640.pdf

Frequency Modulation Atomic Force

Microscopy Reveals Individual

Intermediates Associated with each Unfolded I27 Titin Domain

Michael J. Higgins,* John E. Sader,

y

and Suzanne P. Jarvis*

*Centre for Research on Adaptive Nanodevices and Nanostructures (CRANN), University of Dublin, Trinity College, Dublin 2, Ireland;

and

y

Department of Mathematics and Statistics, University of Melbourne, Victoria 3010, Australia

ABSTRACT In this study, we apply a dynamic atomic force microscopy (AFM) technique, frequency modulation (FM)

detection, to the mechanical unfolding of single titin I27 domains and make comparisons with measurements made using the

AFM contact or static mode method. Static mode measurements revealed the well-known force transition occurring at 100–120

pN in the first unfolding peak, which was less clear, or more often absent, in the subsequent unfolding peaks. In contrast, some

FM-AFM curves clearly resolved a force transition associated with each of the unfolding peaks irrespective of the number of

observed unfolded domains. As expected for FM-AFM, the frequency shift response of the main unfolding peaks and their

intermediates could only be detected when the oscillation amplitudes used were smaller than the interaction lengths being

measured. It was also shown that the forces measured for the dynamical interaction of the FM-AFM technique were significantly

lower than those measured using the static mode. This study highlights the potential for using dynamic AFM for investigating

biological interactions, including protein unfolding and the detection of novel unfolding intermediates.

INTRODUCTION

The reversible unfolding of immunoglobulin (Ig) domains in

the I-band region of the modular protein titin is believed to

function for the passive elasticity of muscle. Along with

other proteins (e.g., barnase) (1), the mechanical unfolding

of the well-defined tertiary structure of the wild-type titin Ig

27 domain (I27) has been studied extensively using atomic

force microscopy (AFM) in the static mode (i.e., the DC-

deflection of a nonoscillating cantilever is measured) (2–6).

By stretching recombinant proteins consisting of multiple

repeats of a single domain using AFM, a suite of information

on protein folding has been revealed, including the mechan-

ical unfolding forces of individual domains, kinetic param-

eters for both unfolding and refolding pathways, determination

of unfolding intermediates, and comparison of forced un-

folding with physiological or chemical denaturant unfolding

(for reviews, see Best and Clarke (7) and Fisher et al. (8)).

Related to this study, an unfolding intermediate due to the

disruption of hydrogen bonds between A and B

-strands of

the I27 domain was initially indicated using steered

molecular dynamic (SMD) simulations (9) and later con-

firmed in AFM measurements (3).

More recently, two major types of dynamic AFM (where

the cantilever is oscillated), amplitude modulated (AM) and

frequency modulated (FM), have been used to study various

biological force interactions, including ligand-receptor inter-

actions (10,11), polysaccharides elasticity (12–14), nucleic

acids/peptides (15,16), and proteins (17–19). These tech-

niques typically involve oscillating the cantilever well

below, or at, the resonance frequency to detect changes in

the amplitude, phase, and/or resonance frequency that occur

in response to changes in the interaction force. Attempts are

then made to quantify the force, though this can be complex

in most cases. In relation to protein unfolding, Okajima et al.

(18) were able to detect a suggested refolding response of the

hydrophobic core in a single monomeric globular protein,

and Janovjak et al. (19) revealed novel unfolding peaks in the

unfolding of bacteriorhodopsin protein from native purple

membrane. Pertinent to this study, Forbes and Wang (20)

used AM-AFM to measure the unfolding response of native

titin from skeletal muscle myofibrils. These authors revealed

the typical periodic sawtooth pattern, though they also de-

tected additional peaks in the stiffness measurements that

were suggested to correspond to structural transitions or in-

termediates during unfolding. Due to the heterogeneity of the

300 globular domains in the native titin and apparent lack

of correlation between the peaks in the simultaneous stiffness

and force measurements, the assignment of the additional

peaks to specific unfolding intermediates was not feasible.

However, the study importantly highlighted the ability of the

dynamic technique to detect transitions that could not easily

be detected in the force measurements alone. The recent

advance toward polymer pulling experiments using dynamic

methods is due to this possibility of achieving a greater force

resolution and ability to obtain additional information on the

dissipative components of the force.

