Single-molecule force spectroscopy reveals amechanically stable protein fold and the rationaltuning of its mechanical stabilityDeepak Sharma*, Ognjen Perisic†, Qing Peng*, Yi Cao*, Canaan Lam*, Hui Lu†, and Hongbin Li*‡

*Department of Chemistry, University of British Columbia, Vancouver, BC, Canada V6T 1Z1; and †Department of Bioengineering,University of Illinois at Chicago, Chicago, IL 60607

Edited by David Baker, University of Washington, Seattle, WA, and approved April 19, 2007 (received for review January 12, 2007)

It is recognized that shear topology of two directly connectedforce-bearing terminal �-strands is a common feature among thevast majority of mechanically stable proteins known so far. How-ever, these proteins belong to only two distinct protein folds,Ig-like � sandwich fold and �-grasp fold, significantly hinderingdelineating molecular determinants of mechanical stability andrational tuning of mechanical properties. Here we combine single-molecule atomic force microscopy and steered molecular dynamicssimulation to reveal that the de novo designed Top7 fold [KuhlmanB, Dantas G, Ireton GC, Varani G, Stoddard BL, Baker D (2003)Science 302:1364–1368] represents a mechanically stable proteinfold that is distinct from Ig-like � sandwich and �-grasp folds.Although the two force-bearing � strands of Top7 are not directlyconnected, Top7 displays significant mechanical stability, demon-strating that the direct connectivity of force-bearing � strands inshear topology is not mandatory for mechanical stability. Thisfinding broadens our understanding of the design of mechanicallystable proteins and expands the protein fold space where mechan-ically stable proteins can be screened. Moreover, our results re-vealed a substructure-sliding mechanism for the mechanical un-folding of Top7 and the existence of two possible unfoldingpathways with different height of energy barrier. Such insightsenabled us to rationally tune the mechanical stability of Top7 byredesigning its mechanical unfolding pathway. Our study demon-strates that computational biology methods (including de novodesign) offer great potential for designing proteins of definedtopology to achieve significant and tunable mechanical propertiesin a rational and systematic fashion.

atomic force microscopy � computational design � mechanical unfolding �unfolding pathway

Protein mechanics is an important aspect of biology. Manyproteins have evolved to sense, generate and bear mechanical

forces (1) in a variety of biological processes, such as cellularadhesion (2), muscle contraction (3), and ligand–receptor interac-tions (4). Elastomeric proteins constitute a unique class of mechan-ical proteins with diverse functions ranging from molecular springsto structural materials of superb mechanical properties. Recentdevelopment in single-molecule force spectroscopy has made itpossible to study the mechanical properties of proteins at single-molecule level (5, 6). These studies not only revealed rich infor-mation about the architectural design of elastomeric proteins andshed light on the biophysical principles underpinning various bio-logical processes, but also revealed promising prospect of usingengineered elastomeric proteins for nanomechanical applications(7–9). However, in comparison to chemical and thermodynamicproperties of proteins, experimental data on mechanical propertiesof proteins remains rather limited and molecular determinants ofmechanical properties of proteins are less well understood. Thesefactors have made it difficult or impossible to predict and tunemechanical properties of proteins in a systematic and rationalfashion. Consequently, the known mechanically stable proteins arerestricted to only a limited number of protein folds, significantly

limiting the potential exploration of elastomeric proteins for nano-mechanical applications.

A number of proteins of different topologies (6, 10) have beenstudied for their mechanical properties, and the importance ofprotein topology to the mechanical stability has emerged (6, 11–13).It is recognized that the vast majority of mechanically stableproteins identified so far share a common shear topology, in whichthe two terminal � strands are arranged in parallel and are directlyconnected to each other by noncovalent interactions (6, 10). Theonly known exceptions are green fluorescent protein (14), a tightlyformed �-barrel protein, and ankyrin (15), a 22–24 repeat protein.Upon stretching of these mechanically stable proteins, the twoterminal � strands are sheared against each other and the nonco-valent interactions connecting the two force-bearing strands pro-vide resistance to mechanical unfolding (6, 11, 12, 16–20). Thesemechanically stable proteins belong to only two distinct proteinfolds: Ig-like � sandwich fold and �-grasp fold. However, it remainsto be demonstrated whether the importance of shear topology onmechanical stability can be generalized to other protein folds. It isunknown whether the direct connectivity between force bearingstrands in a shear topology is a necessary condition for mechanicalstability or any other topological arrangement containing hydrogenbonding interaction between nonadjacent terminal strands may alsooffer significant mechanical stability.

