# Nonkinetic modeling of the mechanical unfolding of multimodular proteins: theory and experiments.

**ABSTRACT** We introduce and discuss a novel approach called back-calculation for analyzing force spectroscopy experiments on multimodular proteins. The relationship between the histograms of the unfolding forces for different peaks, corresponding to a different number of not-yet-unfolded protein modules, is exploited in such a manner that the sole distribution of the forces for one unfolding peak can be used to predict the unfolding forces for other peaks. The scheme is based on a bootstrap prediction method and does not rely on any specific kinetic model for multimodular unfolding. It is tested and validated in both theoretical/computational contexts (based on stochastic simulations) and atomic force microscopy experiments on (GB1)(8) multimodular protein constructs. The prediction accuracy is so high that the predicted average unfolding forces corresponding to each peak for the GB1 construct are within only 5 pN of the averaged directly-measured values. Experimental data are also used to illustrate how the limitations of standard kinetic models can be aptly circumvented by the proposed approach.

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**ABSTRACT:**Atomic force microscopy (AFM) applied to biological systems can, besides generating high-quality and well-resolved images, be employed to study protein folding via AFM-based force spectroscopy. This approach allowed remarkable advances in the measurement of inter- and intramolecular interaction forces with piconewton resolution. The detection of specific interaction forces between molecules based on the AFM sensitivity and the manipulation of individual molecules greatly advanced the understanding of intra-protein and protein-ligand interactions. Apart from the academic interest in the resolution of basic scientific questions, this technique has also key importance on the clarification of several biological questions of immediate biomedical relevance. Force spectroscopy is an especially appropriate technique for "mechanical proteins" that can provide crucial information on single protein molecules and/or domains. Importantly, it also has the potential of combining in a single experiment spatial and kinetic measurements. Here, the main principles of this methodology are described after which the ability to measure interactions at the single-molecule level is discussed, in the context of relevant protein-folding examples. We intend to demonstrate the potential of AFM-based force spectroscopy in the study of protein folding, especially since this technique is able to circumvent some of the difficulties typically encountered in classical thermal/chemical denaturation studies.Archives of Biochemistry and Biophysics 12/2012; · 3.37 Impact Factor - SourceAvailable from: Ionel Popa
##### Article: Nano-mechanics of HaloTag Tethers.

Ionel Popa, Ronen Berkovich, Jorge Alegre-Cebollada, Carmen L Badilla, Jaime Andres Rivas-Pardo, Yukinori Taniguchi, Masaru Kawakami, Julio M Fernandez[Show abstract] [Hide abstract]

**ABSTRACT:**The active site of the Haloalkane Dehydrogenase (HaloTag) enzyme can be covalently attached to a chloroalkane ligand providing a mechanically strong tether, resistant to large pulling forces. Here we demonstrate the covalent tethering of protein L and I27 polyproteins between an AFM cantilever and a glass surface using HaloTag anchoring at one end, and thiol chemistry at the other end. Covalent tethering is unambiguously confirmed by the observation of full length polyprotein unfolding, combined with high detachment forces that range up to ~2000 pN. We use these covalently anchored polyproteins to study the remarkable mechanical properties of HaloTag proteins. We show that the force that triggers unfolding of the HaloTag protein exhibits a four-fold increase, from 131 pN to 491 pN, when the direction of the applied force is changed from the C-terminus to the N-terminus. Force-clamp experiments reveal that unfolding of the HaloTag protein is twice more sensitive to pulling force compared to protein L, and refolds at a slower rate. We show how these properties allow for the long-term observation of protein folding-unfolding cycles at high forces, without interference from the HaloTag tether.Journal of the American Chemical Society 08/2013; · 10.68 Impact Factor

Page 1

Nonkinetic Modeling of the Mechanical Unfolding of Multimodular

Proteins: Theory and Experiments

F. Benedetti,†C. Micheletti,‡§G. Bussi,§S. K. Sekatskii,†and G. Dietler†*

†Laboratory of Physics of Living Matter, Ecole Polytechnique Fe ´de ´rale de Lausanne (EPFL), Lausanne, Switzerland; and‡Scuola

Internazionale Superiore di Studi Avanzati and§Consiglio Nazionale delle Ricerche-Istituto per l’Officina dei Materiali Democritos and Italian

Institute of Technology, SISSA Unit, Trieste, Italy

ABSTRACT

on multimodular proteins. The relationship between the histograms of the unfolding forces for different peaks, corresponding to

a different number of not-yet-unfolded protein modules, is exploited in such a manner that the sole distribution of the forces for

one unfolding peak can be used to predict the unfolding forces for other peaks. The scheme is based on a bootstrap prediction

method and does not rely on any specific kinetic model for multimodular unfolding. It is tested and validated in both theoretical/

computational contexts (based on stochastic simulations) and atomic force microscopy experiments on (GB1)8multimodular

protein constructs. The prediction accuracy is so high that the predicted average unfolding forces corresponding to each

peak for the GB1 construct are within only 5 pN of the averaged directly-measured values. Experimental data are also used

to illustrate how the limitations of standard kinetic models can be aptly circumvented by the proposed approach.