In contrast, for this study, we use FM detection to in-

vestigate the unfolding of tandem repeats of the I27 domain

and make comparisons to static measurements that are also

performed in the study. As mentioned above, an intermediate

in the first unfolded peak has previously been observed using

static mode AFM (3), though the transition in the force due to

Submitted May 16, 2005, and accepted for publication September 27, 2005.

Address reprint requests to Dr. Michael Higgins, Centre for Research on

Adaptive Nanodevices and Nanostructures (CRANN), University of

Dublin, Trinity College, Dublin 2, Ireland. Tel.: 353-608-3088; Fax:

353-608-3027; E-mail: michael.higgins@tcd.ie.

2006 by the Biophysical Society

0006-3495/06/01/640/08 $2.00

doi: 10.1529/biophysj.105.066571

640

Biophysical Journal Volume 90 January 2006 640–647

the intermediate becomes very unclear, or is more often ab-

sent, with subsequent unfolded domains. By performing

dynamic measurements on a well-defined modular protein,

such as titin I27, that has a known intermediate, we were able

to detect corresponding individual unfolding intermediates

for each peak in the force-extension curves. In addition, the

intermediate could clearly be observed in the final unfolded

domain (eighth peak) for an I27 construct with eight do-

mains. This highlights the potential of dynamic techniques

for future studies on protein folding, including the detection

of novel unfolding intermediates.

MATERIAL AND METHODS

Construction of wild-type TI I27 multimer

Recombinant methods used to express and synthesize direct tandem repeats

of wild-type TI I27 consisting of eight individual modules or domains were

performed according to the recent study by Steward et al. (21).

Static mode force measurements on titin protein

Cleaved round 1 cm mica surfaces on Teflon were coated with titin protein

by pipetting 100

Lof30

g/ml protein suspension in phosphate-buffered

saline (PBS) buffer onto the mica surface. The mica surface was immersed

for 15 min to allow for sufficient adsorption of the protein and then washed

by exchanging the protein solution (three times) with fresh PBS buffer. The

protein-coated mica surface was then mounted on the AFM sample stage

and static mode force measurements performed using an Asylum Research

MFP-3D AFM (Santa Barbara, CA). Force measurements were taken using

Mikromasch silicon nitride cantilevers that were calibrated using the plan

view method and had measured spring constants 40–100 pN/nm. Force

measurements were taken with a piezo velocity of 1

m/s and numerous

force curves obtained on different positions on the protein substrate. Anal-

ysis of the force curves, including worm-like chain model (WLC) fitting to

the elastic response of the extended protein, was performed using the Asylum

Research AFM IGOR Pro software (Wavemetrics, Lake Oswego, OR).

Frequency modulation detection (dynamic)

measurements on titin protein

Preparation of cantilevers for magnetic activation

First, a nanosensor silicon cantilever was calibrated as above and had a

measured spring constant of 1.2 N/m. A glass encapsulated NdBFe

(neodymium/boron/iron) particle was then glued (Epotek 41) onto the

back of the cantilever, directly behind the tip, with the aid of an optical

microscope and micromanipulator. The particle was then magnetized using

an impulse magnetizer (ASC Scientific, Carlsbad, CA), Model IM-IO-ZO)

by positioning the cantilever at an angle of 12

from the normal surface (i.e.,

tip angle in AFM holder) and applying a charging voltage of 300 V,

corresponding to a magnetic field strength of 37.3 kG, for 30 s.

