Dual energy landscape: The functional state of the β-barrel outer membrane protein G molds its unfolding energy landscape

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    Dual energy landscape: The functional state of the

    b-barrel outer membrane protein G molds its unfoldingenergy landscape

    Mehdi Damaghi1,2, K. Tanuj Sapra2,3, Stefan Koster4, Ozkan Yildiz4, Werner K .uhlbrandt4

    and Daniel J. Muller1,2

    1 ETH Z .urich, Department of Biosystems Science and Engineering, Basel, Switzerland2 Biotechnology Center, University of Technology, Tatzberg, Dresden, Germany3 Chemistry Research Laboratory, University of Oxford, Oxford, UK4 Max-Planck-Institute of Biophysics, Department of Structural Biology, Frankfurt am Main, Germany

    Received: April 13, 2010

    Revised: May 16, 2010

    Accepted: May 17, 2010

    We applied dynamic single-molecule force spectroscopy to quantify the parameters (free energy

    of activation and distance of the transition state from the folded state) characterizing the energy

    barriers in the unfolding energy landscape of the outer membrane protein G (OmpG) from

    Escherichia coli. The pH-dependent functional switching of OmpG directs the protein alongdifferent regions on the unfolding energy landscape. The two functional states of OmpG take

    the same unfolding pathway during the sequential unfolding of b-hairpins IIV. After theinitial unfolding events, the unfolding pathways diverge. In the open state, the unfolding of

    b-hairpin V in one step precedes the unfolding of b-hairpin VI. In the closed state, b-hairpin Vand b-strand S11 with a part of extracellular loop L6 unfold cooperatively, and subsequentlyb-strand S12 unfolds with the remaining loop L6. These two unfolding pathways in the openand closed states join again in the last unfolding step of b-hairpin VII. Also, the conformationalchange from the open to the closed state witnesses a rigidified extracellular gating loop L6.

    Thus, a change in the conformational state of OmpG not only bifurcates its unfolding pathways

    but also tunes its mechanical properties for optimum function.


    Atomic force microscopy / Interactions / Mechanical properties / Nanoproteomics / pH

    gating / Single-molecule force spectroscopy

    1 Introduction

    Outer membrane proteins (Omps) are found in the outer

    membranes of Gram-negative bacteria, mitochondria, and

    chloroplasts. These b-barrel-forming transmembrane proteinsare imperative to a cells survival owing to their function in

    controlling the transport of solutes in and out of a cell.

    Whereas some Omps, like OmpC, OmpF, and OmpG, are

    non-selective in what they transport through their pores, others

    like LamB, FhuA, BtuB, are solute specific [16]. The gating

    mechanisms of the b-barrel forming Omps attract continuousinterest and remain to be investigated in detail [1]. Several of

    the transmembrane pores formed by Omps of Escherichia coliare pH gated. Low pH induces the closing of the pores of, for

    example, OmpC, OmpF, OmpG, LamB, and PhoE [26]. What

    conformational changes drive pore closure has long been

    debated [1, 7]. In 1999, high-resolution atomic force micro-

    scopy (AFM) imaging, for the first time, showed that at low pH

    the large extracellular loops of the OmpF collapsed onto theAbbreviations: aa, amino acids; AFM, atomic force microscopy;

    DFS, dynamic single-molecule force spectroscopy; F-D curve,

    single-molecule force-distance curve; Omp, outer membrane

    protein; SMFS, single-molecule force spectroscopy; WLC, worm-


    These authors contributed equally to this work.

    Colour Online: See the article online to view fig. 3 in colour.

    Correspondence: Professor Daniel J. Muller, ETH Z .urich,

    Department of Biosystems Science and Engineering, Matten-

    strasse 26, 4058 Basel, Switzerland

    E-mail: daniel.mueller@bsse.ethz.ch

    Fax: 141-61-387-39-94

    & 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

    Proteomics 2010, 10, 41514162 4151DOI 10.1002/pmic.201000241

  • pore entrance [8]. This supported the hypothesis that the Omp

    pores are gated by the conformational changes of the flexible

    extracellular loops. Experiments on the maltoporin LamB from

    E. coli, which is specific for malto-oligosaccharides, corrobo-rated the gating model. When lacking the major extracellular

    loops L4 and L6, LamB failed to close at lower pH [9].

