- 1 - Controlled unfolding and refolding of a single ... · atomic force microscopy ... surface and...

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Kedrov, Ziegler, Janovjak, Kühlbrandt & Müller

Controlled unfolding and refolding of a single sodium-proton antiporter

using AFM

Alexej Kedrov1, Christine Ziegler2, Harald Janovjak1,Werner Kühlbrandt2 & Daniel J. Müller1

1 BIOTEC,

Max-Planck-Institute of Molecular Cell Biology and Genetics,Dresden, Germany.

2 Max-Planck-Institute of Biophysics,

Frankfurt, Germany.

Key words:

Atomic force microscopy, Escherichia coli, folding kinetics, structural motif, membraneprotein, molecular interactions, secondary structure elements

Abbreviations:

aa, amino acid; AFM, Atomic force microscopy; NhaA, sodium-proton antiporter; WLC, worm-like chain;

Running title: Unfolding and refolding single antiporters

Correspondence:Daniel Müller, PhDMax-Planck-Institute of Molecular Cell Biology and GeneticsPfotenhauerstr. 10801307 Dresden, GermanyPhone ++49-351-2102586Fax ++49-351-2102020Email [email protected]

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ABSTRACT

Single-molecule force-spectroscopy was employed to unfold and refold single

sodium-proton antiporters of Escherichia coli (NhaA) from membrane patches. Although

transmembrane α-helices and extracellular polypeptide loops exhibited sufficient stability to

individually establish potential barriers against unfolding, two helices predominantly unfolded

pairwise thereby acting as one structural unit. Many of the potential barriers were detected

unfolding NhaA either from the C- or the N-terminal end. It was found that some molecular

interactions stabilizing secondary structural elements were directional while others were not.

Additionally, some interactions appeared to occur between the secondary structural

elements. After unfolding 10 of the 12 helices, the extracted polypeptide was allowed to

refold back into the membrane. After 5s, the refolded polypeptide established all secondary

structure elements of the native protein. One helical pair showed a characteristic spring like

‘snap in’ into its folded conformation, while the refolding process of other helices was not

detected in particular. Additionally, individual helices required characteristic time periods to

fold. Correlating these results with the primary structure of NhaA allowed us to obtain the first

insights into how potential barriers establish and determine the folding kinetics of the

secondary structure elements.

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INTRODUCTION

Membrane proteins are of fundamental biological importance and form major targets

for drug development. The steadily increasing number of sequenced genes contrasts sharply

with a lack of knowledge about their functional three-dimensional structures and folding

within cell membranes. Protein folding has been of major interest in molecular cell biology

research for many years, but has focused on small water-soluble proteins. Integral

membrane proteins have been largely excluded because they are considerably more difficult

to work with. This is mainly due to difficulties in mimicking the native, anisotropic environment

of the cell membrane, where specific interactions are required for the precise folding of

hydrophobic polypeptides into functional structures 1; 2; 3.

Environmental changes or point mutations may cause structural instabilities in a

membrane protein leading it to unfold or to fold into an alternative conformation, which may

lead to infectious protein misfolding diseases 4; 5; 6; 7. The most prominent of these are

Alzheimer, Creutzfeldt-Jakob (CJD), bovine spongiform encephalophaty (BSE), and some

recently known forms of cancer. Thus, from biological and medical perspectives, pertinent

questions that remain to be answered are: How do polypeptides fold into their three-

dimensional structure? Why do alternative forms of protein structures exist? Which molecular

forces interact between and within biological macromolecules determining their final

biomolecular structures, their stability, dynamics, and functions? Thus, it is tempting to

determine forces that stabilize membrane proteins and their secondary structural elements,

but also to observe how membrane proteins are inserted into a membrane and fold into their

final three-dimensional structure.

To investigate interactions that stabilize membrane proteins, we recently combined

atomic force microscopy (AFM) and single-molecule force spectroscopy 8. This novel

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approach allowed single membrane proteins to be imaged in their native environment and

the stability of their secondary structural elements to be characterized 8; 9; 10. Membrane

proteins are first imaged at a spatial resolution of ~ 0.6 nm, and the terminal end of individual

proteins are then tethered to the stylus of the AFM cantilever. Upon separation of membrane

and stylus the terminal end of the protein is stretched and the increasing force is detected by

the cantilever deflection. This deflection is measured with an accuracy of ~ 0.1 nm while the

detection system exhibits a sensitivity of a few pN (10-12 Newton). By further stretching the

polypeptide, the force exceeds the mechanical stability of the nearest secondary structure

element of the membrane protein. Once this element is unfolded, the next element of the

secondary structural unfolds in a subsequent step. Thus, it is possible to investigate the

structural stability of individual transmembrane α-helices and polypeptide loops. This method

was subsequently applied to study how environmental factors influence molecular

interactions that stabilize individual secondary structural elements of membrane proteins 9; 11.