Modification of AFM for frequency modulation detection

Force measurements were taken using a modified Asylum Research MFP-

3D to enable frequency modulation (FM) detection in liquid. This was

achieved by implementing Magnetic Activation Dynamic (MAD) mode

(22–24), whereby the cantilever with attached magnetic particle was

oscillated by applying an external magnetic field via a solenoid positioned

underneath the sample stage. For this setup, the voltage to the coil was

amplified using a homemade ‘coil driver’, and the solenoid replaced the

position of the objective lens in the AFM base. To regulate the FM detection

scheme, we used a Nanosurf (Liestal, Switzerland) Phase-Loop-Lock

controller/detector (PLL). The PLL used two feedback systems to control the

dynamic force measurements. One feedback system kept the oscillation

amplitude of the cantilever constant by varying the driving voltage to the

coil. A second feedback system shifted the phase signal of the lever response

to 90

, which was then used as the excitation signal to keep the lever

oscillating at its fundamental eigenfrequency. By monitoring changes in the

resonance frequency and excitation amplitude required to keep a constant

oscillation amplitude, we were able to measure frequency shift and

dissipation caused by the tip sample interaction. The AFM was controlled

using a modified version of the Asylum Research software (IGOR Pro,

Wavemetrics).

In contrast to previous dynamical techniques (12–14), a DC-deflection

signal was not acquired simultaneously with the frequency shift and dis-

sipation. This was because the FM-AFM technique in liquid required the use

of stiffer levers (i.e., 1.2 N/m) with higher resonant frequencies to reduce

frequency noise and maintain a stable cantilever oscillation. Thus, the in-

crease in the cantilever stiffness dramatically reduced the DC deflection

sensitivity and made obtaining simultaneous DC measurements difficult.

More importantly, by using relatively large oscillation amplitudes (i.e.,

4.5 nm, 6.2 nm, 11.5 nm, 26.5 nm), the DC deflection probes the average

force experienced by the tip during one oscillation cycle, leading to com-

plexity in its interpretation and comparison to standard DC measurements.

Frequency modulation measurements on titin

and data analysis

Titin protein-coated mica surfaces were prepared for AFM, as above. Before

taking measurements, the resonance frequency of the cantilever (18.47 kHz)

in PBS buffer, with attached magnetic, was obtained by performing a thermal

power spectrum. Frequency shift curves were taken with a piezo velocity of

m/s and varying oscillation amplitudes of 4.5 nm, 6.2 nm, 11.5 nm, and

26.5 nm. Importantly, physisorption of the protein to the cantilever tip could

be achieved only when the tip was set to dwell for 3–5 s during intermittent

contact with the mica surface before retraction.

Frequency shift (volts) versus extension (nanometer) curves were

obtained and converted to frequency shift (hertz) curves using the sensitivity

value (73.4 Hz/V) of the PLL. To convert the observed frequency shift into

an interaction force

;

the formulation recently proposed by Sader and

Jarvis (25) was used,

Þ¼

N

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

;

(1)

where

is the spring constant,

is the oscillation amplitude of the tip,

Þ¼

res

;

res

is the natural resonant frequency of the cantilever

in the absence of an interaction force, and

is the distance of closest

approach between the tip and the surface. Note that

is the conservative

component of the interaction force between tip and sample and does not

include any contribution due to dissipative effects. This formula is valid for

any

and requires that the interaction force be continuous throughout the

measurement. However, our curves contained an apparent discontinuity in

the interaction force at the moment of unfolding of each I27 domain (i.e.,

peak-to-trough transition). To satisfy the requirement of the formula, we

investigated only the region where the force was continuous, which cor-

responded to the elastic response of the unfolded polypeptide. Equation 1

also required that the force decay to zero as the tip-surface distance ap-

proaches infinity, which is not the case in our measurements, i.e., the

interaction force increases with increasing separation, with the maximum

attractive force experienced by the tip being on the retract portion of the

FM-AFM for Unfolding Proteins

641

Biophysical Journal 90(2) 640–647

oscillation cycle. To address this, we effectively treated the elastic response

of the unfolded region as we would for a repulsive interaction that is

experienced during an approach to the surface. In doing so, the data to be

analyzed were reversed so that the peak maximum of the unfolding event

corresponded to the origin of a hypothetical surface. The effect of frequency

noise, exacerbated by a derivative step in the force conversion, was

eliminated by using a polynomial fit of the unfolding region for the analysis.

It is noted that this technique relies on measuring the change in reso-

nance frequency of the cantilever upon application of an interaction force.

