Drug Efflux by a Small Multidrug Resistance Protein Is Inhibited by a Transmembrane Peptide

Abstract

Drug-resistant bacteria use several families of membrane-embedded transporters to remove antibiotics from the cell. One such family is the small multidrug resistance proteins (SMRs) that, because of their relatively small size (ca. 110 residues with four transmembrane [TM] helices), must form (at least) dimers to efflux drugs. Here, we use a Lys-tagged synthetic peptide with exactly the same sequence as TM4 of the full-length SMR Hsmr from Halobacterium salinarum [TM4 sequence: AcA(Sar)₃-VAGVVGLALIVAGVVVLNVAS-KKK (Sar = N-methylglycine)] to compete with and disrupt the native TM4-TM4 interactions believed to constitute the locus of Hsmr dimerization. Using a cellular efflux assay of the fluorescent SMR substrate ethidium bromide, we determined that bacterial cells containing Hsmr are able to remove cellular ethidium via first-order exponential decay with a rate constant (k) of 10.1 × 10⁻³ ± 0.7 × 10⁻³ s⁻¹. Upon treatment of the cells with the TM4 peptide, we observed a saturable ∼60% decrease in the efflux rate constant to 3.7 × 10⁻³ ± 0.2 × 10⁻³ s⁻¹. In corresponding experiments with control peptides, including scrambled sequences and a sequence with d-chirality, a decrease in ethidium efflux either was not observed or was marginal, likely from nonspecific effects. The designed peptides did not evoke bacterial lysis, indicating that they act via the α-helicity and membrane insertion propensities of the native TM4 helix. Our overall results suggest that this approach could conceivably be used to design hydrophobic peptides for disruption of key TM-TM interactions of membrane proteins and represent a valuable route to the discovery of new therapeutics.

INTRODUCTION

Bacteria effectively use membrane-bound efflux transporters to remove cytotoxic compounds as a mechanism of multidrug resistance (25). Among the five families of bacterial multidrug transporters, at least two have been shown to require oligomerization for function: the resistance nodulation division (RND) and the small multidrug resistance (SMR) proteins (6, 22, 25). These families are both found in pathogenic Gram-negative bacteria, such as Escherichia coli, Pseudomonas aeruginosa, and Mycobacterium tuberculosis, while SMRs are also found in Gram-positive bacteria and archaebacteria, such as Staphylococcus aureus and Halobacterium salinarum, respectively (1, 25). Like many effluxers, SMRs use the proton motive force (PMF) to facilitate the removal of various cationic sanitizing agents, dyes, and antibiotics from the bacterial cell (1, 12, 15, 18, 19, 23, 27, 29, 36).

SMR proteins are relatively small compared to the other multidrug efflux families (which often efflux the same molecules, such as ethidium bromide) and are comprised of ∼110 residues that consist of four membrane-spanning (transmembrane [TM]) α-helices with short connecting loops (6). The minimal functional unit of SMRs has been characterized as a dimer, although higher-order oligomerization has also been proposed (4, 9, 33, 36, 37, 39, 40, 42). EmrE from E. coli is the most extensively studied SMR family member and has been characterized by both crystallographic and nuclear magnetic resonance (NMR) methods as an antiparallel homodimer bound to the substrate tetraphenylphosphonium (6, 17, 21). This model shows that TM helices 1 (TM1) through 3 of each monomer surround the substrate, forming a six-helix binding pocket within the membrane bilayer, and utilize the conserved negatively charged residue Glu14 in TM1 to coordinate the protons and/or cationic portions of substrates (6, 23, 36). Several other hydrophobic residues on the interior of the binding pocket are able to variably coordinate aromatic groups frequently present on substrates (6, 17). However, the dimerization propensity per se of SMRs is centered at TM4 via a ⁹⁰GLXLIXXGV⁹⁸ motif (8, 31).