We introduce and discuss a novel approach called back-calculation for analyzing force spectroscopy experiments

INTRODUCTION

During the last decade, single-molecule force spectroscopy

experiments based on optical tweezers or atomic force spec-

troscopy have acquired increasing importance for character-

izing properties of individual proteins, as well as protein

complexes. Among the hundreds of such studies carried out

so far, it is particularly worth mentioning force spectroscopy

investigations of multimodular proteins. These constructs

typically consist of a series of protein modules that are cova-

lentlylinkedattheirends(see,e.g.,(1–12)).Uponpullingthe

constructs at both ends, a series of unfolding events are

observed. The forces at which the unfolding events occur

carry awealth ofinformation about the unfolding mechanics

andkineticsoftheconstructmodules(3,13,14).Customarily,

this information is extracted by analyzing the force distri-

bution obtained by gathering together the succession of un-

folding forces over repeated stretching experiments and

analyzing them with different methods, such as Monte Carlo

simulation andregression tozero force (5,15–19).Thescope

and utility of these commonly employed analysis techniques

can be considerably extended by examining separately the

distribution of the forces associated with the first, second,

etc., unfolding event in the constructs. This approach, which

so far has been applied only limitedly (5,20), is particularly

appropriate and useful whenthe constructconsistsofrepeats

of the same type of globular protein (such as I27 or GB1). In

fact, because of the identical nature of the modules, it is ex-

pected that the forces associated with the various unfolding

events depend on the number of unfolded modules still

present on the molecule, but that the statistical distributions

should nevertheless be tied by a definite relationship, as

pointed out in previous studies (5,20,21,22). To the best of

ourknowledge,suchdependencehasnotyetbeenadequately

exploredorexploitedinexperimentalcontexts.Furthermore,

one may envisage using only the limited information con-

tained in the experimental distribution of one single group

ofunfoldingforcestopredict with highaccuracytheaverage

unfolding forces of all other groups. This issue also has not

been addressed before, and we therefore investigate it in

this study.

The problem is here attacked at two levels. First we

adopt a simplified analytical scheme, which implicitly relies

on a standard kinetic model for the unfolding of the protein

modules (Evans’s theory). This method, which builds on a

treatment introduced in previous studies (20,21,23), com-

bines a transparent analytical formulation with the sim-

plicity of implementation and use. Yet the simplifying

assumptions that allow for the exact analytical treatment

of the model come at a disadvantage, since the predicted

probability distributions for the unfolding forces of the

various peaks can be significantly different from the mea-

sured ones.

This limitation can be overcome by using the alternative

and more general phenomenological approach introduced

and discussed here for the first time that we know of. The

scheme, based on the bootstrap statistics and termed back-

calculation, is parameter-free and does not rely on any spe-

cific kinetic model. The method merely uses the probability

distribution of forces associated with one of the unfolding

events (the first, the second, etc.) and predicts the distribu-

tion of forces of all other events. The method is validated

against data obtained from stochastic simulations (both Lan-

gevin and Monte Carlo) and from atomic force microscopy

(AFM) experiments carried out on multimodular GB1 con-

structs. In all cases, the average forces associated with any

Submitted April 27, 2011, and accepted for publication July 1, 2011.

*Correspondence: giovanni.dietler@epfl.ch

Editor: Laura Finzi.

? 2011 by the Biophysical Society

0006-3495/11/09/1504/9$2.00

doi: 10.1016/j.bpj.2011.07.047

1504Biophysical JournalVolume 101September 20111504–1512

Page 2

unfolding event are well predicted by back-calculation.

Deviations from the experimental measured values are of

only 5 pN, a quantity that is smaller than the uncertainty

typically associated with experimental estimates for protein

unfolding forces.

MATERIALS AND METHODS

Experiment

Our experiments were performed on multimeric constructs consisting of

eight GB1 modules, hereafter denoted as (GB1)8, and dissolved in Tris/

HCl buffer (10 mM, pH 7.5) at a concentration of ~20 mg/ml (1,2,24).

The force-extension curves of (GB1)8 were measured by means of a

commercially available AFM system (Picoforce AFM Nanoscope IIIa,

Bruker, Madison, WI) using a V-shaped silicon nitride cantilever (NP,

Bruker). The spring constant of the lever was measured from thermal fluc-

tuation measurements (25) as part of the AFM calibration procedure and

was found to be equal to 0.0575 N/m. The constructs were pulled along

the x direction at the speed x ¼ 2180 nm/s. Further details of the standard

experimental protocol that was followed can be found in a recently pub-

lished note (21).

A typical experimental force-extension curve is presented in Fig. 1.

Because the AFM tip will not necessarily pick the construct at its free

end, the number of modules trapped between the anchored end and the

AFM tip can be <8. As a matter offact, among the curvespresenting a clear

detachment peak, the most numerous group was the one displaying six

unfolding peaks. We therefore limited considerations to this set of force-

extension curves. The curves were analyzed using Hooke (26) an open-

source software package designed to analyze the force spectroscopy curves.

Hooke was further used to analyze the data from Langevin simulations of

the stretching of multimodular protein constructs (see below).

Numerical simulations

Two different computational approaches, namely Monte Carlo and Lange-

vin simulations, were used to study the mechanical unfolding of multimod-

ular protein constructs. In both cases, the pulled construct is assumed to be

anchored at one end while the other is pulled at fixed speed. The end-to-end

distance of each protein module projected along the pulling direction, x, is

used as an effective order parameter to describe the module state. This

corresponds to considering the system as being effectively one-dimen-

sional, as in the sketch of Fig. 2 a. This is a good approximation, since

for the typical unfolding forces at play in our experiments, the end-to-end

distanceofourconstruct,asobtainedfromawormlike-chain (WLC)model,

FIGURE 1

experiment on a (GB1)8construct. The lower curve shows the force as the

AFM tip approaches the substrate until the contact is established. The upper

curve represents the force while the tip is retracted from the substrate and

shows a series of unfolding events of the construct picked up by the tip.

