Artigo 2- Polyp Rote In GB1 is an Ideal Elastomeric Protein

8/6/2019 Artigo 2- Polyp Rote In GB1 is an Ideal Elastomeric Protein.

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LETTERS

Polyprotein of GB1 is an ideal artificial

elastomeric protein

YI CAO AND HONGBIN LI*

Department of Chemistry, The University of British Columbia, Vancouver, BC, V6T 1Z1, Canada*e-mail:[email protected]

Published online: 21 January 2007; doi:10.1038/nmat1825

Naturally occurring elastomeric proteins function as molecularsprings in their biological settings and show mechanicalproperties that underlie the elasticity of natural adhesives1, celladhesion proteins2 and muscle proteins3. Constantly subject

to repeated stretchingrelaxation cycles, many elastomericproteins demonstrate remarkable consistency and reliabilityin their mechanical performance3,4. Such properties hadhitherto been observed only in naturally evolved elastomericproteins. Here we use single-molecule atomic force microscopytechniques to demonstrate that an artificial polyprotein made oftandem repeats of non-mechanical protein GB1 has mechanicalproperties that are comparable or superior to those of knownelastomeric proteins. In addition to its mechanical stability5,

we show that GB1 polyprotein shows a unique combinationof mechanical features, including the fastest folding kineticsmeasured so far for a tethered protein, high folding fidelity, lowmechanical fatigue during repeated stretchingrelaxation cyclesand ability to fold against residual forces. These fine features

make GB1 polyprotein an ideal artificial protein-based molecularspring that could function in a challenging working environmentrequiring repeated stretchingrelaxation. This study representsa key step towards engineering artificial molecular springs withtailored nanomechanical properties for bottom-up constructionof new devices and materials6.

One of the common features of natural elastomeric proteinsis their tandem modular construction7,8, allowing them to unfoldsequentially when subject to stretching forces9,10. Such a modularunfolding mechanism, which is extensively exploited by nature ina wide variety of materials, conveys high toughness to elastomericproteins and makes them perfect shock-absorbers2,9,11. On removalof stretching force, unfolded proteins can refold back to theiroriginal folded structure to recover their mechanical stability

efficiently. Such features enable elastomeric proteins to maintaintheir mechanical properties during repeated stretchingrelaxationcycles12 and fulfil their mechanical function reliably.

Inspired by naturally evolved elastomeric proteins, researchershave started to explore non-mechanical proteins to expand thetoolbox of elastomeric proteins and construct artificial molecularsprings5,1319. We and others recently identified that the smallnon-mechanical protein GB1 (Fig. 1a), the streptococcal B1immunoglobulin-binding domain of protein G20, and its structuralhomologue protein L, have significant mechanical stability5,14,21. Tomimic the tandem modular design of natural elastomeric proteins,we engineered polyprotein (GB1)8, consisting of eight identicaltandem repeats of GB1 domains (Fig. 1b). As we reported5,stretching (GB1)8 results in forceextension curves of characteristic

saw-tooth pattern appearance (Fig. 1c) with an average unfoldingforce of 18441 pN (mean standard deviation, n= 6,991, at apulling speed of 400 nms1) for GB1 domains, which is comparableto the mechanical stability of the I27 domain from the natural

elastomeric protein titin22. However, having significant mechanicalstability is only one of the prerequisites for a non-mechanicalprotein to function as an artificial elastomeric protein. It is essentialthat artificial elastomeric proteins possess additional features thatare comparable to those of natural ones so that they can functionconsistently and reliably under a continuous stretchingrelaxationenvironment. Because non-mechanical proteins are not evolved formechanical function, it remains to be demonstrated whether theyhave such unique mechanical traits. Here we use single-moleculeatomic force microscopy (AFM) to demonstrate that (GB1)8 isthe first example that an artificial polyprotein can function as anelastomeric protein by showing features that can compete with thatof naturally able ones.

Similar to many naturally occurring elastomeric proteins2,9,2325,

mechanical unfolding of GB1 is a non-equilibrium process(Fig. 1d): there is significant hysteresis between thestretching/unfolding (black) and relaxation (red) curves of (GB1)8(Fig. 1d), indicating that much of the energy invested duringstretching (shaded area) is dissipated as heat in the process ofmechanical unfolding of GB1 domains. Such energy dissipationwill entail high toughness for (GB1)8 and makes it an idealshock-absorber2,9,11.