Accordingly, upon considering strategies by which the drug efflux power of SMRs might be inhibited, we hypothesized that, rather than attempting to inhibit SMR-mediated efflux by binding inhibitors into the substrate binding pocket, which itself is permissive to a wide variety of relatively weakly bound molecules, disruption of the TM4-TM4 helix-helix interaction employed by SMRs for dimerization may be an effective method (5, 30). This method of inhibiting oligomerization and function has been successfully achieved in a few instances (reviewed in references 10 and 24) by using TM peptides that mimic and compete for the native TM-TM interaction. Notably, inhibition of dimer-dependent function has been accomplished by using synthetic peptides to treat mammalian cells in membrane proteins, such as the class II G-protein-coupled secretin receptor (14), the ErbB2 tyrosine kinase receptor (2), and the T-cell receptor (7, 32). In bacteria, TM peptide inhibition of the dimeric aspartate receptor (35) and the trimeric diacylglycerol kinase (28) has also been achieved.

To implement the approach of inhibiting SMR-mediated substrate efflux by disrupting protein oligomerization, in the present work, we utilized a synthetic TM4 peptide with a sequence identical to that of the SMR from H. salinarum (Hsmr). Using an ethidium fluorescence assay to visualize its efflux by Hsmr in intact cells, we report that treatment of these cells with the Hsmr TM4 peptide produces a significant decrease in the rate of efflux. Our findings indicate the validity of this approach for specific peptide targeting of oligomeric membrane proteins.

MATERIALS AND METHODS

Peptide synthesis.

TM4 peptides containing amino acids 85 to 105 of the full-length Hsmr protein (sequences are shown in Table 1) were synthesized using a PS3 peptide synthesizer (Protein Technologies, Inc.) via 9-fluorenylmethoxy carbonyl (Fmoc) chemistry. Sarcosine (N-methyl-glycine) and/or lysine residues were added to both the N and C termini to increase the solubility of the peptide (20). A 0.1-mmol-scale synthesis was used for each peptide with the O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate and N,N-diisopropylethylamine activator pair, with a 4-fold amino acid excess. A low-load (0.18 to 0.22 mmol/g) Fmoc-PAL (aminomethyl-3,5-dimethoxyphenoxyvaleric acid)-polyethylene glycol-polystyrene resin yielded an amidated C terminus upon peptide cleavage. To maintain a neutral charge at the N terminus, peptides (with the exception of K-TM4) were acetylated on the resin prior to cleavage with a 1:1 solution of dichloromethane and acetic anhydride. Peptide cleavage and deprotection were carried out using a solution of 88% trifluoroacetic acid, 5% phenol, 5% ultrapure water, and 2% triisopropylsilane. The cleavage product was precipitated into diethyl ether at −80°C, washed twice with cold ether, dried, and resuspended in ultrapure water. The cleaved peptides were purified by reverse-phase high-performance liquid chromatography on a C₄ preparative column (Phenomenex) with a water-acetonitrile gradient in the presence of 0.01% trifluoroacetic acid. Peptide molecular weights were confirmed by mass spectrometry. The peptides were lyophilized and resuspended in ultrapure water, and the concentration was determined using the Micro BCA assay (Thermo).

Table 1.

Sequences and characterization of designed Hsmr TM4 peptides

Peptide	Sequencea	MMb (Da)	MICc (μM)	Oligomeric stated	Relative efflux rate with 1 μM peptidef
K-TM4	KKK-VAGVVGLALIVAGVVVLNVAS-KKK	2,689	12	Dimere	ND
TM4	AcA(Sar)3-VAGVVGLALIVAGVVVLNVAS-KKK	2,632	>25	Dimer	0.37 ± 0.02g
SCR	AcA(Sar)3-LGVLAVAVVANGVLAVSGVIV-KKK	2,632	>25	Monomer	0.88 ± 0.09
GVSCR	AcA(Sar)3-VAGVVLVGIAGVALVVLNVAS-KKK	2,632	>25	Monomer	0.65 ± 0.04h
d-TM4i	AcA(Sar)3-VAGVVGLALIVAGVVVLNVAS-KKK	2,632	>25	Dimer	0.79 ± 0.03h

^aSolubility tags (set off by hyphens) were added to each peptide. Ac signifies an acetylated N terminus. All peptides contain an amidated C terminus. The dimerization motif is underlined.