Notice that this trace displays six unfolding events. The effect of an aspe-

cific interaction at the beginning of the retracting trace is observed. The

peak forces leading to the unfolding of the various modules are measured

with respect to the background provided by the constant part of the retract-

ing curve.

Typical force-extension curve recorded in an AFM stretching

0510 15

x (nm)

0

5

10

15

20

25

30

U(x)/kB T

xF

xT

x2

xU

x1

GFT

GFU

x0

x1

x2

x3

x4

x5

a

b

FIGURE 2

module of the construct in the Langevin simulation (see Eq. 1). A large

value of A, equal to 100 pN ? nm2, was used in Eq. 1 to enforce the

constraint that the modules cannot be stretched beyond the nominal GB1

contour length, Lc¼ 18 nm. The reference end-to-end separation of the

folded state, xF, is set equal to 4 nm and the the end-to-end separation

between the folded state and the transition (T) state, Dx ¼ xT? xF, is

0.5 nm. The reference end-to-end separation of the unfolded state, xU, is

equal to ðLc? xTÞ=2. Consistent with what has been established in previous

studies, the barrier separating the folded and transition states is set equal to

DGFT¼ 20kBT, whereas the barrier between the folded and unfolded states

has the value DGFU¼ 5kBT, with the temperature, T, equal to 300 K (i.e.,

kBTz4:2pN nm). For simplicity, the curvature, kT, is set equal to kF.The

value of kFin turn is set to 4DGFT=ðxT? xFÞ2¼ 1344 pN/nm to ensure

the continuity of the potential and its derivative at the midpoint,

x1¼ ðxFþ xTÞ=2, where the first two parabolas in Eq. 1 meet. The value

of kUwas set to be much smaller than kF, at kU¼ kF/500 ¼ 2.69 pN/nm.

The value of x2was finally obtained by the requirement of continuity of

the potential. To avoid an excessive parameterization of the model, flexible

linkers in the construct are described as unfolded protein modules. (Inset)

Protein model used in Langevin simulations. (b) A force-extension curve

obtained with the Langevin simulation applied to a model construct that

initially comprised six folded modules intercalated by seven linkers (each

linker has the same length as an unfolded module).

(a) Illustration of the anharmonic spring potential for one

Biophysical Journal 101(6) 1504–1512

Modeling of the Unfolding of Proteins1505

Page 3

is expected to be almost equal to its contour length, so that fluctuations in

the y and z directions can be neglected.

Depending on the value of the end-to-end separation, each module is

considered as being folded (F) or unfolded (U); these two states are sepa-

rated by a barrier of potential energy whose height is modulated by the

applied tensile force. The effective potential energy, U(x), is modeled

explicitly in the Langevin scheme, where one integrates the stochastic

equation of motion for each of the tethered modules in the construct that

is being pulled. By contrast, no explicit representation of the construct is

considered in the Monte Carlo approach. The latter, in fact, is employed

to model the succession of discrete unfolding events occurring at force-

dependent rates.

The two methods clearly embody rather different strategies for simu-

lating the stretching experiments and, also in view of the different parame-

ters used in the corresponding stochastic simulations, are useful to probe

the generality and transferability of the back-calculation method (BC)

proposed here.

A detailed description of the two methods is provided hereafter.

Langevin simulations

With reference to the sketch in the inset of Fig. 2 a, the anchored end of the

construct is located at x0¼ 0, while the other end (x4in the sketch) is

attached to the moving AFM tip. For simplicity, to parallel what is done

in the Monte Carlo scheme below, the latter is modeled as a Hookean spring

(x4–x5in the sketch) with spring constant kAFM¼ 0.01 N/m. Each protein

module behaves as an anharmonic spring; the associated free-energy

profile, U(x), is shown in Fig. 2 a and described by the expression

8

<

>

UðxÞ ¼

A

Lc? xþ

>

>

>

>

>

>

>

>

>

:

1

2kFðx ? xFÞ2if x<x1

DGFT?1

DGFUþ1

2kTðx ? xTÞ2if x1<x<x2

2kUðx ? xUÞ2otherwise

:

(1)

The model parameters are chosen to be consistent with the overall shape

of the potential energy typically found in proteins (27) and are provided in

the caption to Fig. 2. In particular, the contour length of each module is

equal to the nominal contour length of GB1, Lc¼ 18 nm, the reference

end-to-end separation of the folded state is xF¼ 4 nm, and its distance to

the transition state is Dx ¼ 0.5 nm.

In multimodular protein constructs, each protein module is connected to

the next via a short peptidic linker of length 1.5 nm. To keep at a minimum

the number of parameters in the model, we described these linkers, which

clearly do not undergo any transition upon stretching, by unfolded protein

modules. To do so, we initially prepared the pristine construct as a succes-

sion of folded modules with initial end-to-end separation equal to xF, inter-

calated with unfolded modules with initial end-to-end separation equal to

xU.The potential energy barrier separating the F and U states is sufficiently

high that an initially unfolded module will not spontaneously refold over

the short timespan of the model stretching experiment.