Here, we have refined this method to directly observe the folding of a single

polypeptide into the membrane bilayer. We have chosen the sodium-proton antiporter NhaA

from E.coli, since it belongs to a large, diverse group of ion-coupled membrane-transport

proteins found in all organisms, ranging from bacteria to humans 12. Secondary transporters

mediate the reactions of uniport, symport or antiport of ions or small molecules. As they

facilitate solute accumulation and toxin removal against concentration gradients by

converting energy supplied by ion gradients across the cell membrane, this class of

membrane proteins plays a central role in human health and disease 13; 14; 15; 16. The structural

analysis of NhaA reconstituted into two-dimensional crystals revealed that they form dimers.

Each monomer consists of 388 aa, which fold into 12 transmembrane α-helices 17; 18.

Transmembrane helices of NhaA dimers show a remarkably symmetric arrangement in the

cell membrane and provide compelling explanations for the ability to carry out directional

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substrate transport. Both symmetry and dimerization form a common structural motif among

a large class of secondary transporters 19; 20.

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RESULTS AND DISCUSSION

Lipid membranes containing native NhaA were first adsorbed onto freshly cleaved mica

surface and then imaged by high-resolution AFM in buffer solution 8. The crystalline

arrangement and dimeric assembly of NhaA molecules were clearly resolved (Fig. 1). In

accordance with structural insights revealed using electron microscopy, the dimers were

arranged into rows with adjacent dimers showing an alternating up-and-down orientation.

This implies that both the cytoplasmic and the extracellular surfaces of reconstituted NhaA

were exposed to the same side of the membrane 18; 21.

After imaging, the AFM stylus was pushed onto the membrane surface applying a

contact force of ~ 1 nN for ~ 1 second and then withdrawn while recording the cantilever

deflection over the separation. In about 3% of the cases, one terminal domain of the protein

adhered to the stylus, which resulted in a force spectra being extended over more than 95

nm. Taking all aa of the NhaA polypeptide into account it may be concluded that these force-

curves (Fig. 2) reflected the entire polypeptide being unfolded and stretched by the AFM

stylus. The resulting force-spectra exhibited detailed information on the unfolding process,

with each peak representing an internal potential barrier built up by molecular interactions

within the protein.

Unraveling two major unfolding pathways of NhaA

To assign the force peaks to structural elements of the protein, the spectra were

classified. Two main classes were observed (Fig. 2; density plots) and fitted using the worm-

like chain (WLC) model (red lines), which describes the stretching of a polypeptide (polymer

chain) by an external force 8; 22. The first class (Fig. 2A) exhibited a characteristic triple peak

at contour lengths corresponding to a stretched polypeptide of 91, 107 and 125 aa, a strong

peak at 163 aa and a pronounced final double peak at 318 and 328 aa. The second class

(Fig. 2B) exhibited a pronounced peak at 95 aa and a final single peak at 327 aa. Both

classes showed other multiple force peaks. Since the NhaA molecules expose C- and N-

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termini to the same surface, it was not clear which terminal end of the protein attached to the

AFM stylus. In the first attempt, we applied the secondary structure model of NhaA 17 and

assigned possible potential barriers (Fig. 2; bottom panel, green and purple circles)

established by secondary structural elements such as transmembrane α-helices and loops to

the measured force peaks (Fig. 2; bottom). The number of aa obtained from each WLC fit

was subtracted from the primary structure of NhaA and the position marked accordingly to

the secondary structure model. While class (A) fits best to the structural model of mechanical

unfolding from the C-terminal end, class (B) fits best to unfolding from the N-terminal end. It

should be noted here that the unfolding of NhaA from single- or double-layered membrane

sheets (Fig. 1A) did not influence the unfolding patterns observed (data not shown).