^bMM, molecular mass.

^cThe MIC was defined by undetectable growth (Fig. 1).

^dStoichiometry of peptides as determined by migration on SDS-PAGE gels (Fig. 2).

^ePreviously reported (33).

^fDaily experiments were normalized to Hsmr (relative efflux = 1) after a rate constant (k) calculation using first-order exponential decay. See Materials and Methods for details. The mean value of k for Hsmr efflux is 10.1 × 10⁻³ ± 0.7 × 10⁻³ s⁻¹ (n = 10). Cells without the Hsmr vector have an efflux rate of 0.15 ± 0.02 relative to Hsmr. ND, not determined.

^gSignificance of effectiveness of peptide treatment (P < 0.0001).

^hSignificance of effectiveness of peptide treatment (P < 0.05).

ⁱPeptide d-TM4 contains all-d residues but is otherwise identical to TM4.

Antimicrobial peptide assay.

The bactericidal activities of the synthesized peptides were tested using the standard antimicrobial peptide assays (11, 38). Minimal medium A (26) was used instead of Mueller-Hinton broth (MHB) to maintain consistency with our efflux experiments. Briefly, 25,000 CFU of E. coli BL21(DE3) cells (Novagen) harboring the pT7-7 Hsmr vector were grown in 100 μl minimal medium A with 0 to 25 μM peptide in a 96-well clear cell culture plate (Nunc). Cell growth was measured by optical density at 600 nm (OD₆₀₀) on a Gemini EM microplate reader (Molecular Devices) after incubation for 20 h at 37°C. A d-amino acid peptide, d-6kf17, was used as a positive control for antimicrobial activity (38). All experiments were performed in triplicate and were background subtracted.

CD spectroscopy and SDS-PAGE.

Circular dichroism (CD) spectra of peptides were recorded on a Jasco J-720 circular dichroism spectrometer at room temperature. Spectra in SDS (20 mM SDS, 10 mM Tris, 10 mM NaCl, pH 7.2) were recorded using a 1-mm-path-length cuvette at a peptide concentration of 20 μM. All spectra were background subtracted and converted to mean residue molar ellipticity (MRE [degree cm² dmol⁻¹ × 10⁻⁴]). The mean residue ellipticities shown are the averages of three separate scans, and statistical differences at 222 nm between all peptides were determined by t tests. SDS-PAGE was performed using materials and protocols from Invitrogen. Purified peptide (0.5 to 1 μg) was loaded onto a 12% NuPAGE Bis-Tris gel with Mark12 protein standards and stained using GelCode Blue stain (Thermo).

Ethidium bromide efflux assay.

Ethidium efflux was measured as previously described (26). E. coli BL21(DE3) harboring the pT7-7 Hsmr or pT7-7 EmrE vector was grown at 37°C to an OD₆₀₀ of ∼0.4 before expression was induced with 0.5 mM IPTG (isopropyl-β-d-thiogalactopyranoside) for 2 h with shaking. The cells were pelleted by centrifugation, resuspended in minimal medium A, and treated with 40 μM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 5 min at room temperature before the addition of 1 μg/ml ethidium bromide and 0 to 1.5 μM peptide. The cells were incubated at 37°C with shaking for 1 h, pelleted by centrifugation, and resuspended in minimal medium A, and fluorescence was quickly measured with stirring on a Photon Technology International C-60 spectrofluorimeter for 1200 s at 1-s intervals (excitation wavelength [λ_ex], 530 nm, 2-nm slit width; emission wavelength [λ_em], 600 nm, 4-nm slit width). The data were normalized to the highest fluorescence point and analyzed using GraphPad Prism fitted to the first-order exponential-decay equation: F = F₀e^−kt, where F is fluorescence at any time, F₀ is the initial fluorescence value at time zero, t is time in seconds, and k is the decay rate constant (s⁻¹).