The total potential energy ofthe homomeric modulechain composedof n

protein modules, ‘ linkers, and the AFM tip is given by

Hðx1;x2;::xn;xnþ‘þ1Þ ¼

X

þ1

nþ‘

i¼1

Uðxi? xi?1Þ

2kAFMðxnþ‘þ1? xnþ‘Þ2:

(2)

The time evolution of the key construct positions, xi¼1;.nþ‘; follows the

overdamped Langevin dynamics:

g_ xi ¼ ?vH

vxiþ hðtÞ;

(3)

where g ¼ 4.4 ? 10?5pN s/nm is the friction coefficient appropriate to

yield (according to Kramer’s theory) a spontaneous unfolding rate (at

zero applied force) equal to koff¼ 10?2s?1. hðtÞ is a Gaussian white noise

with zero mean and variance equal to 2kBTg (kBis the Boltzmann constant

and T ¼ 300 K is the temperature). Notice that the derivative of the poten-

tial U (entailed by the derivative of H) is not continuous in x2.

The stochastic equations of motion were integrated numerically with a

time step of 1 ns. After an initial equilibration, the position of the AFM

tip, xnþ‘þ1 is moved at constant velocity, xnþ‘þ1ðtÞ ¼ vt, with n ¼

500 nm/s. This velocity value is commonly employed in stretching simula-

tions and falls in the typical range of pulling velocities used in experiments

(28). The typical time span required to unfold all the n ¼ 6 modules in the

constant-velocity simulation was 0.25 s.

The force/extension curve of the system is obtained by recording the

restoring force experienced by the AFM tip, f ¼ kAFMðxnþ‘þ1? xnþ‘Þ,

as a function of the AFM tip position, xnþ‘þ1, as shown in Fig. 2 b. Several

hundred such curves were collected and analyzed with Hooke after per-

forming a time average over windows of duration 0.15 ms to mimic the

finite time resolution of a typical experiment.

Monte Carlo simulations

As anticipated at the beginning of the section, the Monte Carlo approach

(here implemented as in studies by Rief and colleagues (28,29) and Zinober

and colleagues (30)) provides a phenomenological approach to the kinetics

of mechanical unfolding. The advantage of its transparent formulation is

balanced by the highly simplified nature of the model. In particular, by

contrast with the Langevin modeling of biopolymer stretching employed

here and in other approaches (31), no explicit representation of the module

constructs is considered, and the linkers are not accounted for. In addition,

the pullingactionis assumedto act equallyon all the nmodules,causingthe

same steady increase of the end-to-end separation for each of them. Notice

that because of the limitedsound velocity in the chain, this condition is only

approximately realized in Langevin schemes and experiments (where other

effects, such as viscosity, can be at play). In any case, the lower the pulling

rate the better the approximation is expected to be.

Within the above assumption, the end-to-end distance (equal to zero at

the initial time, t ¼ 0) of each one of the n modules at time t is equal to

ðyt ? FðtÞ=kAFMÞ=n. In this study, we considered n ¼ 6 and v ¼ 500 nm/s,

and the effective spring constant of the AFM tip is set to kAFM¼ 0.01 N/m.

Notice that kAFMis smaller than the nominal spring constant of the tip used

in our typical stretching experiments. This is because kAFMstands for an

effective spring that, in addition to the AFM tip, accounts for stiffness of

the folded modules, which are not explicitly included in our Monte Carlo

scheme. With this simplified description, the loading rate is not dependent

on the number of folded modules, which brings the Monte Carlo closer to

the BC assumptions, as discussed later. We underline that our goal here is to

provide a benchmarkfor the BC and notto reproducethe experimental data,

so a qualitative picture is satisfactory at this stage. The instantaneous force

experienced by each module is computed from the theoretical force-exten-

sion curve, fWLC(x) of an equilibrated WLC with contour length Lc¼ 18 nm

(appropriate for GB1) and a persistence length of lp¼ 0.4 nm. The progres-

sive loading of the modules is followed at time increments of duration

Dt ¼ 1.6 ? 10?5s. At the (discrete) time, t, the probability that one of

the modules yields and becomes unfolded is computed within the Evans

approximation (13) disregarding the refolding probability:

?fWLCðntÞ ? Dx

pðtÞ ¼ koffexp

kBT

?

Dt;

(4)

where kBis the Boltzmann constant and T ¼ 300 K is the system tempera-

ture. The effectivevalues koff¼ 0.11s?1and Dx ¼ 1.44A˚are obtained from

Biophysical Journal 101(6) 1504–1512

1506 Benedetti et al.

Page 4

a fit of the experimentaldata usingEvans’s theory as in Benedetti et al. (20).

The fittingprocedureensuresthatthe unfoldingforcesfall in a rangesimilar

to the experimental ones, although a precise match is neither expected nor

sought. The Monte Carlo scheme consists of drawing a random number,

uniformly distributed in the [0,1] interval, for each of the n protein modules

and comparing it with p(t). An unfolding event occurs when one of the n

random numbers is smaller than p(t). The associated unfolding force is

recorded and the calculation is next repeated with the n – 1 modules. The

statistical distribution of the unfolding forces for each value of n was ob-

tained from 1000 repeats of the Monte Carlo unfolding simulations.

Analytically solvable model

Simple analyticalexpressions for the probabilitydistributionsof the unfold-

ing forces, and the associated meanvalues and variance, as a function of the

numberofdomains,n,canbeobtainedbyintroducingafurthersimplification

besides the ones introduced for the Monte Carlo scheme. Specifically, each

proteinmoduleistreatedasaharmonicspring(asintheLangevinapproach)

ratherthanaWLC,andtheunfoldingprocessfollowstheBell-Evanstheory.