Assigning C- and N-terminal unfolding pathways

To confirm this assignment, the C-terminal end and the loop connecting

transmembrane helices VIII and IX of NhaA were cleaved using trypsin (Fig. 3A). The

truncated protein was unfolded and four spectral classes occurred reproducibly (Figs. 3 B-

E). Again, all force peaks were fitted using the WLC model (red lines). The first class of force

curves (Fig. 3B) exhibited the same characteristic triple peak observed with native NhaA

(Fig. 2A, region separated by ~ 30 nm from the membrane surface). Therefore, it was

assumed that these proteins were attached with their C-terminal end to the AFM stylus. As

the C-terminal end was shortened after proteolytic cleavage (Materials & Methods; Fig. 3A),

a shift of the force peaks was expected. Taking a shift of ~ 10 – 14 aa into account every

force peak of this fraction correlated to the unfolding spectra of the intact protein.

It was not possible to correlate the class shown in Fig. 3C to any of the unfolding

pathways observed for the intact protein. However, the secondary structure model allows

assigning these force peaks to an unfolding pathway of a small fraction of four

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transmembrane α-helices by pulling on the peptide end of helix IX. Most interestingly, the last

force peak of the third class (Fig. 3D) exhibited a double peak similar to that observed for the

intact protein (Fig. 2A). Therefore we believe that this unfolding pathway represents the large

protein fragment by grabbing its polypeptide end located at helix VIII (Fig. 3D). This

assignment was supported by the fact that the potential barriers (Fig. 3D; schematic

drawing) are located to positions similar to those in the intact protein (Fig. 2A). The force

peaks of the fourth class (Fig. 3E) are precisely located at positions of those detected for the

intact protein (Fig. 2B). Therefore, this unfolding pathway was assigned to unfolding the

protein from its N-terminal end.

Secondary structural elements tentatively unfold in pairwise associations

Force-spectroscopy of proteolytically cleaved NhaA demonstrated that the two

unfolding pathways observed on the intact protein represented the unfolding from the C-

terminal (Fig. 2A) and the N-terminal (Fig. 2B) end. The data suggested that the protein

fragments retained secondary structure and molecular interactions of the native protein.

Otherwise potential barriers established by individual secondary structure elements would

have changed which was not observed. Both unfolding pathways observed for the functional

protein exhibited five main unfolding peaks. In most cases (88 ± 14 %; average ± SD), these

main peaks occurred without side peaks indicating the unfolding of two transmembrane α-

helices and of their connecting polypeptide loop in a single step. This finding supports the

concept that pairwise association of transmembrane α-helices by independently stable

helices builds a key feature of their assembly into the functional structure 23. This model,

which was developed for bacteriorhodopsin 24, holds also for NhaA.

Directional potential barriers?

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The force spectroscopy data (Fig. 2) revealed, that every main peak had a distinct

probability of exhibiting side peaks. In most cases, a side peak represented the unfolding of a

single helix or a loop 9. The potential barriers were located at the aqueous interfaces of

predicted secondary structural elements 17. In less frequent cases, side peaks suggested the

stepwise unfolding of a single secondary structure element. The positions of these potential

barriers were sometimes located to areas inside the hydrophobic part of the protein or to

regions of loops. This suggests, that specific interactions locally stabilized regions of

secondary structures.

Since the force spectroscopy data directly revealed the length of the stretched

polypeptide, the potential barriers built by the protein may be correlated to its sequence (Fig.

4). Superimpositions of the potential barriers showed, that their positions sometimes depend

upon NhaA was unfolded from the C- or the N-terminus. Obviously, individual potential

barriers were detected if the NhaA peptide was mechanically pulled in one direction, but not

when pulled in the opposite direction. In apparent contrast, some internal potential barriers of

NhaA did not depend on the pulling direction. Since potential barriers are established by

inter- and intramolecular interactions, it may be concluded that these barriers can be

established in a directional manner. Examples of interactions that stabilize protein structures

in a directional way are hydrogen bonds, dispersion forces, steric and electrostatic

interactions. However, it may also be assumed that some specific interactions simply cannot

occur after secondary structural elements have been removed before others. In other words,

potential barriers may occur differently because secondary structural elements were

removed in another sequence if unfolding NhaA from the N- or C-terminus. Answers to both

the hypotheses were given by the unfolding experiments performed on the truncated form of

NhaA (Fig. 3). The experiments showed, that some NhaA fragments (Fig. 3 B,D,E)

established identical potential barriers as those observed on unfolding the entire protein. This

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finding, which is in agreement with similar force-spectroscopy experiments performed on

native and truncated bacteriorhodopsin 8, supports the hypothesis that these potential

barriers were intrinsic for individual and groups of secondary structural elements (Fig. 2). It is

in good agreement with the two stage folding model of membrane proteins suggesting that

individual secondary structural elements of membrane proteins independently build stable

subunits in the membrane bilayer, which then associate into higher ordered structures 23.