Equation 1 unifies previously established theoretical work (26) by allowing

for the unequivocal determination of the interaction force, for any oscillation

amplitude. The constant-amplitude approach implicitly decouples the in-

fluence of conservative and dissipative force. Equation 1 thus gives a direct

connection between the change in frequency and the true interaction force,

with no ambiguity, and is a dynamic analogy of Hooke’s law for FM-AFM

constant amplitude measurements. The above analysis procedure has been

explained in detail previously (11) and used to calculate unbinding forces of

single ligand receptors interactions using a flexible linker.

RESULTS

Static mode force curves revealing protein

unfolding and force transitions

Static mode measurements performed to explain the prin-

ciple of force-induced modular protein unfolding, and for

comparison with FM detection, revealed that the protein had

sufficiently adsorbed to both the cantilever tip and mica to

enable the unfolding of the I27 domains during extension.

Typical force-extension curves for the protein were observed

(Fig. 1

), whereby the sawtooth pattern with periodic

spacing between adjacent peaks indicated the sequential

unfolding of modular domains (six domains in this case)

within the polymer construct. The mean spacing between

peaks was found to be 25.4 nm

2.56 nm (mean

SD,

52), indicating the nonfully extended length of the unfolded

polypeptide. The error in the spacing may be due to the

measurements being taken on a mica surface compared to

a gold surface that is typically used to promote good binding.

A reduction in the binding on the mica would lower the

probability of observing an idealized protein unfolding curve

(many curves are convoluted by multiple protein interac-

tions) and thus may increase the measured error. WLC fitting

to the elastic response of the unfolded polypeptide regions in

Fig. 1, using the persistence length

as an adjustable pa-

rameter, gave an average

of 0.29 nm and change in contour

length

of 28.5 nm for each unfolding event. It is noted

that the elastic response in the first peak does not represent an

unfolded polypeptide, but the elastic properties of the poly-

mer construct with fully folded domains (i.e., no domains

have yet unfolded). The

and

were in agreement with the

stretching of a single titin I27 molecule and were consistent

throughout all curves that showed similar force-extension

profiles. The average peak force was measured to be 208

43 pN (

93), which was in the range for unfolding forces

previously measured at similar tip velocities. As previously

reported, the elastic response of the first unfolding peak was

not well described by the WLC due to a known force

transition, observed as a ‘hump’, occurring at

;

100 pN (Fig.

and

). This force transition was regularly observed in

the first peak (Fig. 1

) but became less clear, or was mostly

absent, with increasing peak number (data not shown). For

the force curve shown in Fig. 1

, the transition could not be

observed past the first peak. Fig. 1

shows an expanded

image of the first unfolding peak from Fig. 1

, which

consists of two WLC fits, one to the elastic response of the

fully folded polymer construct before the force transition

(WLC 1) and after the transition (WLC 2). With

set at

0.4 nm (3),

between both fits was measured to be 3.9 nm.

The analysis of the intermediate using two WLC fits was

performed according to previous work first describing the

intermediate (3), and the

value obtained is used to de-

scribe the accumulated lengthening of the folded domains in

their native state during extension. Our

value fell on the

linear regression slope for the previously observed relation-

FIGURE 1 (

) Typical force-extension curve for the unfolding of

repeating titin I27 domains in recombinant protein. The sawtooth pattern

with periodic spacing of 25.4 nm

2.56 nm for all recorded peaks (

52)

indicated the sequential unfolding of modular domains. WLC fitting to the

elastic response of the unfolded polypeptide region (with the exception of

the first peak) of each peak in the figure gave a mean persistence length of

0.29

0.04 nm and change in the contour length of 28.5

1.1 nm. The first

peak (

leftmost

peak beginning at zero extension) was not fitted well by the

WLC due to the force transition (

arrow

) occurring at a force of

;

100 pN.

The force transition was not observed in the subsequent unfolded domains.

(

) Expanded image of the first unfolded peak from

clearly showing the

occurrence of the force transition. WLC fitting, with the persistence length

set at 0.4 nm, to the elastic response of the polymer construct with fully

folded domains before the transition (WLC 1) and after the transition region

(WLC 2) gave contour lengths of 16.4 nm and 20.3 nm, respectively. A

previous relationship shows that the

for these two WLC fits to the first

unfolded peak increases with an increase in the observed number of

unfolded domains. A

of 3.9 nm for the WLC fits in the figure agrees with

the described relationship for the observed unfolding of six domains, as in

642

Higgins et al.