To examine the above possibilities and explore the feasibility ofrational tuning of mechanical stability, here we combine single-molecule atomic force microscopy (AFM), steered molecular dy-namics simulation (SMD), and protein engineering techniques todemonstrate that a de novo designed protein Top7 (21) representsa mechanically stable protein fold that is distinct from Ig � sandwichand � grasp folds. SMD simulations revealed a unique substructure-sliding unfolding mechanism for Top7 and the existence of twopossible unfolding pathways with different height of energy barrier.Such molecular insights into unfolding mechanism have enabled usto rationally tune the mechanical stability of Top7 by redesigning itsmechanical unfolding pathway. Thus, we now have a mechanicalcontrol method beyond the conventional tuning of key interactionsalong the unfolding pathway. Our results demonstrate the greatpotential of using computational methods (including de novodesign) to engineer proteins of significant and tunable mechanicalproperties, and open up avenues toward exploring the designprinciples of protein mechanics and engineering.

Author contributions: D.S., H. Lu, and H. Li designed research; D.S., O.P., Q.P., Y.C., C.L., andH. Li performed research; D.S. and O.P. analyzed data; and D.S., O.P., H. Lu, and H. Li wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Abbreviations: AFM, atomic force microscopy; SMD, steered molecular dynamics simulation.

‡To whom correspondence should be addressed. E-mail: hongbin@chem.ubc.ca.

This article contains supporting information online at www.pnas.org/cgi/content/full/0700351104/DC1.

© 2007 by The National Academy of Sciences of the USA

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ResultsStructural Analysis of Top7. Top7 is a de novo designed protein thatconsists of 92 residues and shows significant thermodynamic sta-bility yet complex kinetic behavior (21, 22). Top7 is a �/� proteinand shows a novel protein topology (21) that has not been sampledby nature through evolution. Top7 is made of two � helices and one� sheet consisting of five � strands (21). The two terminal force-bearing strands (strands 1 and 5) are arranged in parallel, consti-tuting a shear topology. However, the two terminal strands areconnected to �-strand 3 without being directly connected to eachother. Therefore, Top7 potentially has more degrees of freedom torespond to mechanical stress than previously studied mechanicallystable proteins, such as I27 (23). To better understand its responseto force, Top7 structure can be described as being made of threeinterconnected substructures (Fig. 1). Substructure A (blue) in-cludes two � strands (residues 1–22) and one � helix (residues23–43). Substructure B (yellow) is sole � strand in the middle of thestructure (residues 44–54). Substructure C is structurally similar tosubstructure A and consists of one � helix (residues 55–74) and two� strands (residues 75–92). Substructure A and C have similarhydrogen bonding pattern, whereas substructure B is connected toboth A and C via backbone hydrogen bonds.

Top7 Shows Significant Resistance to Mechanical Unfolding. To in-vestigate the mechanical stability of Top7 using single-moleculeAFM, we constructed a polyprotein chimera (GB1)4-(Top7)2-(GB1)4, in which (Top7)2 is flanked by (GB1)4 at both ends (Fig.2A). In this polyprotein chimera, the well characterized proteinGB1 (8, 12), whose mechanical unfolding is characterized by acontour length increment �LC of �18 nm and an unfolding forceof �180 pN (at pulling speed of 400 nm/s), serves as an internalmarker for identifying single-molecule stretching events as well aspinning down the signature of the mechanical unfolding of Top7.

Stretching (GB1)4-(Top7)2-(GB1)4 results in force-extensioncurves showing characteristic sawtooth patterns (Fig. 2B), in whichthe individual force peaks correspond to the mechanical unravelingof the individual protein domains being stretched (5, 23). Theseforce-extension curves display two populations of unfolding forcepeaks with different peak-to-peak distance. For example, in the firstforce-extension curve, the eight unfolding events marked in redhave similar peak-to-peak spacing of �16 nm, whereas the twounfolding events marked in green have similar peak-to-peak spac-