Within these assumptions, the probability distribution of unfolding forces

has been previously worked out both for single-chain stretching (see, e.g.,

Hummer and Szabo (23)) and for multimodular constructs (20,21). For

completeness, and for the purposes of better discussing the phenomenolog-

ical BC method, an analogous derivation is provided here.

Let us consider a model construct consisting of n0þ ‘ harmonic springs:

the n0initially folded modules have spring constant equal to kF, whereas the

remaining ‘ have a smaller spring constant, kU,as appropriate for unfolded

modules. The model construct is subject to the AFM pulling action (the

AFM tip is again modeled as a harmonic spring with constant kAFM).

Because the tip is pulled at constant velocity, v, the tensile force experi-

enced at time t by each construct is equal to

nt

n=kFþ ðn0? n þ ‘Þ=kUþ 1=kAFMhkeffnt;

where keffis the effective spring constant of the construct in series with the

AFM tip and its inverse decreases with n as k?1

k?1

the completely unfolded construct, and A is a correction term that describes

the dependence of the spring constant on the number of folded modules.

Following Evans’s theory, the survival probability that any one module

has remained folded up to time t is equal to (23)

fðtÞ ¼

(5)

eff¼ ðn0? n þ ‘Þk?1

Uþ

AFMþ nk?1

Fhk?1ð1 ? AnÞ. Here k?1is the inverse spring constant of

S1ðtÞhexp

"

?

ZfðtÞ

0

koffe

vkeff

fDx

kBT

df

#

:

(6)

The probability that all the n modules have remained folded up to time t,

or equivalently up to the loading force fðtÞ ¼ nt keff, is simply obtained by

raising the above expression to the power n,

SnðtÞ ¼ S1ðtÞn:

(7)

By differentiating Snwith respect to f, one obtains the probability distri-

bution, pnðfÞ, for the force at which the first unfolding event occurs in

a chain of n modules.

The sought expression is

0

B

pnðfÞf exp

B

@

fDx

kBT?nð1 ? AnÞkoff

Dxkv

kBT

e

fDx

kBT

1

C

C

A;

(8)

where the proportionality factor, containing the normalization of the prob-

ability distribution, was omitted.

Since the function above is typically nonnegligible only for positive f, we

can compute its average and variance integrating over ½?N;þN?, which

leads to the analytical result

2

Dxkn

hfin¼ ?kBT

Dx

664g þ log

koff

kBT

þ log?n ? An2?

3

775

(9)

s2

n¼

p2

6

?Dx

kBT

?2

(10)

where g ? 0:577 is the Euler-Mascheroni constant.

We remark here that the variance is independent of the number of folded

modules,n,intheconstruct.Thisresultisrelatedtotheempiricalobservation

that in typical stretching experiments of a single protein construct, the vari-

ance of the unfolding force is largely independent of the loading rate (23).

If the dependence of the springconstant on the number offoldedmodules

can be neglected, the average unfolding force acquires a particularly simple

expression:

hfin¼ ?g

a?log½bn?

a

;

(11)

where the parameters a and b are obtained from the average force and

variance for a given n: a ¼ p=

Finally, we notice that for all values of n, the expression of Eq. 8 corre-

sponds to a Gumbel extremal distribution (32) with the fat tail extending

toward low values of the force, f. Accordingly, theviability of the analytical

model to capture the statistical properties of the unfolding forces measured

for a given value of n can be ascertained by checking whether the forces

follow the Gumbel distribution. To address this point, we employed the

Anderson-Darling test and computed the significance level to which one

can support the null hypothesis that the data originate from a Gumbel distri-

bution. According to custom, the threshold of 5% statistical significance

was used to accept or reject the null hypothesis.

ffiffiffiffiffiffiffi ffi

6s2

p

and b ¼ exp½?g ? ahfin?=n.

Back-calculation

The previous analytical results rely on a definite kinetic model (Evans’s

theory) and on the harmonic modeling of the elastic response of the AFM

tip and the protein modules. These effects could be included in a more

general theoretical framework which, however, would not yield simple

analytical calculations.

This difficulty can be circumvented using a simple and physically

appealing phenomenological approach, which we term the back-calculation

method, described hereafter. The method is parameter-free, as it relies on

the knowledge of the empirical probability distribution of the unfolding

forces at one particular value of n. This reference distribution can be

used straightforwardly to predict the average value of the force and its vari-

ance at all other values of n. The scheme is best illustrated assuming that the

reference distribution is the one for n ¼ 1, pn¼1ðfÞ. This distribution is

directly obtained from the data gathered in the stretching experiments or

from the stochastic simulations (Fig. 3). In the same spirit of the Monte

Carlo and the analytically solvable model, we assume that the loading

rate is sufficiently low that at any given time, all modules experience the

same instantaneous tensile force applied at their ends, f, and that each of

them can unfold independently from the others. We also assume that the

stiffness of the construct, defined as the derivative of f with respect to its

length, is not dependent on the number of folded constructs, n. This is

equivalent to considering A ¼ 0 in the analytical model, and it is realistic

for investigated cases (for counterexamples, see King et al. (21)). Under

Biophysical Journal 101(6) 1504–1512

Modeling of the Unfolding of Proteins1507

Page 5

these assumptions, without resorting to any kinetic model or lengthy

stochastic simulations, the average unfolding force associated with the

nth peak, hfin, is computed by drawing n random numbers distributed ac-

cording to pn¼1ðfÞ and taking the smallest of them as the force at which

one of the n modules first unfolds. The averagevalue of the unfolding force,

hfin(and its variance), is clearly obtained by repeating the batch force

sampling process several times.