RESULTS

Peptide design and characterization.

With the purpose of using synthetic peptides to specifically inhibit the oligomerization capabilities and subsequently the substrate efflux of Hsmr, five peptides with a residue composition identical to that of wild-type Hsmr TM4 were designed and synthesized. To aid in water solubility, these synthetic hydrophobic TM segments were supplemented during synthesis with poly(Lys) residues at one or both termini of the peptide (20); this design is typified by peptide K-TM4 (Table 1), with three Lys residues at each terminus. To achieve the possible benefit of an uncharged solubility tag at the N terminus that might aid in peptide insertion into the cellular membrane, we further elaborated our tags to include an acetylated N terminus containing Ala(Sar)₃ while retaining the three Lys residues at the C terminus. Sarcosine residues have been characterized as aiding in the solubilization and maintaining the helicity of hydrophobic TM segments (20). Further peptides consisted of a d-amino acid version of TM4 (d-TM4) and two scrambled peptides, one with a random sequence over the whole segment (SCR) and another in which only the dimerization sequence, encompassing residues 90 to 98 (GVSCR), is scrambled. In peptide SCR, the sequence was further chosen for omission of known oligomerization motifs (24).

Assay for antimicrobial activity.

Since hydrophobic segments with greater than three positively charged residues may have the propensity for causing bacterial membrane disruption and hence are categorized as cationic antimicrobial peptides (3, 13), we first assayed the bactericidal activity of each peptide to optimize our design for membrane insertion without disrupting its integrity (Fig. 1). Cells were grown in minimal medium in the presence of up to 25 μM peptide, and among our designed peptides, only K-TM4 (with a +7 charge) caused cell death (measured as the MIC) at 12 μM and greater. A known cationic antimicrobial peptide, 6kf17, composed of d-amino acids, was used as a control for lysis and displayed an MIC of 3 μM in the present system, similar to its previously reported value of 2 μM in MHB medium (38). Cells were able to grow at all tested concentrations of the other TM4 peptide variants, and although the MIC of K-TM4 is much higher than those used in the in vivo assays, it was excluded from further investigation. The assay was also performed using relatively enriched MHB medium, resulting in essentially identical results (data not shown).

Fig 1.

Antimicrobial peptide assay. Cells were incubated in minimal medium at 37°C for 20 h in the presence of 0 to 25 μM peptide, and growth was measured by absorbance at 600 nm. Lines of best fit are shown. The cells were able to grow at all concentrations of the TM4, d-TM4, SCR, and GVSCR peptides, while cell death occurred in the presence of the K-TM4 peptide at concentrations ≥12 μM. A known antimicrobial peptide, 6k-f17 (with all-d chirality), was used as a control and caused cell death at 3 μM, similar to the previously reported value (38). The error bars represent the standard errors of the mean (SEM) of at least three experiments. a.u., arbitrary units.

Secondary structures of designed peptides.