ing of �24 nm. Fits of the worm-like chain model (WLC) ofpolymer elasticity (blue line) to the experimental data revealed thatthe unfolding events in red have �LC of �18 nm, whereas the twounfolding events in green have �LC of �29 nm. Because themechanical unfolding of GB1 is characterized by �LC of �18 nmand unfolding force of �180 pN, we can readily recognize theunfolding events with �LC of 18 nm as the mechanical unfolding ofGB1 domains in the polyprotein chimera. Because (Top7)2 aresandwiched between two (GB1)4 in our designed polyprotein, if weobserve five or more unfolding events of GB1 in a force-extensioncurve, we are certain that both Top7 domains have been stretchedand the force-extension curve must contain the signature of themechanical unfolding of both Top7 domains. Indeed, in all of theforce-extension curves of (GB1)4-(Top7)2-(GB1)4 that contain fiveor more GB1 unfolding events (red), we always observe twoadditional unfolding events with �LC of �29 nm (green). There-fore, we can assign the unfolding events of �LC of 29 nm to themechanical unfolding of Top7 domains without any ambiguity. Ahistogram of �LC for the mechanical unfolding of Top7 measuresan average �LC of 28.8 � 0.8 nm [supporting information (SI) Fig.5]. Top7 consists of 92 aa residues and is 33.1 nm long when it isunfolded and fully extended (0.36 nm/aa � 92 aa). The distancebetween the N and C termini of the folded Top7 is � 3.0 nm. Assuch, the complete mechanical unfolding of Top7 will result in �LC

Fig. 1. Structural features of Top7. Top7 is composed of five � strands andtwo � helices, and can be divided into three substructures: substructure A(residues 1–43), substructure B (residues 44–54), and substructure C (residues55–92). Cyan bars indicate the backbone hydrogen bonds connecting sub-structure B to substructures A and C.

Fig. 2. Force extension relationships of (GB1)4-(Top7)2-(GB1)4 polyprotein chi-mera. (A) A schematic of the designed (GB1)4-(Top7)2-(GB1)4 polyprotein chi-mera. (Top7)2 is sandwiched between two (GB1)4 on both ends in the chimera.GB1 domains are shown in red and Top7 in green. (B) Force-extension curves of(GB1)4-(Top7)2-(GB1)4. Two populations of unfolding events were observed: un-folding events (colored in red) of GB1 domains are characterized by �LC of �18nm and unfolding forces of �180 pN, whereas the unfolding events of Top7domains (green) are characterized by �LC of �29 nm. Blue curves correspond tothe worm-like chain fits to the force-extension curves. (C) Unfolding force histo-gram of Top7 (n � 425). The red line corresponds to the Monte-Carlo simulationresult of the mechanical unfolding of Top7 using �0 of 0.06 s�1 and �xu of 0.21 nmat a pulling velocity of 400 nm/s. (D) The unfolding forces of Top7 depend onpulling speed. The experimental data (black symbols) can be well described byMonte Carlo simulations (red line) using �0 of 0.06 s�1 and �xu of 0.21 nm.

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of 30.1 nm (33.1–3.0 nm), which is in close agreement with theexperimentally determined value of �LC, suggesting that the ex-perimentally observed unfolding of Top7 domains corresponds tothe complete unfolding of the folded Top7 domains. In addition,WLC model fits well the force-extension relationship of Top7 andthere is no significant deviation of the experimental data from thefits. These results indicate that the mechanical unfolding of Top7 isan apparent two-state process and there is no stable unfoldingintermediate state accumulating during its mechanical unfolding.

Because two Top7 domains are linked in series with eight GB1domains in the chimera, all of the domains will be subject to thesame stretching force and unfold sequentially according to theirintrinsic mechanical stability: the weakest one unfolds first, and thestrongest one unfolds last. Our designed polyprotein chimera allowsus to directly compare the mechanical stability of Top7 with that ofGB1 (8, 12). For the force-extension curves of the polyproteinchimera, we observed that the mechanical unfolding events of Top7scattered among the GB1 unfolding events: the mechanical unfold-ing of Top7 can occur before any GB1 unfolding event or quiteoften after some of the GB1 domains have been unfolded. Thisresult indicates that the mechanical stability of Top7 is comparableto that of GB1. Indeed, the unfolding force histogram of Top7measures an average unfolding force of 155 � 36 pN (Fig. 2C),which is slightly lower than that of GB1 (178 pN) (12). Suchmechanical stability is comparable with that of typical elastomericproteins, such as ubiquitin (13) and I27 from titin (23). This resultstrongly indicates that Top7 has significant mechanical stability.

