domains isobserved (Fig. 6D). These results indicate that the I27 domainsfunction as a reporter for motor domain misfolding. UNC-45 seemsto prevent the interference between the motor domain and the I27units, presumably by binding to the myosin motor domain andpreventing its misfolding [115]. This approach should be applicable



Fig. 6. Probing the effects of osmolytes and molecular chaperones on protein folding and stability. (A) Typical force–extension traces for a polyPKDd1-I27 protein obtained indifferent conditions: 1 M urea, 1 M urea + 1 M sorbitol, and 1 M urea + 1 M TMAO. (B) Plot of the unfolding forces for PKD domains as a function of the sorbitol/urea ratio. Theunfolding forces of PKDd1 steadily increases from 78 at 0 M, 71 at 0.1 M, 98 at 0.5 M, 161 at 0.7 M, to 193 at 1 M. The line represents a linear fit to the experimental dataobtained below 1.0 sorbitol/urea ratio. (C) (Left) Diagram of a titin-derivatized myosin motor domain. Myosin (blue) was derivatized with a mechanical reporter (eightrepeats of the titin I27 domain) carrying an N-terminal cysteine residue and a C-terminal His6 tag. (C) (Right) Single derivatized myosins were subjected to repeated cycles ofmechanical stretching, separated by a waiting time of 10 s at zero force. The characteristic I27 sawtooth pattern is seen in the initial unfolding trace (1). It is not present in thefollowing traces (2 and 3), indicating that the unfolded myosin interferes with refolding of the I27 domains. The low-force plateau (around 30 pN) represents the unfolding ofthe myosin rod domain showing that it refolds independently from the motor domain. (D) Plot of the refolding probability of titin I27 domains as a function of the stretching/relaxation cycles. Single S1-I27 proteins were subjected to repeated cycles of unfolding in the absence of chaperone (squares) or in the presence of 1 lM UNC-45 (circles). As acontrol, the same experiment was performed using the I27 polyprotein (triangles). Reproduced with permission from [32,33].


to elucidate chaperone–substrate interactions of different biologi-cal systems.

7. Perspectives

Single-molecule force spectroscopy methods are providing uswith fundamental information on how proteins fold and work. Thisfield has grown at an impressive rate during the last 20 years andSMFS methods are becoming an indispensable tool used in bio-physics and biochemistry. We have gained essential informationon protein-based elasticity, mechanism of protein–protein interac-tions, unfolding/refolding reactions, chaperone action as well asenzymatic catalysis. A key challenge of future experiments is to ap-ply SMFS methods to study unfolding and refolding reactions ofproteins in environments that more closely mimic those found inliving cells. This will require the combination of SMFS with sin-gle-molecule fluorescence techniques (such as fluorescence reso-nance energy transfer) which should enable us to look deeperinside into the mechanism of protein folding and misfolding.

Acknowledgements

This work was funded in part by NIH grant R01DK073394, bythe UTMB Claude Pepper Older Americans Independence Center


NIH/NIA Grant P30 AG024832 and the John Sealy MemorialEndowment Fund for Biomedical Research.

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tracking unfolding and refolding reactions of single proteins using atomic force microscopy methods

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