In contrast, unfolding the NhaA fraction (helices IX to XII, Fig. 3C), showed that these

four helices established different potential barriers than those observed for the intact protein

(Fig. 2B). Thus, it may be assumed that these potential barriers may depend on the

presence of helices I to VIII which have been removed in one case (Fig. 2B) but not in the

other (Fig. 3C). Further experiments exploring the energy landscape of these potential

barriers by dynamic force spectroscopy experiments 25 may provide more detailed insights

into the nature of the observed interactions and of interactions possibly not detected yet.

Controlled refolding of individual secondary structural elements

After characterizing the unfolding pathways of NhaA, the single-molecule force-

spectroscopy experiments were modified to allow controlled refolding of individual secondary

structures. Once distinct helices and loops had been unfolded, the whole system was relaxed

for a given time period allowing the polypeptide to refold. Repeated recording of a force

spectrum detected whether the polypeptide refolded to its native secondary structure. An

example of such an experiment is shown in Fig. 5. First, NhaA was unfolded until only

helices II and I remained anchored in the membrane (Fig. 5 A1; and schematic drawing A).

The force peaks of the unfolding spectra indicated that the protein was unfolded from its C-

terminal end. The AFM stylus was then lowered to the membrane (Fig. 5 B1; and schematic

drawing B). Interestingly, the reverse force spectroscopy curves reproducibly showed a

characteristic ‘snap in’ occurring at a contour length of ~ 202 aa (Fig. 5 B1 – B5; circles).

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This event may indicate a first refolding step occurring as soon as the polypeptide has been

relaxed by approaching the AFM stylus towards the membrane surface for ~ 30 nm. Once

the system had been relaxed for 5s (Fig. 5 C1), the AFM stylus was separated from the

membrane to re-stretch the polypeptide. The force spectrum contained detailed information

on the repeated unfolding process (Fig. 5 A2). All major peaks (potential barriers) observed

during the initial unfolding were detected. This indicated that the secondary structures

observed for native NhaA were refolded and supports the postulation that unfolding and

folding of transmembrane helices may be fully reversible 26. However, in comparison to the

first unfolding spectra some side peaks disappeared, indicating that the protein followed

different pathways in its second unfolding process. Analyzing ~ 50 refolding experiments on

different NhaA (data not shown) demonstrated that the refolded polypeptides exhibited the

same major unfolding peaks as detected in the initial unfolding process but showed a

variation in their side peaks. Sometimes side peaks were absent, in other experiments they

appeared although they had not been detected in the preceding unfolding experiment. This

suggests that a distinct unfolding pathway followed by given structural elements may be

probability driven and not an intrinsic feature of the individual protein 25.

Individual secondary structural elements require individual refolding times

Examples of repeated refolding events at different conditions (Fig.5 B2-5)

demonstrated that NhaA is not completely folded within shorter time scales. However, all

refolding experiments performed so far have shown the characteristic ‘snap in’ occurring at

202 aa and the unfolding barrier at 202 aa. This suggests that the secondary structure

establishing this potential barrier folds within the smallest given time range of 0.1s. In

contrast to this unfolding event, other force peaks did not occur within the shorter refolding

time. This observation is in agreement with the recent biochemical experiments suggesting

that individual secondary structural elements may exhibit individual folding rates 27. Judged

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from the NhaA structure (Fig. 4), the helical pair (helices V and VI) establishes the potential

barrier at ~ 202 aa. This helical pair and its short connecting loop contain no proline residues.

In contrast, the other helical pairs and their connecting loop contain several proline residues

(Fig. 4). This finding suggests that the proline residues may play a role in determining the

folding kinetics of the structural motifs of a transmembrane α-helical pair and loop. In folded

proteins the cis-isoform of proline may be stabilized by the tertiary structure. In agreement

with the folding experiments of other proteins 28; 29; 30, it may be concluded that proline

residues determine the rate limiting process of folding secondary structural elements of

NhaA.