The Transition State for the Mechanical Unfolding of Top7 Is HighlyNative Like. To fully characterize the mechanical unfolding of Top7,we examined the unfolding kinetics of Top7 by carrying out theexperiments at different pulling speeds. As shown in Fig. 2D, theaverage unfolding force for Top7 was found to depend on pullingspeeds. Because there is no detectable unfolding intermediate statein the force-extension curves, we modeled the mechanical unfoldingof Top7 as a two-state process with force-dependent unfolding rateconstant. Using standard Monte Carlo simulation procedures (23),we reproduced the force-extension curves, as those shown in Fig.2B, of polyprotein (GB1)4-(Top7)2-(GB1)4 at different pullingspeed to estimate the unfolding rate constant at zero force �0 andthe unfolding distance �xu between the folded state and transitionstate for Top7. We found that, by using a combination of �0 of 0.06s�1 and �xu of 0.21 nm, we can adequately describe the unfoldingforce histogram (Fig. 2C, solid line) as well as the pulling speed-dependency of the unfolding forces for Top7 (Fig. 2D, red line). The�xu for Top7 is similar to that of several other mechanically stableproteins (11, 13, 23). A small value of �xu indicates that transitionstate for the mechanical unfolding of Top7 is structurally verysimilar to the native state. In addition, we also observed that themechanical unfolding of Top7 is reversible, and Top7 folds back toits native state at zero force at a rate of �2.6 � 0.4s�1 (SI Figs. 6and 7).

Molecular Origin for the Mechanical Stability of Top7. Although thetwo terminal force-bearing � strands of Top7 constitute the sheartopology, unlike other mechanically stable proteins, the two ter-minal � strands are not directly connected. Instead, they are spacedby a third � strand and these three � strands are interconnected bya network of hydrogen bonds (Fig. 1). Stretching of Top7 willinvolve the deformation of hydrogen bonding network in the middlethree � strands and is much more complex than the scenario inwhich I27 is stretched and unraveled. Therefore, it is not straight-forward to predict the mechanical stability or the unfolding mech-anism of Top7 a priori.

Significant mechanical stability displayed by Top7 indicates thatthe direct connectivity of the two terminal force-bearing strands isnot a prerequisite for mechanical stability in the shear topology.Here we carried out SMD simulations of the mechanical unfolding

of Top7 to understand the molecular origin for the mechanicalstability as well as the unfolding mechanism of Top7. Moleculardynamics simulations have been extensively used to investigate themolecular mechanisms underlying the mechanical unfolding ofproteins, and atomic level pictures revealed by SMD have beenshown to correlate well with single molecule AFM results (17–20,24). We carried out SMD simulations in constant velocity pullingmode, which closely mimics AFM experiments, with several dif-ferent pulling velocities, ranging from 5 to 100 m/s. Due tocomputational cost, we performed five simulations at 10 m/s tocollect better statistics and only performed one simulation at 5 m/s.In the pulling velocity of 10 m/s, we also attempted to pull theprotein from N (three times) and C (twice) termini. Three of theseforce extension curves for the 10 m/s SMD are shown in Fig. 3A.Due to the shorter time scale accessible to SMD, the force peaksobserved in SMD are much higher than the ones observed in AFMexperiments. However, simulated Top7 force-extension curvesshare qualitative features that are consistent with those observed inAFM experiments.

The force-extension curves in Top7 SMD trajectories showedthat there is a single dominant force peak at �900–1,100 pNbetween 7 and 9 Å in pulling extension, and after that the proteinunfolds easier. This finding is in agreement with the AFM resultsthat each domain has one dominant force peak. Before reaching themain force peak, the stretching force increased rapidly accompa-nied by a relatively small gain in extension. During this process, thedistance between N and C termini increased gradually by �7 Å,whereas Top7 maintained its overall stable structure as well as itsbackbone hydrogen bonds within the � sheet. As the stretchingprocess continued, Top7 structure started to break down, giving riseto the main unfolding event of Top7: the backbone hydrogen bondsconnecting � strands 1 and 3 break concurrently, and substructureA and substructure B/C slide past each other (Fig. 3B). Thisobservation indicates that the hydrogen bonding pattern in Top7,especially those between � strands 1 and 3, plays crucial roles indetermining the mechanical stability of Top7. In most simulations

Fig. 3. Constant velocity SMD simulations of the mechanical unfolding ofTop7. (A) Force-extension curves of Top7 obtained from SMD (pulling velocity:10 m/s). For comparison, SMD simulations were also carried out on I27 (greentrace). Similar to AFM results, Top7 unfolds at slightly lower force than I27. (B)Comparison of Top7 structure before and after the main unfolding force peak.Dashed lines indicate the backbone hydrogen bonds in the � sheet.