Biophysical Journal 90(2) 640–647

ship between

in the first unfolding peak and number of

unfolded domains observed in the force-extension curve (3).

A full statistical analysis is still required to make a com-

parison with the previous work mentioned, as the first peak is

known to be convoluted by the effects of various protein-

surface and protein-protein adhesive interactions. For the

purpose of this work, we give only an example of this WLC

fitting procedure to provide background detail on the inter-

mediate.

Frequency modulation detection revealing

individual force transitions of each module

FM detection measurements revealed sawtooth patterns in

the frequency shift curves, indicating that the dynamic tech-

nique was also able to detect the sequential unfolding of

domains in the I27 protein construct (Fig. 2

A, light gray

trace

). The mean spacing between adjacent peaks in curves

for

values of 4.5 nm, 6.2 nm, and 11.5 nm was found to be

25.2

2.9 nm (

35), 25. 3

3.4 nm (

61), and 24.6

3.1 nm (

13), respectively. Thus, irrespective of

, the

mean spacing agreed with the nonfully extended length of

the unfolded polypeptide region measured in the static force

measurements. In addition to the main frequency peaks

representing the onset of an unfolded domain, a second

smaller peak was also observed to be associated with each of

the main unfolding peaks (Fig. 2

). These secondary peaks

were very difficult to observe in the raw frequency shift

curves due to the significant noise level. A smoothed curve

of the frequency shift (

dark line

) also revealed the secondary

peaks, which appeared as a small increase in the frequency

shift to

;

15–18 Hz to form a discontinuity in the initial

frequency shift (Fig. 2

arrows

), followed by a further

increase in the frequency shift corresponding to the main

unfolding peak. This discontinuity in the frequency shift of

the main unfolding peaks appeared to indicate the presence

of a force transition, presumably that observed in the static

measurements. However, we stress that the secondary peaks

observed in the smoothed frequency curve do not represent

the true structure of the transition and must be further

validated, as the binominal smoothing process applied

consists of both the feature of the transition and frequency

noise. To validate the true presence and structure of the

transition for the different unfolded domain numbers, we

performed an averaging process for all measured peaks

obtained for

of values 4.5 nm and 6.2 nm and according to

their unfolding number (Fig. 2

). This is possible because

the frequency noise had a mean of zero and is additive to the

true signal (26). This property enabled the noise to be

minimized by averaging multiple measurements of the same

interaction to leave the underlying true signal undistorted.

The averaged frequency shifts for the different folded

domains are shown in Fig. 2

. We emphasize that these

measurements were highly reproducible and independent of

the number of averages taken. In addition, averaging a greater

number of peaks reduced the noise level without distorting

the underlying curve and thus demonstrated the validity of

this procedure. Fig. 2

revealed that the intricate structure of

the transition appeared to be hump-like or a plateau that

occurred at a consistent frequency shift of

;

15–18 Hz and

had a slight tendency to increase in length with an increase in

the unfolding number. In some curves, the intermediate was

not always present for every unfolded domain in the

frequency shift curves, as highlighted in the first few peaks

for the top curve (4.5 nm) in Fig. 3

. The absence of the

intermediate may have been due to a reduction in the signal

because of the apparent frequency noise highlighted in Fig. 2

. Interestingly, the intermediate was always clearly resolved

in the final unfolded domain for the I27

construct.

FIGURE 2 (

) Frequency shift versus extension curve (

light gray trace

)