The TM4 and analog peptides were characterized for helicity and oligomerization by circular dichroism and SDS-PAGE, respectively (Fig. 2). All the peptides display α-helical CD spectra in the SDS membrane mimetic environment with minima (or maxima, as mirrored by d-TM4) at 208 and 222 nm, secondary structure consistent with the conformation of the TM segments of native SMR proteins (36) (Fig. 2A). A comparison of the absolute values of MREs at 222 nm determined that only the SCR peptide is significantly different from the other three peptides (P < 0.05). The identical CD experiments of the peptides in buffer lacking SDS display spectra associated with a “random-coil” structure, as illustrated for the TM4 peptide. SDS-PAGE revealed that the two peptides that contain the native dimerization sequence ⁹⁰GLxLIxxGV⁹⁸ (31)—TM4 and d-TM4—are dimeric, while the two scrambled peptides, GVSCR and SCR, run as monomers (Fig. 2B). The SCR peptide migrates on SDS-PAGE more quickly than GVSCR, a finding consistent with its lower helicity and thus reduced SDS binding (34a). Several attempts were made to dissociate purified dimeric Hsmr with the peptides in SDS, but changes in Hsmr and peptide gel migration were not observable, likely due to the ability of SDS to compete for tertiary and/or quaternary interactions and of (at least some) peptides to form antiparallel dimers in micelles (data not shown).

Fig 2.

Characterization of peptides related to Hsmr TM4. (A) Circular dichroism spectra of 20 μM peptide samples in the membrane mimetic detergent SDS. The spectra are color coded as shown, with the dotted line representing TM4 without SDS present. As the d-amino acid version of TM4, d-TM4 displays a spectrum that is the mirror image of that of TM4. (B) Representative Coomassie-stained SDS-PAGE of 0.5 to 1 μg purified peptides. The locations of Mark12 molecular mass standards are indicated on the left of the gel. The TM4 and d-TM4 peptides run as apparent dimers, while the GVSCR and SCR peptides are monomeric.

Ethidium efflux of peptide-treated cells.

Hsmr inhibition was measured using an in vivo ethidium efflux assay where ethidium fluorescence was measured over 20 min. The decrease in fluorescence is proportional to its efflux from the cell, as the emission intensity of ethidium is ∼25-fold greater when interacting with intracellular DNA (17a). E. coli cells expressing Hsmr extrude ethidium (Fig. 3A) at a rate consistent with first-order exponential decay (R² > 0.99) with a mean rate constant (k) of 10.1 × 10⁻³ ± 0.7 × 10⁻³ s⁻¹. Upon treatment of Hsmr-containing cells with TM4 peptide, the rate of efflux is decreased (k = 3.7 × 10⁻³ ± 0.2 × 10⁻³ s⁻¹), while control cells lacking Hsmr have a basal decrease in fluorescence (k = 1.5 × 10⁻³ ± 0.4 × 10⁻³ s⁻¹) (data not shown). The normalized rate constants are displayed relative to the efflux by Hsmr (Table 1), with single representative decay curves shown in Fig. 3B. Upon treatment with 1 μM additions of each peptide, only the SCR peptide displays an efflux rate statistically equivalent to that of Hsmr (k = 0.88 × 10⁻³ ± 0.09 × 10⁻³). Importantly, the TM4 peptide had the most significant decrease in its efflux rate, with an ∼60% reduction of the decay rate constant (k = 0.37 × 10⁻³ ± 0.02 × 10⁻³; P < 0.0001). Although the d-TM4 and GVSCR peptide treatments also reduce efflux by Hsmr (k = 0.79 × 10⁻³ ± 0.03 × 10⁻³ and 0.65 × 10⁻³ ± 0.04 × 10⁻³, respectively; P < 0.05), they are both statistically equivalent to the SCR peptide and different from TM4 (P < 0.0001). The rate constant decrease was further found to be proportional to the concentration of TM4 (Fig. 4), with efflux inhibition becoming saturated at 1 μM peptide, although the relative rate constant does not reach the basal efflux rate of E. coli cells.

Fig 3.

TM4 peptide inhibition of Hsmr ethidium bromide efflux. (A) Fluorescence measurements of E. coli cells harboring Hsmr actively effluxing ethidium with (+ TM4) and without 1 μM TM4 over 20 min. The curves shown represent the average of 8 to 10 measurements in each case. At 0 s, the medium yields basal fluorescence levels, and as cells containing ethidium are added at 30 s, the fluorescence signal becomes maximal and decreases in proportion to ethidium efflux from the cell. (B) Representative single traces of ethidium efflux data fitted to the first-order exponential decay equation. Hsmr efflux is shown after incubation without peptide (Hsmr) and with (+) 1 μM peptide. The relative rate constants are given in Table 1.