For nanomechanical applications, it is an important requisitethat a candidate protein can fold fast to efficiently recover itsmechanical stability and avoid mechanical fatigue/ageing. GB1is a fast folder via a biphasic kinetics in water26 with foldingrate constants of2,000 s1 and 700s1 for the fast and slowphases, respectively. However, tethering both termini of proteins

on stretching may restrict the degrees of freedom for proteinsand consequently slow down the folding kinetics22. For example,the folding rate constant of I27 on tethering is 30 times slowerthan that of I27 free in solution22. Hence, it is unknown whetherGB1 can fold efficiently on tethering. Here we used a standarddouble-pulse protocol4,22 to measure the folding kinetics of GB1on tethering (Fig. 2a inset, see the Methods section for details).We observed that the folding probability Nrefold/Ntotal at zero forcedepends exponentially on relaxation time t, where Ntotal is thetotal number of domains in the polyprotein chain and Nrefold isthe number of domains that refold after relaxation (Fig. 2b). Itis evident that 80% of GB1 domains refold within 2 ms andalmost 100% of GB1 domains refold when t is longer than 15 ms.This result suggests that the folding reaction of GB1 on tethering

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18.2 nm

18.0 nm

50 nm 20 nm

Extension Extension

Force

Force

200

pN

10

0pN

a b

c d

F

F

2

1

4

3

Figure 1 Polyprotein (GB1)8 has significant mechanical stability. a, The three-dimensional structure of the non-mechanical protein GB1. GB1 is an /-protein with the

two terminal -strands (strands 1 and 4) arranged in parallel; these are bonded by a series of backbone hydrogen bonds (indicated by lines) and form mechanical resistance

to unfolding. b, A schematic diagram of polyprotein (GB1)8. Eight identical GB1 monomers are joined in tandem by connecting the N- and C-termini. c, Typical

forceextension curves of (GB1)8 polyproteins. These forceextension curves show a characteristic saw-tooth pattern, with equally spaced force peaks, which result from the

mechanical unravelling of each individual GB1 domain in the polyprotein chain. The last peak in each forceextension curve corresponds to the detachment of the protein

from either the AFM tip or substrate. The forceextension curves can have up to eight unfolding force peaks. The forceextension curves can be well described by the

worm-like-chain (WLC) model of polymer elasticity. WLC fits (red lines) to the consecutive individual unfolding force peaks measure a contour length increment Lc of

18.00.5 nm (mean standard deviation, n= 472) for the unfolding of the GB1 domain. d, Mechanical unfolding of GB1 is a non-equilibrium process. A pair of typicalstretching (black line) and relaxation (red line) curves of a polyprotein GB1 at a pulling speed of 400nm s1. The stretching curve shows a saw-tooth pattern with six GB1

unfolding peaks. In contrast, the relaxation curve shows only a nonlinear entropic elastic behaviour. The hysteresis between the stretching and relaxation curves (shaded

area) reflects the energy dissipated during the mechanical unfolding of GB1 domains.

proceeds very fast. Because the unfolding reaction is significantlyslower than the folding reaction at zero force, we treated the foldingof GB1 at zero force as a first-order reaction,

Nrefold/Ntotal = 1exp(0 t), (1)

where 0 is the folding rate constant at zero force. For comparison,

we plotted equation (1) using 0 of 2,000 s1

and 200 s1

in Fig. 2b.Evidently, the folding kinetics of GB1 is much faster than 200 s1

but slower than 2,000 s1. Fitting equation (1) to our data estimatesthe folding rate constant of 720 120s1 for GB1 being tethered.However, owing to the lack of measurements in the range of02ms, 0 of 720 s

1 has to be regarded as somewhat approximate.Improvements in temporal resolution will be required to achieve amore accurate measurement of the folding rate constant of GB1 ontethering. Nonetheless, such fast folding of GB1 outperforms anynatural elastomeric protein2,22,25 and makes GB1 the fastest folderreported so far under tethering: 600 times faster than I27 and threetimes faster than the fast folder filamin22,25. This result indicates thattethering does not significantly impede the folding of GB1 and it ispossible for a non-mechanical protein to fold quickly on tethering,

making (GB1)8 a perfect candidate for artificial nanomechanicalsprings, in that it can regain its mechanical stability efficiently.