CONCLUSIONS

We have identified potential barriers established within NhaA against mechanical

unfolding. Some of these barriers showed a strong dependence on the unfolding direction

which occurred either from the N- or the C-terminal. Experimental setups allowing to

determine the energy potential of barriers acting directional, non-directional and of barriers

being established between secondary structural elements will provide important insights into

the nature of these inter- and intramolecular interactions. In the future, mapping of potential

barriers established by different membrane proteins may be used to learn and determine the

crucial areas of molecular interactions determining membrane protein stability 1; 2.

It was shown here for the first time how a single polypeptide of a membrane protein

folds into the membrane. The folding event of transmembrane α-helices V and VI showed a

spring-like snap-in, while other helices refolded in a ‘silent’ process which was not detected

in the folding curves. Additionally, individual secondary structural elements required different

folding times, despite belonging to the same class of secondary structures. Thus, it may be

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assumed that an intrinsic hierarchy may exist determining which structural elements fold

before the others to finally assemble into the functional protein structure.

Unfolding and refolding experiments can be performed under various physiological

conditions. Future studies applying assays to screen multiple parameters will allow the

characterization of factors that influence the inter- and intramolecular interactions within a

membrane protein. This provides novel avenues to increase the understanding of how

membrane proteins fold into their functional three-dimensional structure and allows

investigation of protein misfolding diseases in molecular detail.

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MATERIALS AND METHODS

NhaA preparation

NhaA was overexpressed with an 8 aa long spacer and a His6-tag fused to the C-

terminal domain in E.coli, BL21(DE3), from a pET construct 31 as described 21 and purified

using Ni-NTA affinity chromatography. Two-dimensional crystals were obtained as described

21 and the NhaA concentration was generally lowered to 0.5 mg/ml and the E. coli polar lipids

(Avanti) were solubilized in 0.15% dodecyl maltoside (Sigma) in the starting crystallization

mixture. Activity measurements of the reconstituted protein showed that NhaA was still active

within the two-dimensional crystals. For trypsin cleavage, 300 µg of NhaA crystals were

resuspended in 300 µl buffer (100 mM KCl, 700 µM EDTA, 1 mM CaCl2, 50 mM Tris/HEPES,

pH 8.5). Trypsin was added up to enzyme:protein ratio 1:50 (w/w) and the sample was

incubated for 1 h at 37°C. The enzymatic treatment was stopped adding trypsin inhibitor

(Sigma). Treated samples were dyalized in slide-a-lysers (Pierce) against crystallization

buffer 21 overnight at 4°C. Control experiments by cryo–electron microscopy showed that the

digested remained their dimensions and crystallinity identical of the membrane protein

patches.

SDS-PAGE of intact NhaA and tryptic fragments was done according to 32. The

unstained Coommassie gel was incubated for 5min in transfer buffer (25 mM Tris-Hcl, pH 8,

10 mM glycine, 10% methanol and 0.25 mM DTT) and blotted on a PVDF membrane (0.45

µm, 26.5 x 3.75 cm, Millipore Immobilon-P) at 3 mA/cm2 in transfer buffer between several

layers of wet filter paper (Whatman). The membrane was transferred to blocking buffer (200

mM Tris, pH 7.5, 1.5 M NaCl and 5% milk powder) and blocked overnight at 4˚C. After

incubation with the primary antibody (mouse anti-(His)6 IgG, Sigma) for 2 h at room

temperature and 3 washing steps, the membrane was exposed to the secondary antibody

(rabbit anti-mouse IgG, Sigma) coupled with alkaline phosphatase for 1h at room

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temperature followed by 3 washing steps. Antibodies were dissolved in blocking buffer at

concentrations of 1:10000 and 1:20000 respectively, and blocking buffer was used for

washing. The membrane was then transferred into developing buffer (100 mM Tris-HCl, 100

mM NaCl, 5 mM MgCl2, pH 9.5 adjusted with NaOH) and developed by adding 100 µg of

NBT. The color reaction was stopped by water and 20 mM EDTA. It was shown that intact

NhaA contained well-stained band at 32 kDa, while tryptic fragments were not stained. This

implies the C-terminal shortening.