Fig 4.

Dose dependence of TM4 inhibition of ethidium bromide efflux. Shown are normalized decay rate constants relative to Hsmr efflux in the absence of peptide after incubation with various concentrations of the TM4 peptide. The data were obtained by measurement of efflux by ethidium fluorescence over 20 min as in Fig. 3. The red line connects the data points for clarity. The diagram indicates that inhibition becomes saturated at 1 μM peptide. The dashed line represents the relative efflux rate of ethidium from cells without Hsmr (k = 0.15). The error bars represent SEM of at least three experiments.

DISCUSSION

Because Hsmr and related SMRs are “small,” it is physically improbable that these proteins can function in vivo as drug efflux pumps in a monomeric form, particularly with their known activity against relatively “large” molecules, such as ethidium bromide, tetraphenylphosphonium, and benzalkonium (1). Also, attempts to inhibit their functions by binding specifically designed inhibitors into a pocket prospectively formed by dimers must confront the fact that such pockets are dynamic, as they must transport—not permanently bind—their substrates and must adjust to the demands of a wide variety of substrate structure and stereochemistry. It therefore seemed most plausible to address SMR inhibition at the source of its function, viz., by relegating the protein molecules to a monomeric form. The SMR dimerization motif of ⁹⁰GLXLIXXGV⁹⁸ within the TM4 sequence is both evolutionarily conserved and required for efflux function, as shown in the two family members EmrE and Hsmr (8, 31). Although SMR proteins are structurally flexible and use a variety of residues on TM1 to TM3 to accommodate the various compounds that imbue SMRs with their multidrug efflux capabilities, TM4 appears rigid and sensitive to minor changes in the motif (17, 30). Accordingly, we designed peptides to mimic and compete for this functionally dependent TM4-TM4 interaction between two Hsmr monomers with the goal of causing inhibition. These principles underlie the present work, as shown schematically in Fig. 5, where the initially dimeric SMR protein interacts with the designed TM4 peptide to form inactive SMR-TM4 1:1 complexes. Since SMRs function as antiparallel dimers (6, 17, 21), the TM4 peptide (which is unable to form a dimer with itself due to its unidirectional N terminus in-C terminus out orientation in the membrane) could theoretically interact only with the N terminus in-C terminus in SMR subunit.

Fig 5.

Proposed inhibition mechanism of drug efflux. The TM4 peptide (gray) converts from a random coil to an α-helix upon insertion in the bacterial membrane (the neutral N terminus and positively charged C terminus are shown). The TM4 peptide competes for the functional TM4-TM4 native SMR interaction (represented by the EmrE antiparallel dimer [6], Protein Data Bank [PDB] ID, 3B5D, with each monomer colored red or blue), thereby preventing its efflux of ethidium bromide (molecule shown) by producing a TM4-EmrE monomer complex via antiparallel association with TM4 plus an (inactive) EmrE monomer.