for the sequential unfolding of titin I27 domains taken using a resonance

frequency

of 18.5 kHz and oscillation amplitude

of 6.5 nm. The mean

spacing for

values of 4.5 nm, 6.5 nm, and 11.5 nm were 25.2

2.9 (

35), 25.3

3.4 (

61), and 24.6

13), respectively, indicating the

successful unfolding modular domains using this dynamic technique. The

curve shows 10 peaks with each peak recording a positive frequency shift of

25 Hz. The first two peaks, which are slightly greater in magnitude than the

remaining eight, may be due to multiple binding of proteins (i.e., the peaks

correspond to an additional protein molecule on the tip). During extension,

the increasing stiffness of the unfolded polypeptide region restricts the

cantilever movement, causing an increase in the effective spring constant

of the cantilever. Thus, an increase in

causes a direct increase in the

cantilever resonance frequency, which is observed as a positive frequency

shift in the unfolding peaks. A smoothed curve (

dark line

) clearly shows

a discontinuity (

arrows

) in the positive frequency shift of the main unfolding

peak, indicating the unfolding intermediate. (

) Graph showing averaging of

all peaks showing intermediates (from

4.5 nm and 6.5 nm) and

according to their unfolded domain number, which was done by

superimposing and aligning each peak at their peak maximum. The distance

between peaks is negligible, and the noise in each peak increases with the

unfolded domain number due to the fewer numbers of samples averaged.

The distance from the beginning of the force transition (

arrow

) to the peak

maximum showed a slight increase with an increase in the unfolded domain

number. These distances were calculated to be 5.6 nm (peak 1), 6.6 nm (peak 2),

7.9 nm (peak 3), 8.1 nm (peak 4), 10.9 nm (peak 5), and 14.4 nm (peak 6).

FM-AFM for Unfolding Proteins

643

Biophysical Journal 90(2) 640–647

In comparison to smaller

values of 4.5 nm and 6.2 nm,

force transitions were not observed in the unfolding peaks,

or were albeit very unclear, for the

of 11.5 nm (Fig. 3),

whereas only some irregular peaks were observed for the

of 26.5 nm (Fig. 3). In addition, frequency shifts were greater

for smaller amplitudes, which was expected due to

1.5

, as given in Eq. 1 (Fig. 3). Conversion of the frequency

shift curves into an interaction force, as outlined in the

Material and Methods section and shown in Fig. 4,

and

surprisingly resulted in much lower unfolding forces com-

pared to static mode measurements (Fig. 4

). Average peak

unfolding forces were found to be 75

19.9 pN (

37),

63.1

20.2 pN (

54), and 63.9

7.9 (

14) pN for

values of 4.5 nm, 6.2 nm, and 11.5 nm, respectively,

indicating that the same force law was recovered irrespective

of the oscillation amplitude used.

DISCUSSION

The Ig I27 domain in native titin or as tandem repeats in

recombinant polypeptides is unique compared to other mod-

ular proteins in that individual domains unfold via an in-

termediate when under an applied force. SMD simulations

(9) show that this intermediate results from the rupture of two

hydrogen bonds bridging the A and B

-strands, which is

expected to proceed before the detachment of the A

and G

strands. The latter process presumably renders the C- and

N-terminal free to unravel, causing the domain to completely

unfold. This intermediate has been directly observed pre-

viously using AFM (3) with similar results repeated in this

study for comparison with dynamic techniques. In these

static mode AFM studies, the intermediate is observed as

a prominent ‘hump’ or force transition in the first unfolding

peak occurring at

100–120 pN that subsides, or is not

present, in subsequent unfolding events. Data showing an

increase in the change of contour length in the first unfolding

peak, as described in Fig. 1

, as a function of an increase in

the number of observed unfolded domains are used to

explain a 15% lengthening or 6.6 A

extension of a resting

domain during the force transition (3), which agrees with

previous SMD observations (9). It is suggested that the force

transition in the first peak corresponds to the accumulation

transitional lengthening of all the folded domains, whereas

the reappearance of smaller transitions in subsequent peaks

describes the process as being reversible before the next

FIGURE 3 Comparison of unfolding peaks obtained using a range of

oscillation amplitudes. Peaks for

4.2 nm and 6.2 nm mostly show a clear

unfolding intermediate (

arrows

) in the frequency shift (the intermediate is

not clear for three peaks in

4.2 nm,

no arrows

) compared to

11.5

nm. For

26.5 nm, the peaks do not correspond to a specific unfolded

domain number, as measurements for this amplitude showed irregularities in

the peak spacing. The frequency shift of the peaks increases with a decrease

according to

1.5

, as given in Eq. 1.