The folding rate of elastomeric proteins can be significantlyslowed down by residual force acting on the proteins duringfolding. We used a modified double-pulse protocol to investigatewhether GB1 can fold against considerable force. As shown inFig. 3a (inset), instead of being relaxed to zero extension, the

unfolded polyprotein was relaxed partially to a shorter extension,x, and allowed to refold for 10 ms. For x> 0, a residual force willact on the polyprotein chain owing to the entropic elasticity of thepolymer chain, which can be calculated using the WLC (ref. 27)model of polymer elasticity. It is clear that, when the polyproteinwas relaxed only partially (x> 0), the number of domains thatrefolded within 10 ms decreased sharply as x increased. Theprobability of refolding (Nrefolded/Ntotal) versus the residual force(F), which was assumed constant during relaxation and calculatedusing the WLC model, shows a reverse sigmoid shape (Fig. 3b) (seethe Supplementary Information). It is of note that the folding ofGB1 is almost completely inhibited in a time window of 10 ms bya force of12 pN. On increasing the observation time window t,it is anticipated that the probability of refolding at a given force

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t= 2.5 ms

t= 5 ms

t= 34 ms

20 nm

2

00

pN

t

Time

Z

0

0

0.2

0.4

0.6

0.8

1.0

10 20 30 40 50t (ms)

a

b

Nrefolded

/Ntotal

Figure 2 The fast folding kinetics of GB1. a, The folding kinetics of GB1 is probed

by AFM using a double-pulse protocol (inset). The polyprotein is first stretched to

unfold all the GB1 domains in the chain and count the total number of domains that

are available in the polyprotein chain, Ntotal (upper traces), and then the unfolded

polyprotein is quickly relaxed back to its original length within 2ms. After a

relaxation time t, the protein is stretched again to count the number of domains that

have refolded during relaxation, Nrefolded (lower traces). b, Plot of the refolding

probability, Nrefolded/Ntotal, versus t. Error bars represent standard deviation. Because

the unfolding rate constant is negligible at zero force, the folding kinetics can be

described adequately using a simple first-order kinetic equation,

Nrefolded/Ntotal (t)= 1exp(0 t), using 0 = 720120 s1 (black solid line). The

data point at zero waiting time was added to facilitate the fitting. For comparison,

we also plotted equation (1) using 0 of 2,000s1 (grey solid line) and 200s1 (grey

dashed line), the fast-phase folding rate constant of GB1 in water and the fastest

folding rate constant reported so far for tethered proteins, respectively.

F will increase. Indeed, when t was increased to 1 s, the observed(Nrefolded/Ntotal)F curve shifted towards higher force. These resultsindicate that GB1 can fold at a considerable rate even at a residualforce up to 15 pN. Such ability for GB1 to fold against considerable

Z

x

Lo

t

Time

First pull

x/LO = 0

x/LO = 0.25

x/LO = 0.5

0

0

0.2

0.4

0.6

0.8

1.0

5 10 15 20 25 30F (pN)

Nrefolded

/N

total

a

b

Figure 3 GB1 can fold in the presence of residual forces. a, Folding kinetics of

GB1 under different forces is probed by a modified double-pulse protocol (inset).

First, the protein is stretched to a certain length (L0 ) to unfold all the GB1 domains in

the polyprotein chain. Then, it is rapidly relaxed to a shorter length (x) and held for a

fixed time period (10ms). A second pull of the protein measures the number of

domains refolded (Nrefolded ) during the waiting time under the force. The total number

of peaks in the first pulling curve between the lengths xand L0 is counted as Ntotal.

b, Plot of Nrefolded/Ntotal versus residual force. Black squares correspond to the data

obtained with t= 10ms, and grey squares correspond to the data obtained with

t= 1 s. Error bars represent the standard deviation of each independent

measurement. The residual force acting on the unfolded polyprotein chain slowed

down the folding process of GB1 significantly. Solid lines correspond to the curves

fitted by the function Pf (F)= Nrefolded/Ntotal = 1exp(t0)exp(Fxf/kBT),

where t is the relaxation time, F is the force acting on the protein, xf is the folding

distance, kB is the Boltzmann constant and Tis the absolute temperature. The black

line was generated using xf = 2.1nm, 0= 720 s1 and t= 10ms, and the grey

line was generated using xf = 2.1nm, 0= 720 s1 and t= 1 s.

residual forces matches similar traits shown by natural elastomericproteins2,4,22, although GB1 is not evolved for mechanical function.This result reveals the great potential of GB1 polyprotein as anartificial elastomeric protein.