To experimentally test the ability of our TM4 peptide to inhibit Hsmr, we used an in vivo efflux assay that takes advantage of the fluorescence capabilities of an SMR substrate, ethidium bromide. Upon treatment of E. coli cells harboring Hsmr with ethidium bromide and the ionophore CCCP, the interiors of cells become saturated with ethidium, as the abolishment of the PMF prevents efflux, yielding a high fluorescence signal due to the cellular DNA-ethidium interaction (Fig. 3). As CCCP is removed, the cells are able to regain the PMF, resulting in the active removal of cellular ethidium, lowering the fluorescence signal (26). The efflux data are then fitted to first-order exponential decay, and the decay rate constant for Hsmr was determined to be 10.1 × 10⁻³ s⁻¹. When the TM4 peptide was incubated with the cells before efflux, the activity of Hsmr was reduced to a decay constant of 3.7 × 10⁻³ s⁻¹ (P < 0.0001). Importantly, the “control” peptide, d-TM4, SCR, and GVSCR, were significantly less effective in this assay (Table 1), reinforcing the notion that the presence of the native dimerization sequence motif and correct chirality underlie the specificity of TM4 inhibition. Interestingly, the control peptide treatments did result in a minor amount of efflux inhibition. Since the d-TM4 peptide should have a propensity identical to that of the TM4 peptide to insert into the bacterial membrane, such insertion may generally impede bacterial function and/or have a measurable effect on membrane integrity. Although d- and l-TM peptides with identical sequences from the bacterial aspartate receptor (Tar-1) have been shown to form d/l heterodimers despite opposite chirality, structural models of the heterodimer demonstrated that a modification involving helix tilting was required to satisfy the hydrogen-bonding interactions of the native l/l homodimer (35). A possible explanation for the lack of a d-TM4 interaction with Hsmr is that the specific van der Waals interactions required for proper TM4-TM4 packing are not as permissive, or as strong, as the polar interactions between the Tar-1 peptides, as further supported by a hydrophobic residue scan of Hsmr TM4 in which TM-TM interactions could be readily disrupted (30). Further design and investigation of effective d-peptide TM4 inhibitors would nevertheless be useful, since d-peptides are resistant to proteolytic degradation compared to l-peptides (reviewed in reference 3). Although effective at preventing Hsmr efflux over a 20-min period (Fig. 3), at this point in our design, the TM4 peptide is not yet able to significantly reduce cell growth over extended periods in the presence of ethidium or benzalkonium due to the inevitable degradation of the l-peptide (data not shown).

When a membrane protein dimerizes or assembles to higher quaternary structure, this self-association is mediated primarily by sequence motifs and van der Waals packing opportunities within the protein TM domain(s). For example, single membrane-spanning proteins, such as glycophorin A and bacteriophage M13 coat protein, form strong TM-TM dimers by this pathway; the calcium channel regulator phospholamban assembles into pentamers in a similar manner (34). The observation that the designed wild-type TM4 peptide and its d-TM4 analog migrate as dimers on SDS-PAGE (Fig. 2B) is a strong indication that these peptides retain their functional helix-helix interaction motifs. In contrast, the two scrambled peptides were unable to form dimers, since they lack the native Hsmr dimerization sequence found in the TM4 and d-TM4 peptides. Furthermore, the design of this series of TM4 and analog peptides with uncharged N termini would be expected to enhance their ability to insert into the bacterial membrane, perhaps in a “corkscrew” manner, with the neutrally charged Ac-Ala(Sar)₃ (Sar = N-methylglycine) N-terminal moiety acting as a membrane entry segment. All four peptides display a random-coil structure in aqueous buffer and large amounts of α-helicity in the membrane mimetic SDS detergent system, with only SCR having a significantly lower helicity. The latter observation may be attributable to the midsequence Asn residue in SCR, as it has been shown that a polar side chain at this location may elicit reduced helicity and quicker gel mobility in vitro (41), as well as a reduced propensity to insert into mammalian cellular membranes (16).

Conclusions.

For the first time, to our knowledge, specific TM peptide inhibition of a bacterial efflux protein (Hsmr) in vivo has been demonstrated through use of a peptide with a sequence identical to that of its native TM4 that includes a deduced TM4-TM4 dimerization motif (31). Although the TM4 peptide does not achieve complete abolishment of Hsmr efflux function, further investigation and development of peptide analogs of TM4 could lead to a high-functioning SMR inhibitor. In the broader view, this approach could conceivably be used to design hydrophobic peptides for disruption of key TM-TM interactions of membrane proteins that arise in many organisms and, as such, represent a valuable route to the discovery of new therapeutics.