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after the main unfolding event (after the breakage of A from B/C),substructure A will keep unraveling, whereas substructures B andC stay together and remained rather stable. This result is in goodagreement with a recent report that substructures B and C form astable miniprotein by itself (25). Upon further stretching, Top7unfolded gradually and the strands unraveled one by one in anunzipping fashion; this explains why no intermediate state isobserved in AFM experiments despite that B/C is autonomouslystable. For comparison, we also carried out SMD on I27. Ascompared with the force-extension curve of I27 (Fig. 3A, greencurve), the unfolding force peak for Top7 (800–1,100 pN) is slightlylower than that of I27 (1,400 pN), in good agreement with theAFM data.

The substructure-sliding unfolding mechanism revealed by SMDunderlines the molecular origin of the mechanical stability of Top7.This is a truly novel unfolding mechanism that has not been seenbefore and is significantly different from that for I27 or othermechanically stable proteins. The topology of Top7 is such that, forthe domain to be extended from both termini, the � sheet has to bebroken at certain point. Unlike the I27 unfolding, where there isonly one way to break the sheet: rupture of strand A� and G (18,19), in Top7 there are two possible ways to break the sheet: eitherrupture between substructures A and B or between substructuresB and C. Therefore, the mechanical unfolding of Top7, in principle,should have two possible unfolding pathways: in one of the unfold-ing pathways Top7 unfolds by sliding substructure A against B/C,whereas in the other one, substructure C slides against A/B totrigger unfolding. However, in every SMD simulation, the forcepeak correlates with the rupture of hydrogen bond patch betweensubstructure A and B, indicating that the patch of hydrogen bondslinking � strands 1 and 3 are easier to rupture than those between� strands 3 and 5. Therefore, sliding substructure A against B/Cconstitutes a lower energy barrier for the mechanical unfolding ofTop7, whereas sliding substructure C against A/B is of a higherbarrier. Hence, sliding substructure A against B/C dominates themechanical unfolding of Top7. When comparing the hydrogenbonds alone, the patch between � strands 1 and 3 are not weakerthan that between 3 and 5. Thus the difference is in overallinteractions between AB and between BC. Indeed, there arenoticeable differences in the hydrophobic patterns in the interfacesbetween AB and between BC (data not shown), indicating thatinteractions other than hydrogen bonding may also contribute tomechanical stability of Top7.

Tuning the Mechanical Stability of Top7 by Rationally Redesigning ItsMechanical Unfolding Pathway. SMD simulations revealed two pos-sible mechanical unfolding pathways for Top7, and the mechanicalstability of Top7 is determined by the pathway of the lowermechanical unfolding barrier. Such unique unfolding mechanismoffers unprecedented opportunity enabling us to rationally modu-late the mechanical stability of Top7 by redesigning its mechanicalunfolding pathway. If the normal unfolding pathway of Top7, thesliding of substructure A against B/C, were blocked by covalentlylinking � strand 1 to 3, Top7 would have to unfold by following thepathway of the higher energy barrier by sliding substructure C pastA/B. Such shift of unfolding pathway will result in an increase of themechanical stability of Top7. Based on this hypothesis, we usedMODIP (modeling of disulfide bridges in proteins) (26) to identifypotential sites for introducing disulfide bond between � strands 1and 3. Among identified potential sites, we selected positions 3 and51 because they are very close to the N terminus and a disulfidebridge will completely block the sliding of substructure A againstB/C. We engineered a double cysteine mutant Q3C/T51C-Top7(Q3C/T51C) and constructed a polyprotein chimera (GB1)4-Q3C/T51C-(GB1)4 for AFM experiments. The formation of a disulfidebond in the oxidized Q3C/T51C will result in a shorter �LC, whichmay affect the effective loading rate for the polyprotein. To avoidsuch complications, we incorporated only one copy of Q3C/T51C

in the designed polyprotein chimera. Because �Lc of Q3C/T51Coccurs only after Q3C/T51C unfolds, the �Lc of Q3C/T51C will notaffect the effective loading rate for the only Q3C/T51C present inthe engineered polyprotein chimera. Instead, it only affects theeffective loading rate for the GB1 domains that unfold afterQ3C/T51C. Hence, comparing the unfolding force of the oxidizedand reduced Q3C/T51C will directly illustrate the effect of shiftingunfolding pathway on the mechanical stability of Top7.