    Among the pH-gated Omps, the structure and function

    relationship of the OmpG from E. coli represents possibly thebest-studied example. The OmpG structure solved by X-ray

    crystallography [10, 11] and NMR [12] comprises 14 b-strands(S1S14) that form a transmembrane b-barrel. On the peri-plasmic side the b-strands are connected by six short poly-peptide turns (T1T6). On the extracellular side the b-strandsare connected by seven longer loops (L1L7) that exhibit

    enhanced intrinsic flexibility [11, 12]. A pH-dependent gating

    controls the flux of small molecules through the OmpG pore

    [6]. X-ray structures obtained from three-dimensional OmpG

    crystals grown at neutral (pH 7.5) and acidic (pH 5.6) pH

    provide insight into the conformational changes that may

    guide the gating mechanism [10]. At low pH, the largest

    extracellular loop L6 folds into the pore, thereby constricting

    its entrance. However, the three-dimensional crystals of

    solubilized OmpG grown at different pHs showed different

    packing arrangements, and some extracellular loops formed

    crystal contacts with adjacent OmpG molecules. Thus, it may

    be assumed that the conformations observed may not

    represent those that naturally occur in the gating mechanism

    of OmpG. To test this hypothesis, OmpG was reconstituted

    in native E. coli lipids and imaged by high-resolution AFM inbuffer solution at room temperature [13]. The AFM topo-

    graphs confirmed that the pH-dependent gating mechanism

    suggested from the X-ray structures indeed occurred in

    physiological conditions.

    Besides the gating mechanisms of the Omps, the

    mechanisms that guide their folding and unfolding are of

    pertinent interest [1420]. So far most experiments investi-

    gating the folding of Omps have first denatured Omps in

    detergent and/or urea and then characterized the refolding

    into a lipid bilayer or in a detergent [14, 2124]. Such bulk

    unfolding experiments suggest that OmpG unfolds and

    refolds reversibly [22]. The folding process of Omps is

    described as being coupled with membrane insertion [17]. A

    folding and insertion process has been recently described for

    the b-barrel transmembrane protein PagP from E. coli [25].PagP solubilized and denatured in 10 M urea is found to

    adsorb to the lipid headgroups of the bilayer, where it forms a

    transition state that tilts and inserts into the lipid membrane

    to complete the folding process. Apparently, these models

    contrast with the results from single-molecule force spectro-

    scopy (SMFS) in which single OmpG molecules have been

    mechanically stressed to induce their unfolding from the

    native lipid membrane in a buffer solution [26]. These single-

    molecule experiments clearly show that OmpG molecules

    unfold via many sequential unfolding intermediatesdescribing a detailed unfolding pathway. The unfolding step

    of a single b-hairpin characterizes the transition from one

    unfolding intermediate to the next one. However, the differ-

    ences between chemical denaturation and refolding and

    mechanical unfolding experiments may have different

    origins. First, one may assume that the refolding mechanism

    in the absence of any external force does not reflect unfolding

    under an applied force. Second, it may be that the experi-

    mental conditions alter the unfolding and folding pathways

    chosen by b-barrel membrane proteins. In case of a-helicaltransmembrane proteins it has been shown that alterations in

    the temperature and buffer solution within the physiological

    relevant range can considerably modify their unfolding

    pathways [27, 28]. Therefore, it is not surprising that the

    exposure of membrane proteins to urea, detergent, and

    mechanical stress may force them along very different

    unfolding and folding pathways.

    SMFS has been particularly successful in characterizing

    the unfolding pathways of membrane proteins and to

    quantify the interactions and energies of the intermediates

    in the unfolding pathways [28, 29]. SMFS provides detailed

    insights into the nature of molecular interactions and most

    importantly allows to locate and quantify these interactions

    structurally with an accuracy of E26 amino acids (aa). Inthis work we have performed dynamic SMFS (DFS) to probe

    the strength of the interactions that stabilize the unfolding

    intermediates of OmpG at different loading rates (applied

    force over time). The dependence of these interaction

    strengths on the loading rate allows quantifying the

    unfolding energy barriers of the intermediates [30, 31].

    These measurements provide the position of the transition

    state, the transition rate of the intermediate from the folded

    to the unfolded state, and the energy of activation to cross

    the transition barrier. Because the sensitivity of SMFS

    permits to directly determine the sequence at which the

    unfolding barriers are located along an unfolding pathway,

    we can chart the unfolding energy landscape of OmpG in

    the two pH-dependent conformational and functional states.

    The energy landscapes reveal detailed insights into how

    interactions can change the unfolding pathways and the

    gating mechan