OnemayusetheorderedlistofNmeasurementstoconstructacumulative

probability distribution interpolated linearly between consecutive measured

values. The cumulative distribution is next straightforwardly used (see

Chapter 7.3 in Press et al. (33)) to sample, with the correct weight, the n

force values. Describing the process in terms of the cumulative distribution

has also the following important advantage. It is possible to exploit the

simple relationship of Eq. 7 (which is based on the assumption of indepen-

dence and hence valid regardless of the specific underlying kinetic process)

to generate data for unfolding forces of the nth peak starting from the data

obtained for a peak with a different order, say mth.

In fact, indicating by

Zf

?N

the cumulative distribution for the unfolding forces of the mth peak, it can

be determined that the corresponding cumulative distribution for the nth

peak is

QnðfÞ ¼ 1 ? ð1 ? QmðfÞÞn=m:

QmðfÞ ¼

df0pmðf0Þ

(12)

It is importantto stressthat the aboverelationshipsare ofhigh conceptual

and practical interest for recovering the distribution of unfolding forces of

one peak, say n ¼ 1, starting from a peak of higher order, say m ¼ 2.

A detailed description of how this backward extrapolation can be practi-

cally implemented in a numerical scheme is provided in the Appendix,

and the results are provided in the Supporting Material. The results dis-

cussed hereafter are produced with a more refined method where the prob-

ability pn¼1ðfÞ is obtained from fitting the histogram of the raw force

measurements with a convolution of Gaussians using the kernel density

estimation (KDE) (34) (Fig. 3). Data are sampled according to this distribu-

tion using either the cumulative distribution, or the rejection scheme (see

Chapter 7.3 in Press et al. (33)).

RESULTS AND DISCUSSION

Forall the three systems ofinterest (the GB1 experiment and

the Monte Carlo and Langevin simulations), we analyzed

the data of the force-versus-extension (or equivalently

force-versus-time) curves. In all three cases, the data per-

tained to the stretching of constructs of n0¼ 6 modules,

and therefore, the few curves that did not display a clear

presence of six force peaks were discarded.

The peaks were indexed in an inverse order with respect

to their order of appearance in the stretching experiment.

Specifically, the peak of order n ¼ 6 corresponds to the

peak observed first (when six folded modules were present

before the unfolding event), whereas peak order n ¼ 1 corre-

sponds to the unfolding event for which only one module

was present before the unfolding event and occurring imme-

diately before the construct detachment from the support.

The peak force data for each value of n were next considered

(see Benedetti et al. (20) for details on the automated peak

division procedure) and used to compute the histograms

reflecting the force distribution. The probability distribution

is obtained with a convolution of Gaussians using the KDE

method mentioned in the Methods section. The resulting

normalized distribution of the forces, pn(f), at which a single

module unfolds in the Monte Carlo scheme and GB1 exper-

iments is shown in Fig. 3. The best-fit Gaussian convolu-

tions were used to obtain a robust estimate of the average

unfolding force and its standard deviation (SD) at each value

of n. The results are provided in Tables 1–3 and Figs. 4–6.

The best-fit distribution for the last surviving peak, n ¼ 1,

was typically used as input for the back-calculation and

analytically solvable methods to obtain predictions for the

average unfolding forces at all values of n. For the case of

highest practical interest, namely, the GB1 experiment, the

distribution of unfolding forces of all other peaks,

n ¼ 2; 3; 4; 5; 6, was also used to predict the unfolding

forces of other peaks (see Table S1 in the Supporting

Material).

a

b

FIGURE 3

for the n ¼ 1 peaks (i.e., near the detachment point) obtained for (a) Monte

Carlo simulation and (b) GB1pulling experiments. The continuous line in

both cases represents the Gaussian KDE estimated from the raw data.

The histograms are both normalized.

Normalized probability distribution of the unfolding forces

TABLE 1Unfolding forces from Monte Carlo simulations

Comparison with n ¼ 1 back-calculated values

n654321

Average, Monte Carlo data

Average, n ¼ 1 BC

SD, Monte Carlo data

SD, n ¼ 1 BC

All values are given in pN.

142

138

40

29

146

142

35

31

150

148

35

32

157

156

35

31

166

168

37

33

187

—

35

—

Biophysical Journal 101(6) 1504–1512

1508 Benedetti et al.

Page 6

Monte Carlo data

We start by discussing the application of the method to data

generated using the Monte Carlo procedure. Of the three

sets of data (from experiment and Langevin and Monte

Carlo simulations), this set is the one that is expected to

be most appropriately captured by back-calculation. The

Monte Carlo scheme indeed builds on the identical kinetic

status of all the modules, and during this process, only the

total contour length changes, with very mild effect on the

loading rate.

By using the n ¼ 1 data, it is indeed seen in Table 1 that

the mean values of the predicted and measured unfolding

forces are in good agreement for all peaks n ¼ 2.6, with

differences always <5 pN. The agreement is readily

perceived in Fig. 4, where it is seen that the BC data up to

n ¼ 4 fall within the statistical uncertainty of the Monte

Carlo data, and only the forces predicted at n ¼ 5 and n ¼

6 present SDs of ~2.5 from the Monte Carlo data.