A typical force-extension curve of (GB1)4-Q3C/T51C-(GB1)4 inthe presence of reducing agent DTT is shown in Fig. 4A. In thereduced form, the disulfide bond does not form and hence Q3C/T51C will behave similarly as wt-Top7. Indeed, the mechanicalunfolding of reduced Q3C/T51C is characterized by �Lc of �30 nm(Fig. 4A, green, and Fig. 4B Inset). The average unfolding force ofreduced Q3C/T51C is 140 � 28 pN (Fig. 4B), slightly lower than thatof wt-Top7 due to cysteine mutations. Similar to wt-Top7, the

Fig. 4. Tuning the mechanical stability of Top7 by redesigning its mechanicalunfolding pathway. Force-extension curves and cartoon representations ofdesigned Top7 mutants are shown in A, C, and E. (A) Mechanical properties ofreduced Q3C/T51C-Top7. In the presence of DTT, the disulfide bond does notform. The force-extension curves show unfolding events of reduced Q3C/T51Cwith �LC of �30 nm (green). (B) The average unfolding force is 140 pN (n �299) and �LC is 31.0 � 2.0 nm (Inset). Solid lines are Gaussian fits to theexperimental data. (C) The mechanical stability of oxidized Q3C/T51C in-creased due to the shifting of the unfolding pathway. Upon oxidation, 3C and51C form a disulfide bond that covalently links � strands 1 and 3, blocking theunfolding pathway of sliding substructure A against B/C. The unfolding ofoxidized Q3C-T51C results in unfolding events with �LC of �13 nm. (D) Theaverage unfolding force of oxidized Q3C/T51C is 172 pN (n � 218), �30 pNincrease as compared with the reduced Q3C/T51C (B), and �LC is 13.5 � 1.7 nm(Inset). Red lines are Gaussian fits. (E) G90P-Top7 unfolds at lower forces. G90Pmutation selectively disrupted the hydrogen bonds linking strands 3 and 5 anddestabilized substructure C. The unfolding of G90P mutant results in unfold-ing events with �LC of �29 nm (green). (F) The average unfolding force forG90P is 126 � 29 pN (n � 326), weaker than wt-Top7 (blue line). Red line is aGaussian fit to the experimental data. (Inset) The pulling speed-dependenceof unfolding forces of G90P (red line and symbols), which has a similar slopeas that of wt-Top7 (black line). The unfolding force of G90P is lower than thatof wt-Top7 at all of the pulling speeds.

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mechanical unfolding of reduced Q3C/T51C is presumably due tothe sliding of substructure A against B/C. In contrast, stretching ofoxidized Q3C/T51C results in unfolding events with �Lc of �13 nm(Fig. 4C, colored in green and Fig. 4D Inset). The fully stretchedoxidized Q3C/T51C is 16.2 nm long [(92 aa � 47 aa) � 0.36 nm/aa]and the distance between N and C termini is 3 nm, resulting in �Lcof 13.2 nm upon unfolding of the oxidized Q3C/T51C, in goodagreement with our measurements. This result indicated that, in theoxidized form, a disulfide bond indeed formed between 3C and 51C(Fig. 4C). As such, � strand 1 is covalently linked to strand 3, makingthe sliding of substructure A against B/C impossible. Hence,oxidized Q3C/T51C will have to unfold by sliding substructure Cagainst A/B, a shift of the unfolding pathway as compared withwt-Top7. As predicted, the unfolding force for oxidized Q3C/T51Cis indeed increased: the average unfolding force is 172 � 32 pN (Fig.4D), �30 pN increase as compared with that of reduced Q3C/T51C(Fig. 4B). To our best knowledge, this is the first example ofincreasing the mechanical stability of a protein via rational design,demonstrating the power of combining mechanistic study withcomputational design for tailoring the mechanical properties ofproteins.