AFM imaging and Single-molecule force-spectroscopy

The AFM (PicoForce, di-Veeco) used was equipped with 200 µm long Si3N4 AFM

cantilevers (di-Veeco). Spring constants k (~ 0.06 N/m) were calibrated in solution using the

thermal noise technique 33. All experiments were performed in buffer solution 150 mM KCl,

50 mM NaCl, 20 mM citric acid, pH 3.8. The NhaA membranes were imaged using contact

mode AFM at a force of ≈ 100 pN applied to the cantilever. To prevent possible sample

perturbations, differences between topographs scanned in trace and retrace directions were

minimized by adjusting scanning speed, feedback loop and applied force. After imaging of

the membrane an unperturbed area was selected at which individual proteins were unfolded

at pulling velocities of 120 nm/s.

Attachment of NhaA to the AFM stylus

In previous studies, different strategies have been developed to attach the terminus of

membrane proteins to the stylus 8. Here we used the nonspecific attachment in combination

with subsequent imaging and force trace classification as this approach allows a much higher

throughput 9.

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Data Analysis

A clear criterion was required that distinguishes curves of NhaA attached to the AFM

stylus with different regions of their polypeptide backbone. One suitable criterion is the

overall length of the force curve, which reflects the stylus-sample distance at which the last

force peak occurs. It is evident that a protein attached to the cantilever by one of its loops

results in a force curve with smaller overall length than a protein attached by one of its

termini. From the primary structure it can be concluded that force extension curves exhibiting

an overall length between 95 and 105 nm result from completely unfolded and extended

NhaA molecules which were attached with one of their terminal ends to the AFM stylus 9. All

force curves exhibiting these overall lengths were selected, classified into two classes and

aligned at their force peaks. To avoid statistical difficulties, we analyzed only relative

positions of the peaks. We used identical procedures and criteria to align each data set. To

analyze the side peaks, however, we superimposed every main peak separately (Fig. 2).

Every single peak of these superimpositions was fitted using the WLC model using a

persistence length of 0.4 nm 22 and a monomer length of 0.36 nm. We calculated the number

of unfolded aa at each peak using the contour length as obtained from the WLC model (see

also legend of Fig. 2).

ACKNOWLEDGEMENTS

We thank Jens Struckmeier for technical assistance and Hermann Gaub, Jonne

Helenius, Tanuj Sapra, Kate Poole, Max Kessler and Evan Evans for stimulating discussions.

Volkswagenstiftung, European Community and State of Saxony supported this work.

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33. Florin, E. L., Rief, M., Lehmann, H., Ludwig, M., Dornmair, C., Moy, V. T. & Gaub, H.(1995). Sensing specific molecular interactions with the atomic force microscope.

Biosens. Bioelectron. 10, 895–901.34. Rothman, A., Gerchman, Y., Padan, E. & Schuldiner, S. (1997). Probing the

conformation of NhaA, a Na+/H+ antiporter from Escherichia coli, with trypsin.Biochemistry 36, 14572-6.

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FIGURE CAPTIONS

Figure 1. High-resolution AFM topographs of NhaA. A , AFM topograph showing a

double-layered membrane of reconstituted NhaA. During adsorption onto the supporting

mica the tubular crystals collapsed and the upper membrane adsorbed onto the lower one.

B, membrane surface imaged at high-resolution. The correlation average (inset) of B shows

structural details of the membrane. Topographs were recorded in buffer solution (200 mM

NaCl, 20 mM Tris-HCl, pH 7.8) and exhibit full gray levels corresponding to vertical ranges of

50 nm (A) and 3 nm (B and C).

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Figure 2. Mechanical unfolding pathways of NhaA. A and B, scattered spectra represent

superimpositions of two classes of force curves. Every class consists of 30 force spectra

each revealed on a single NhaA. Superimpositions highlight common features of the

unfolding events and reduce deviations, which may occur in individual spectra. Red lines

represent WLC fits of individual with their numbers indicating the contour length of the

stretched polypeptides. To compare the polypeptide length derived from the WLC fits with

the NhaA structure we have chosen the secondary structure model of Rothman et al. 17. The

first clear peaks of class A occurred as a characteristic triplet at distances corresponding to

stretched polypeptides of 91, 107 and 125 aa. As shown in the magnified region (green

outlined circle) the main peak at 202 aa sometimes decayed over a side peak at 225 aa

before the peptide was stretched again to a length of 258 aa. The last force peaks were split

into a characteristic doublet (purple outlined circle) at 318 and at 328 aa. Bottom, potential

barriers of NhaA assuming unfolding from the C-terminal end. Filled green circles assign