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20 40 60 80 100

1

33

51

101

124

210

246

Extension (nm)

Force

200

pN

0 100 200 300 400

Unloading force (pN)

Frequency

ofevents

0

0

0

200

20 40 60 80

0

0

0.5

1.0

0.5

1.0

100 200

0

0.5

1.0

200

300

100 200

Number of cycles

Folding

probability

a

c

b

300

200

300

Unfolding

force

(pN)

Figure 4 Polyprotein (GB1)8 does not show noticeable mechanical fatigue. a, Forceextension curves of polyprotein (GB1) 8 during a repeated stretchingrelaxation

experiment. The number above each curve indicates the number of cycles that the polyprotein has been subject to. b, The unfolding force and folding probability of GB1

remain unchanged during repetitive stretchingrelaxation cycles. The top panel was measured from the same molecule as shown in Fig. 4a. The middle and bottom ones

were measured from two additional molecules that were subject to repeated stretchingrelaxation. The blue diamonds and red squares correspond to the average folding

probability and unfolding force of GB1, respectively, in ten consecutive cycles. Error bars represent standard deviation. Solid lines indicate the overall average values of the

folding probability and unfolding force over all the stretchingrelaxation cycles for the polyprotein under investigation. c, Repeated stretchingrelaxation cycles do not weaken

the mechanical stability of GB1. The unfolding force histogram compiled from the unfolding events of GB1 during the repeated stretchingrelaxation cycles for the same

polyprotein (red bars) is indistinguishable from the unfolding force histogram obtained by stretching different individual (GB1) 8 polyproteins (black bars) as shown in Fig. 1c.

Force-induced unfoldingrefolding cycles may be part ofthe natural life of natural elastomeric proteins2,9. It is notsurprising that they can maintain mechanical stability after manyrepeated stretchingrelaxation cycles without showing significantfatigue4,12 . Can a molecular spring made of the non-mechanicalprotein GB1 function in the challenging working environmentand survive as many stretchingrelaxation cycles as its naturalcounterparts? To address this question, we subject a single (GB1)8molecule to repetitive stretchingrelaxation for as many cyclesas possible before the protein detaches from either the AFMtip or substrate. Figure 4a shows forceextension curves afterdifferent numbers of stretchingrelaxation cycles from such an

experiment, in which the protein survived a total of 276 cyclesbefore it detached. Between consecutive cycles, the protein wasrelaxed at zero extension for 15 ms. The folding probabilityof GB1 remained constant throughout the experiment (Fig. 4b,top panel, blue symbols), indicating that almost all the GB1domains in the chain can regain their mechanical stability afterrelaxation, regardless of the number of stretchingrelaxation cyclesthey have undergone. This result demonstrates that there isno noticeable mechanical fatigue preventing GB1 domains fromfolding. Furthermore, GB1 did not show significant fatigue in theform of reduced mechanical stability. As shown in Fig. 4b (toppanel, red symbols), the average unfolding forces of GB1 remained



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constant (180 pN) throughout the experiment. Similar resultswere observed on additional polyprotein GB1 molecules that weresubject to repetitive stretchingrelaxation cycles (Fig. 4b, middleand bottom panels). The overall unfolding force histogram ofGB1 compiled from repetitive stretchingrelaxation cycles (Fig. 4c,red) is almost identical to that obtained by stretching individual(GB1)8 (Fig. 4c, black). These results indicate that GB1 retainedits mechanical stability during repeated stretchingrelaxation cyclesand no mechanical fatigue is present, at either individual moleculeor ensemble level (see the Supplementary Information). Such aproperty is similar to that of projectin (an insect flight muscleprotein)4, and clearly outperforms mammalian titin12,28, at least atthe single-molecule level.

Misfolding can occur for elastomeric proteins and will resultin altered mechanical response29. A recent ensemble chemicalunfolding/folding study suggested that tandem modular proteinswith high sequence identity are prone to misfolding andaggregation due to the effective high local protein concentrationin the vicinity of constituting domains in the tandem modularproteins30. An implication of this study is that homopolyproteins,such as (GB1)8, are potentially prone to misfolding and assuch their mechanical properties will be compromised. Indeed,misfolding events were reported for tandem modular proteins

with varied frequency (2% for polyprotein (I27)12 and 4%for a recombinant fragment of tenascin29). To explore whethermisfolding of GB1 in the polyprotein could potentially jeopardizethe mechanical performance of GB1, we monitored the foldingfidelity of (GB1)8 using repeated stretchingrelaxation protocols.The contour length increment on domain unfolding (Lc)is sensitive to misfolding and has been used to monitor theformation of misfolded state29. GB1 has a Lc of 18.0 0.5nm.We found that more than 99.8% of the unfolding events ofGB1 domains in repetitive stretchingrelaxation cycles show Lcof 18 nm, identical to those in single-pulling experiments(Fig. 1c). Moreover, the average unfolding forces of GB1 domainsin repetitive stretchingrelaxation cycles remained unchanged(Fig. 4b). These results strongly indicated that the folding reaction

of GB1 proceeded with exceptionally high fidelity, which is evensuperior to some natural elastomeric proteins29.