We also explored the possibility of lowering the mechanicalstability of Top7 by shifting its unfolding pathway. Sliding substruc-ture C against A/B is the pathway of higher energy barrier forwt-Top7. If we destabilize the B/C interface while keeping A/Binterface intact, it is possible that sliding C against A/B will becomemuch easier than sliding A against B/C. Hence, the unfolding ofTop7 will be initiated by sliding C against A/B and occur at lowerforces. Hydrogen bonds are known to play important roles indetermining the mechanical stability of proteins. Therefore, weattempted to use proline mutagenesis to selectively disrupt thehydrogen bonds joining substructure B and C to weaken the B/Cinterface. We mutated Gly-90 in � strand 5 to proline to disrupt thebackbone hydrogen bond between Gly-90 and Val-46 in � strand 3,as well as the local � sheet structure of region C. We did not mutateany residue of �-strand 3 to avoid any structural perturbation ofregion A/B. G90P mutant was well folded as confirmed by far-UVCD spectroscopy (SI Fig. 8). The mechanical unfolding of G90P ischaracterized by �LC of �29 nm and readily identified from theforce-extension curves of (GB1)4-G90P-(GB1)4 (Fig. 4E and SI Fig.9). The average unfolding force of G90P is 126 � 29 pN (n � 326,Fig. 4F), well below the average unfolding force for wt-Top7 (155pN). We also measured pulling-speed dependence of unfoldingforce for G90P (Fig. 4F Inset). It is evident that the averageunfolding force of G90P is lower than that of wt-Top7 at all pullingspeeds, indicating that the mechanical unfolding of G90P is distinctfrom wt-Top7. Because substructures A and B are intact in G90P,we attribute the lower unfolding force for G90P to the sliding of thedestabilized substructure C against A/B. These results clearlydemonstrate the feasibility to systematically modulate the mechan-ical stability of Top7 by shifting its mechanical unfolding pathway.

DiscussionProtein topology, not evolved function, is known to be importantin determining mechanical stability of proteins. Previous studiesshowed that shear topology is an important feature shared bymost mechanically stable proteins (11–13). However, theseproteins belong to either Ig � sandwich fold or �-grasp fold (withthe exception of GFP and ankyrin), in which shear topology ispresent and two terminal � strands are directly connected. Top7is computationally designed and contains sequence and topologythat has not yet been seen in biological machinery (21). Althoughnot evolved nor designed for mechanical purposes, Top7 showssignificant mechanical stability and contains features shown bynatural elastomeric proteins. As such, Top7 represents a me-chanically stable protein fold that is distinct from Ig-like �sandwich and �-grasp folds, thus adding a member to the familyof mechanically stable proteins. These results demonstrate that

the direct connectivity between the two force-bearing � strandsin a shear topology is not a prerequisite for mechanical stability.Although the two terminal force-bearing � strands of Top7 arenot directly connected, the sliding of substructures against eachother provides Top7 with the resistance to mechanical unfolding.This finding enriches our understanding of the design of me-chanically stable proteins and illustrates a new structural featurethat is important for mechanical stability and yet may be presentin a large number of proteins that are not necessarily evolved andoptimized for mechanical purposes. Hence, our findings offergreat opportunities in searching proteins with diverse mechan-ical features in a much broader protein fold space, and willgreatly expand the pool of proteins of significant mechanicalstability. Furthermore, our study suggests that not all of theprotein folds that can bear forces have been sampled by natureand, therefore, success of protein mechanics does not dependentirely upon naturally gifted materials.

Because the molecular determinants for mechanical stabilityof proteins remain elusive, it is still a great challenge tomodulate the mechanical properties of proteins in a rationalfashion. Although there has been some elegant work attempt-ing to tune the mechanical stability (27–33), most of theseefforts are largely trial-and-error in nature. By combiningsingle-molecule AFM and SMD, we found a substructure-sliding unfolding mechanism for Top7 and revealed the exis-tence of two possible such unfolding pathways with apparentlydifferent height of energy barrier. Such molecular insightsenabled us to devise a rational methodology to tune themechanical stability of Top7 by redesigning its unfoldingpathway. Using computationally designed disulfide mutant, wewere able to specifically block one unfolding pathway andforced Top7 to unfold via the pathway of higher energy barrier,hence increased the mechanical stability of Top7. Loweringthe mechanical stability of a given protein has been attemptedwith great success (27, 29, 34); however, it has been challengingto rationally increase the mechanical stability of a protein. Thesuccessful example demonstrated here represents a uniqueapproach toward this challenge, and illustrates the power andgreat potential of simulation and computational biology intailoring the mechanical properties of proteins in a systematicand rational way. Although the approach demonstrated here isdevised based on Top7, the concept of tuning mechanicalstability by redesigning unfolding pathway may be general andcan be applicable to other proteins. We anticipate that such astrategy will serve as one of the important criteria to compu-tationally design novel proteins with tunable mechanical sta-bility that can be further modulated via environmental stimuli,such as redox potential.