- 22 -


potential barriers to the secondary structure. For this, the number of aa obtained from the

WLC fit of an individual force peak (red lines) was subtracted from the C-terminal end. When

pulling the polypeptide from the cytoplasmic surface, the anchor of the peptide sometimes

was located at the opposite extracellular surface. In this case, the membrane thickness (~ 4

nm) had to be considered and 11 aa (11 x 0.36 nm ≈ 4 nm) were added to the aa number

determined using the WLC model 9. For these cases, the entire rupture length of the

stretched polypeptide is given in brackets. In contrast to the first class of force spectra, the

second class B showed a strong major peak at 95 aa, which sometimes declined over a side

peak before the peptide was stretched to 157 aa (green outlined circle). The last rupture

event occurred as a single force peak at 327 aa. The blue outlined circle shows minor peaks

corresponding to early unfolding events at a distance < 30 nm. Bottom, potential barriers of

NhaA assuming unfolding starting from the N-terminal end. Filled purple circles mark

locations of individual barriers. Positions of potential barriers were located to the topological

model by fitting the force peaks using the WLC model (red lines). Positions of potential

barriers occurring on the extracellular surface were compensated by the membrane offset

and given in brackets.

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Figure 3. Single-molecule force spectroscopy curves of trypsin-cleaved NhaA. A,

Trypsin cleavage sites of NhaA. Two cleavage sites were detected: At the loop connecting

helices VIII and IX 34 and at the C-terminal end. SDS-PAGE was performed with native (lane

1) and trypsin-treated (lane 2) NhaA. The trypsin cleaved sample was dialyzed against

crystallization buffer with a cut off filter of 10 kDa to remove the enzymes. Native NhaA

migrated at 32 kDa as a single band. Trypsin cleaved NhaA (lane 2) migrated in two bands at

13 and 20 kDa 34. However, certain amount of NhaA molecules remained unaffected by the

trypsin treatment as observed by their unchanged migration. B , C , D and E show force

curves observed in force spectroscopy experiments performed on trypsin-treated NhaA

samples. Similarities in peak positions between the four fractions and force spectra of native

NhaA confirm the hypothesis of two unfolding pathways, one starting at C-, and the other at

the N-terminal end of the molecule.

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Figure 4. Locating potential barriers of NhaA. Potential barriers are indicated on the

primary structure of NhaA. Tentative regions of transmembrane α-helices 17 are under- and

upperlined. Green areas mark barriers detected while unfolding NhaA from the C-terminal

end. Purple areas mark barriers, detected unfolding NhaA from its N-terminal end. The

barrier width, which is determined by the error of the experimental procedure and analysis

corresponds to ± 7 aa (~ ± 2 nm). The number of aa counted from N-terminal and C-terminal

ends are superscripted and subscripted, respectively. Proline residues are encircled red.

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Figure 5. Unfolding and refolding of single NhaA. The experiment scheme is shown on

the right. After the C-terminal end of a single NhaA was tethered to the AFM stylus, the

polypeptide was stretched until parts of the protein unfolded (A ) except the last two

transmembrane α-helices which were left in the membrane. Thereafter, the AFM stylus was

lowered towards the membrane surface at a speed of 20 nm/s (B). The system was relaxed

(C) allowing the peptide to refold. After a certain time, NhaA was unfolded again detecting

secondary structural elements that had refolded. Left, experimental data obtained during the

process. Red force curves marked An (n = 1, 2, … 6) correspond to unfolding of NhaA up to a

polypeptide length of about 80 nm. After this, the polypeptide chain (black curves marked as

Bn) was relaxed and a distinct time (Cn) allowed the peptide to refold (C1 = 5 s, C2 = 1 s, C3 =

0.8 s, C4 = 0.5 s, and C5 = 0.1 s). In the experimental setup shown, structural elements of

NhaA refolded at a high probability of > 90% if the time allowed for refolding was extended to

5 s. Shorter time periods revealed insights into the faster folding of secondary structural

elements.

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