A more challenging quantity to compare is the second

moment of the distribution, which is the variance or, equiv-

alently, the SD. For the latter quantity, the agreement is still

good. The deviation of the Monte Carlo and back-calculated

values, jsBC? sdataj=ðsBCþ sdataÞ, is typically within 10%

and is worst for the last peak, n ¼ 6, for which it is 16%.

The results of the analytical model present an accord with

the Monte Carlo data that is comparable with their agree-

ment with the BC. This is illustrated by the dashed line in

Fig. 4, which reports the analytical predictions based on

the Monte Carlo data for n ¼ 1 (data for this case and other

values of n are provided in the Supporting Material). The

good accord is nontrivial in view of the fact that the simpli-

fied analytical treatment describes the folded protein

domains as harmonic springs, whereas the Monte Carlo

data were generated employing a WLC model for each

domain. We carried out the Anderson-Darling statistical

test described in the Methods section and established that

the Monte Carlo data for n ¼ 1 (and higher values, too)

are compatible with an underlying Gumbel distribution.

This reinforces the applicability of the simplified analytical

scheme in the model Monte Carlo context.

Langevin data

The same analysis was repeated for the data generated using

the Langevin scheme, which contains several differences

from the Monte Carlo scheme. Specifically, the Langevin

scheme does not enforce either Evans’s kinetics or the

same precise behavior of all folded modules in the chain.

In addition, it accounts for the presence of model linkers

between the folded modules, and finally, values of Dx and

koffare appreciably different from those in the Monte Carlo

case.

FIGURE 4

Carlo (circles) and BC (diamonds) from the distribution of unfolding forces

of the peak order n ¼ 1 stemming from the Monte Carlo simulation and

kinetic model (dashed line). The statistical error (mean 5 SD) is the

same size as the symbols, ~0.5 pN.

Average unfolding force versus peak order for the Monte

FIGURE 5

(circles) and BC (diamonds) from the distribution of unfolding forces of

the peak order n ¼ 1 stemming from the Langevin simulation and kinetic

model (dashed line). The statistical errors (mean 5 SD) are shown with

error bars for the Langevin simulation, whereas for the BC data, the errors

are the same size as the symbols (~0.5 pN).

Average unfolding force versus peak order for the Langevin

TABLE 2 Unfolding forces from Langevin simulations

Comparison with n ¼ 1 back-calculated values

n654321

Average, Langevin simulation

Average, n ¼ 1 BC

SD, Langevin simulation

SD, n ¼ 1 BC

All values are given in pN.

70

70

16

11

73

72

14

11

74

74

13

12

78

78

13

13

84

83

14

14

92

—

15

—

TABLE 3 Unfolding forces for GB1

Comparison with n ¼ 1 and n ¼ 2 back-calculated values

n654321

Average, experiment

Average, n ¼ 1 BC

Average, n ¼ 2 BC

SD – experiment

SD, n ¼ 1 BC

SD, n ¼ 2 BC

Accuracy in prediction of the SD is improved when data from n ¼ 2 are

used. All values are given in pN.

124

121

123

25

17

21

128

125

127

30

18

21

129

128

131

31

19

22

137

134

137

31

21

24

146

143

—

30

25

—

162

—

160

31

—

36

Biophysical Journal 101(6) 1504–1512

Modeling of the Unfolding of Proteins1509

Page 7

As is visible from Table 2 and Fig. 5 also for the Langevin

context, the performance of the back-calculation method is

good and, with the exception of the point for the fourth

peak (which compared to the trend of the other data points

appears to be an outlier), the average predicted values of un-

folding forces are all within about one SD of the Langevin

data. As for the Monte Carlo data, the predicted SDs are

also consistent with the measured ones, and the largest rela-

tive error, again found for the peak with the largest extrap-

olation, n ¼ 6, is 14%.

As shown in Fig. 5, the performance of the analytical

model based on the n ¼ 1 data is not dissimilar from that

of the back-calculation (the detailed results are again re-

ported in the Supporting Material). Indeed, also in this

context, the Anderson-Darling test indicates that distribu-

tions of the unfolding forces are compatible with a Gumbel

distribution.

Experimental data on (GB1)8

Finally, we turned to the experimental data, which clearly

represent the greatest challenge. Because of the complex

interplay of the several factors that impact on the stretching

process, and because the pulling rate is not particularly low,

it may not be expected a priori that the system unfolding

response might be well captured by the back-calculation.

In particular, it is not obvious a priori that the unfolding

events of various peaks in the chain can be appropriately

described as statistically independent events. In fact, corre-

lations can arise in nearby protein moduli because of the

limited sound velocity in the chain or because of contact

interactions. Moreover, given the small number of experi-

mental samples, 47 measurements for each force peak, it

is not simple to obtain a reference histogram from the exper-

iment or to pin a distribution, even when using the KDE

interpolation scheme. Thus, any defect in the starting distri-

bution is consequently amplified by the back-calculation

method.

Despite these caveats, the predictive capability of the

back-calculation method for the average unfolding forces

was found to be very good also in this case. The level of

agreement can be appreciated by examining Table 3 and

Fig. 6. The increasing underestimation, as a function of n,

of the sample SD (predicted from the n ¼ 1 peak) is prob-

ably ascribable to the fewer-than-expected measurements

at low forces. This is readily demonstrated by starting the

back-calculation from the second peak, n ¼ 2, which by

covering lower values of unfolding forces can reproduce

very well not only the mean unfolding forces at all other

values of n, but also the corresponding SDs.