Recent de novo design of proteins has made tremendousprogress and proteins with novel sequence and fold have beendesigned with atomic level accuracy (21). Although de novodesign of enzymes with well defined functions remains challeng-ing, computationally designing proteins of significant mechani-cal stability may be within the reach of the current computationalbiology, as mechanical stability is a largely structural property.Being a mechanically stable protein, Top7 actually represents thefirst example in this aspect, although the original design of Top7was not intended for mechanical purposes. We anticipate thatthe combination of de novo design, SMD, and single-moleculeAFM will make it possible to de novo design proteins of definedtopology and unfolding pathways to achieve significant andtailored mechanical properties, which will be an importantmilestone toward using engineered elastomeric proteins for welldefined nanomechanical applications.

Materials and MethodsProtein Engineering.Plasmid containing Top7 gene was obtainedas a kind gift from David Baker. Top7 gene was amplified via

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PCR using forward and reverse primers containing restrictionsites BamHI and BglII followed by KpnI, respectively. Geneencoding Q3C/T51C-Top7 was constructed from wt-Top7 usingmegaprimer approach (33). G90P-Top7 was constructed usingsite-directed mutagenesis. The sequences of the constructedgenes were confirmed by direct DNA sequencing.

To investigate mechanical properties of a protein in detailusing single-molecule AFM, it is generally desirable to constructa polyprotein containing identical tandem repeats of the proteinof interest. However, possibly due to DNA recombination, wewere unable to make Top7 polyprotein gene bigger than (Top7)2even in the recombination defective strain BLR(DE3).

To overcome this hurdle, we constructed a chimeric polypro-tein (GB1)4-(Top7)2-(GB1)4, in which (Top7)2 was flanked by(GB1)4 at both ends, for AFM experiments. Based on theidentity of the sticky ends generated by BamHI and BglIIrestriction enzymes, pQE80L-(GB1)4-(Top7)2-(GB1)4 was con-structed using well established methodology (12, 23). Similarly,pQE80L-(GB1)4-Q3C/T51C-(GB1)4 and pQE80L-(GB1)4-G90P-(GB1)4 were constructed. The polyproteins were ex-pressed in DH5� strain and purified using Ni-NTA affinitychromatography. For reduced Q3C/T51C, 10 mM DTT wasadded to the solution. Oxidized form of Q3C/T51C was obtainedby air oxidation.

Single-Molecule AFM. Single-molecule AFM experiments werecarried out on a custom built AFM, which was constructed as

described (35). The unfolding of Top7 was described as atwo-state process with force-dependent rate constants. MonteCarlo simulations were carried out on (GB1)4-(Top7)2-(GB1)4according to published procedures (23). The number of domainsin a polyprotein affects the measured mechanical stability, butthis effect is small (36, 37). Hence, our measured mechanicalstability of Top7 using a heteropolyprotein does not differ fromthe value expected from a polyprotein (Top7)8.

SMD Simulations. Top7 was subject to a simulated equilibrationand constant velocity stretching in SMD. The aqueous environ-ment was modeled using explicit water representation, i.e.,protein was solvated in a water box with periodic boundaryconditions. The water box was large enough for equilibration andfor the first 50 Å of stretching (length 110 Å, width 50 Å, height55 Å). The whole protein–water system contains �31,000 atoms.

The velocities used in SMD simulations were in the rangebetween 5 and 100 m/s. The model preparation and data analysiswere performed with VMD and MD simulation with NAMD asdescribed (19). During the 1-ns equilibration, the protein is rea-sonably stable from the initial Protein Data Bank structure 1QYS,with the rmsd in the range of 2Å. That final structure was thestarting point used in the constant velocity pulling SMD.

This work is supported by Natural Sciences and Engineering ResearchCouncil of Canada, Canada Research Chairs program, and CanadaFoundation for Innovation (to H. Li) and National Institutes of HealthGrant P01AI060915 (to H. Lu).

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