FIGURE 4 (

) Diagram showing method for the conversion of the

frequency shift curves to a force. The elastic response of the unfolded

polypeptide region in the frequency shift curves was fit to a polynomial

(

smooth trace

), which was then reversed so that the peak maximum occurred

at zero extension distance that corresponded to the origin of a hypothetical

surface. (

) The force (

solid line

) was then calculated from values of the fit

using Eq. 1. The mean forces were 75

19.9 pN (

37), 63.1

20.2 pN

(

54), and 63.9

7.9 (

14) pN for

values of 4.5 nm, 6.2 nm, and

11.5 nm, respectively. The intermediate unfolding forces were typically half,

or just below half, of the unfolding force, as observed in the force curve. The

corresponding reversed frequency shift curve (

dotted line

) is plotted for

comparison and shown to reflect the force profile.

644

Higgins et al.

Biophysical Journal 90(2) 640–647

unfolding event takes place. However, the latter point is still

vague, as a review of the literature reveals that it is very

difficult to discern the transition past the second unfolding

peak, or third peak at most, even when many domains are

observed to have unfolded. This observation is also apparent

in the static mode curves measured in this study. To address

this issue, we have shown here that FM detection is able to

detect individual transitions associated with each domain

regardless of the number observed in the force-extension

profile. This is perhaps in most part due to the FM detection

technique being more sensitive to the force in the

direction

compared to the static mode. The transition cannot be

explained due to refolding due to the following: partial

refolding (i.e., the entire domain is not relaxed) would only

be possible on the downswing of an oscillation cycle and

where the cantilever spends most of its time at the bottom of

the cycle. However, the time period for a complete

oscillation cycle (1/

s) is much smaller than the

time required for domain refolding (5). In addition, a greater

opportunity for refolding should be the case for the larger

amplitude of 11.5 nm due to sampling farther back (i.e.,

during the downswing of an oscillation) along the refolding

pathway, though these measurements showed only peaks

corresponding to the unfolding of the domains, indicating

that refolding was not evident.

The ability to detect both discrete unfolding events and

their intermediate in titin I27 is dependent on the size of the

being smaller than the length scale of the interactions in-

tended for measurement. In this case, the predominant length

scales for an individual unfolding event and its intermediate

are

;

25 nm and 10–15 nm, respectively. For amplitudes

much greater than these length scales, such as the 26.5 nm

used here, it is apparent that the detected frequency shifts

become smaller and less clear. This decrease in the frequency

shift is expected as the interaction force of a single unfolding

domain is averaged over a larger distance in comparison to

smaller amplitude measurements, i.e., a shorter portion of the

oscillation cycle experiences the interaction force for larger

amplitudes. Furthermore, in the case of modular proteins,

a single oscillation cycle with large amplitude may contain

different components of the unfolding force profile, in-

cluding unfolding one domain while stretching another. This

can lead to discontinuities in the force and make inter-

pretation of the data difficult. For an

value of 11.5 nm,

discrete frequency shifts can be detected for each unfolded

domain, though the intermediate is smoothed out due to a

decrease in the frequency shift when sampling this com-

parable length scale. In contrast, both of the smaller

values

(4.5 nm and 6.2 nm) are most sensitive to both the length

scales in question. Due to the complexity of protein structure

and composition, the length scales of interactions de-

termining unfolding pathways will be highly variable and

be protein specific. Therefore, the ideal situation for these

types of measurements would be to use the smallest achiev-

able amplitude to account for all possible interactions. Here

in this study, the use of smaller amplitudes was limited due to

the stability of the oscillation and size of the frequency noise,

though further AFM modifications are being made in an

attempt to reduce amplitudes to subnanometer values.