In light of this consideration, thevery good consistency of

the back-calculation data with the measured distribution is

very remarkable, and testifies to the robust applicability of

the method.

It is particularly instructive to discuss the performance

of the analytical method as well. Neither the average un-

folding forces nor their SDs are dissimilar from the experi-

mental ones (see Fig. 6 and Supporting Material). However,

unlike in the cases for the Monte Carlo and Langevin data,

thisagreementdoesnotstanduptocloser statisticalscrutiny.

In fact, the Anderson-Darling statistical test indicates that

the experimental data do not follow the Gumbel extremal

statistics entailed by the analytical model at each value of

n (see Eq. 8). In fact, the null hypothesis for the n ¼ 1

peak is supported with a confidence level of <1%. The

same applies for the n ¼ 2 peak as well (in spite of the

fact that a more and more pronounced Gumbel-like char-

acter is expected as n increases).

The above observations demonstrate the utility of the

back-calculation approach in the context of practical in-

terest. Indeed, the phenomenology of systems such as multi-

modular constructs of GB1 can be too rich to be well

accounted for by Evans’s theory. In such contexts, a good

control/prediction of the unfolding forces for varying

numbers of surviving modules can be made only starting

from the phenomenological distribution.

CONCLUSIONS

We present a systematic investigation of the statistical

properties of the forces associated with the first, second,

etc., unfolding events in a multimodular construct. We intro-

duced a phenomenological scheme, termed back-calcula-

tion, which, using as sole input the distribution of the

forces associated with a certain unfolding event (e.g., the

first), predicts the force distribution of all other events. We

stress that the method follows a bootstrap approach starting

from the raw force-extension measurements. In particular,

it does not rely on any model of mechanical response for

protein unfolding kinetics.

At a general level, it is shown that the standard procedure

of analyzing experimental stretching data by grouping

together forces associated with all unfolding events, could

FIGURE 6

imental data (circles) and BC (diamonds) from the distribution of unfolding

forces of the peak order n ¼ 1 stemming from the experiments and kinetic

model (dashed line). The statistical errors (mean 5 SD) are shown with

error bars, whereas the BC data have errors of the same value as the size

of the symbols (~0.5 pN).

Average unfolding forceversus peak order for the GB1 exper-

Biophysical Journal 101(6) 1504–1512

1510Benedetti et al.

Page 8

be more profitably replaced by considering the events sepa-

rately with equal order of appearance. To the best of our

knowledge, the possibility of applying such a scheme to

analyze experimental data has not been explored before.

Second, a comparison of the experimental distributions of

unfolding forces with that predicted by standard kinetic

models reveals appreciable discrepancies, thus preventing

their use as reliable descriptors of the mechanical unfolding

process. This fact is consistent with previous independent

investigations (27).

In addition, the approach has several implications for the

design/analysis of stretching experiments of multimodular

constructs. First, its simplicity makes the back-calculation

particularly appealing as a simple and transparent scheme

for the interpretation of experimental data. In this respect,

an interesting applicative avenue is offered by heteroge-

neous multimodular constructs, for which the back-cal-

culation can offer a term of reference apt for highlighting

composition-dependent modulations of the mechanical re-

sponse. Second, it offers a simple, parameter-free phenom-

enological approach for predicting the distributions of the

various unfolding peaks using a negligible computational

effort. In this respect, it presents major advantages com-

pared to the more computationally intensive stochastic

(Monte Carlo or Langevin) numerical approaches. Finally,

it can be applied to the design of biomaterials starting

from their molecular modular components (e.g., choosing

an appropriate number of repeats), with unfolding forces

falling in a desired range, or to precondition a pulling exper-

iment (choice of pulling speed, stiffness of the AFM tip) so

that the mechanical response is profiled with a desired

resolution. A study of the latter aspects is underway.

The numerical implementations (C programming lan-

guage) of the back-calculation techniques are available

upon request from the authors.

APPENDIX

The procedure used to predict the force distribution for peak n given a set of

experimental measurements for peak m is discussed here in detail. As a first

step, an ordered table FðmÞ

i

, with i ¼ 1;.;N, is built that contains the N

measured forces for peak m.

To resample new data from the same distribution, the procedure is as

follows:

Extract a uniform random number r ˛½0;1?.

Find i such that i<Nr<i þ 1.

The extracted force is computed as ði ? Nr þ 1ÞFðmÞ

The last step is based on a liner interpolation of the cumulate of the

distribution.

To extract data corresponding to the distribution of a different peak n,

which can be larger or smaller than m, the procedure has to be modified

as follows:

i

þ ðNr ? iÞFðmÞ

iþ1.

Extract a uniform random number r ˛½0;1?.

Compute r0¼ 1 ? ð1 ? rÞm=n.

Find i such that i<Nr0<i þ 1.

The extracted force is computed as ði ? Nr0þ 1ÞFðmÞ

i

þ ðNr0? iÞFðmÞ

iþ1.

SUPPORTING MATERIAL

One figure and one table are available at http://www.biophysj.org/biophysj/

supplemental/S0006-3495(11)00938-6.

This work was supported by the Swiss National Science Foundation (grant

No. 205320-131828) and by the Italian Ministry of Education (MIUR).

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