In summary, we demonstrate that artificial GB1 polyproteinexhibits a unique combination of mechanical features, includingfast, high-fidelity folding kinetics, low mechanical fatigue andability to fold against residual force. These properties allow theartificial GB1 polyprotein to recover its mechanical stability moreefficiently and help to reduce mechanical fatigue over long periodsof continuous stretchingrelaxation cycles. These mechanicalfeatures make GB1 polyprotein an ideal artificial elastomericprotein. Because GB1 is not naturally evolved for mechanicalfunction, the superior mechanical properties shown by GB1polyprotein reveal promising prospect for engineering elastomericproteins using non-mechanical proteins. It is anticipated that

the mechanical properties of GB1 can be further finely tunedusing protein engineering techniques, an important step towardstailoring the mechanical properties of elastomeric proteins tomeet the requirements of different working environments andintegrating artificial elastomeric proteins into nanomechanicaldevices and/or constructing materials (such as hydrogels) withsuperior mechanical properties.

METHODS

PROTEIN ENGINEERING

The plasmid encoding GB1 protein was generously provided by David Baker of

the University of Washington. GB1 monomer, flanked with a 5 BamHIrestriction site and 3 BglII and KpnI restriction sites, was amplified by

polymerase chain reaction and subcloned into pQE80L expression vector. On

the basis of the identity of the sticky ends generated byBamHI and BglIIrestriction enzymes, the (GB1)8 polyprotein gene was constructed using an

iterative approach of cloning GB1 monomer into monomer, dimer into dimer

and tetramer into tetramer22. The sequence of the polyprotein (GB1)8 is

MetArgGlySer(His)6-GlySer(GB1-ArgSer)8-CysCys, where the linker sequence

ArgSer between GB1 domains resulted from the hybrid sites ofBamHI andBglII. (GB1)8 was overexpressed in DH5 strain and purified by Ni

2+-affinity

chromatography. The purified polyprotein sample was at a final concentration

of740g ml1

, and was kept at 4

C in PBS buff

er with 5 mM dithiothreitol(DTT) to prevent the dimerization of(GB1)8 via the two C-terminus

cysteine residues.

SINGLE-MOLECULE ATOMIC FORCE MICROSCOPY

Single-molecule AFM experiments were carried out on a custom-built AFM.

The details of single-molecule AFM experiments have been described

elsewhere5. In our AFM, we used a high-speed, high-performance PicoCube

XYZ piezo stage (P-363) from Physik Instrumente (Karlsruhe, Germany). This

actuator is equipped with capacitive sensors for all three axes and has a high

resonant frequency in the zaxis (9.8kHz).

To measure the folding kinetics of GB1 at zero force, we used a

double-pulse protocol22: (GB1)8 was first extended to unfold all the GB1

domains in the chain. Then the unfolded polyprotein was quickly relaxed to

zero extension (within 2 ms, which sets the shortest relaxation time for the

folding experiments) before it detached from either the AFM tip or the

substrate. The number of unfolding force peaks measures the total number of

GB1 domains available in the polyprotein chain, Ntotal . After relaxation at zero

extension for a variable period of time, t (from 0 to 50ms), the polyprotein wasstretched again by the second pulse. Because a few GB1 domains refolded

within t, we observed the characteristic sawtooth pattern again. The number ofdomains that refolded (Nrefold) within the waiting time t can be counted from

the number of unfolding force peaks in the second forceextension curve.

Received 24 July 2006; accepted 12 December 2006; published 21 January 2007.

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AcknowledgementsThis work was supported by the Natural Sciences and Engineering Research Council of Canada, theCanada Research Chairs programme and the start-up fund from the University of British Columbia.Y.C. is partially supported by the Laird Fellowship.Correspondence and requests for materials should be addressed to H.L.Supplementary Information accompanies this paper on www.nature.com/naturematerials.

Competing financial interestsThe authors declare that they have no competing financial interests.

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Artigo 2- Polyp Rote In GB1 is an Ideal Elastomeric Protein