As predicted by Eq. 1, similar unfolding peak forces were

obtained, irrespective of

, which supports the validity of the

approach. Thus, it remains unclear why the measured forces

(

70 pN) are significantly lower than the forces obtained in

static force measurements (

208 pN). This observation is in

contrast to previous dynamic studies where measured forces

are comparable to the statically measured forces of the same

system. For example, two different dynamic approaches were

able to measure the chair-to-boat transition forces (700–1000

pN) of single dextran polysaccharides (12,14), which were

comparable to those measured independently in earlier static

mode studies (27). For the latter dynamic studies, the forces

were obtained by filtering out the dynamic signal to obtain

the static deflection of the cantilever. In contrast, here, the

force is determined directly from the dynamic signal

(frequency shift). We now discuss possible reasons for the

lower forces observed with our dynamic technique. Errors in

the measured frequency shift may arise if the interaction

response of the molecule occurs on a shorter timescale than

the time constant of the PLL. In which case, a positive fre-

quency shift would be underestimated (as too the force) due

to the delayed response of the PLL not being able to track the

frequency shift increase. However, for the measurements

here, the 400 Hz bandwidth of the PLL is sufficient to

correctly track significantly larger frequency shifts during the

35 ms timescale of the elastic response of the unfolded poly-

peptide. Therefore, the frequency shifts recorded here would

pertain to being real. The converted forces were obtained

using a recently developed arbitrary amplitude formula (Eq. 1)

(25) that unifies two previous well-established formulas that

describe the relationship between the frequency shift and force

(26,28,29). The fact that the unfolding forces scale correctly

with respect to

validates the unification of the two previous

formulas within Eq. 1. This arbitrary amplitude formula has

also been experimentally validated by quantifying the struc-

tural forces of liquids whereby the forces scaled appropriately

with

and, importantly, agreed with the forces previously

measured for the same liquid using a different technique (i.e.,

surface force apparatus) (30). Without any previous constant

amplitude FM measurements on modular proteins for

comparison, we speculate that the lower unfolding forces

may relate to the discontinuity in the force interaction during

a single oscillation cycle of the tip as the domain unfolds (e.g.,

during the intermediate). This discontinuity in the force may

occur if bonds are broken during the upswing of an oscillation

cycle and are unable to reform on the timescale of the

cantilever oscillation, even when the domains are allowed to

relax during the downswing. As a result, the oscillating can-

tilever would be repeatedly sampling through the force

interaction of already unfolded domain regions, with the

effective averaging of the force resulting in potentially lower

FM-AFM for Unfolding Proteins

645

Biophysical Journal 90(2) 640–647

values. In addition, the potential for the force to vary as

a function of the measurement technique has also recently

been observed for the folding and refolding of individual

proteins (31). To alleviate possible discontinuities in the force,

appropriate modifications to the force analysis have been

outlined in the Material and Methods section and other studies

(11), though further investigation is required to assess the

possible additional effects due to the disruption of hydrogen

bonds during the intermediate.

As mentioned, previous dynamic AFM studies have shown

that extended single polysaccharide chains also display force

transitions that are comparable to those previously observed

in static mode measurements (12–14), and novel transitions

and intermediates have also been reported in recent experi-

ments on proteins. Both Mitsui et al. (17) and Okajima et al.

(18) measured an out-of-phase response in the cantilever

deflection signal for a partially unfolded protein and attrib-

uted the response to refolding. Recently, Janovjak et al. (19)

dynamically unfolded single bacteriorhodopsin proteins from

native purple membranes to reveal novel force peaks that

were ascribed to an intermediate involving kinks in the

-helices, though the possibility of refolding due to relax-

ation was noted.

CONCLUSION

The aim of this study was to investigate the possibility of

detecting unfolding intermediates in I27 domains using FM

detection, as the dynamic technique was proposed to be more

sensitive in regions where tip instabilities normally occur for

typical static mode measurements and to changes in the force

gradient along the force-extension profile. Although the pro-

tein unfolding response in dynamic measurements was more

complex compared to static measurements, it was clearly

shown that the unfolding intermediate could be observed for

each unfolded domain. Thus, this approach opens up the

possibility of detecting novel unfolding intermediates in

similar protein studies. Future work is ongoing to make a full

statistical analysis of the effects of the oscillation amplitude

and resonance frequency on the unfolding response of the

protein. In addition, the implementation of subangstrom

oscillation amplitude measurements is currently being de-

veloped and modified for operation in the liquid environment.

We gratefully acknowledge Annette Steward and Jane Clarke (University of

Cambridge, UK) as the source of the wild-type TI I27 multimer protein. We

also thank Irene Revenko (Asylum Research) for her assistance.

This research was supported by Science Foundation Ireland Research Grant

(01/PI.2/C033) and the Human Frontier Science Program.

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