Negatively Charged Phospholipids Trigger the Interaction of a

Jan 20, 2012 - ABSTRACT: Folded proteins can be translocated across bio- logical membranes via the Tat machinery. It has been shown in vitro that thes...
0 downloads 0 Views 2MB Size
Article pubs.acs.org/Langmuir

Negatively Charged Phospholipids Trigger the Interaction of a Bacterial Tat Substrate Precursor Protein with Lipid Monolayers Tina Brehmer,† Andreas Kerth,† Wenke Graubner,‡ Miroslav Malesevic,§ Bo Hou,‡ Thomas Brüser,‡ and Alfred Blume*,† †

Institute of Chemistry - Physical Chemistry, Martin-Luther-University Halle-Wittenberg, Von-Danckelmann-Platz 4, 06120 Halle, Germany ‡ Institute of Microbiology, Leibniz University Hannover, Schneiderberg 50, 30167 Hannover, Germany § Max Planck Research Unit for Enzymology of Protein Folding, Weinbergweg 22, 06120 Halle, Germany ABSTRACT: Folded proteins can be translocated across biological membranes via the Tat machinery. It has been shown in vitro that these Tat substrates can interact with membranes prior to translocation. Here we report a monolayer and infrared reflection−absorption spectroscopic (IRRAS) study of the initial states of this membrane interaction, the binding to a lipid monolayer at the air/water interface serving as a model for half of a biological membrane. Using the model Tat substrate HiPIP (high potential iron−sulfur protein) from Allochromatium vinosum, we found that the precursor preferentially interacts with monolayers of negatively charged phospholipids. The signal peptide is essential for the interaction of the precursor protein with the monolayer because the mature HiPIP protein showed no interaction with the lipid monolayer. However, the individual signal peptide interacted differently with the monolayer compared to the complete precursor protein. IRRA spectroscopy indicated that the individual signal peptide forms mainly aggregated β-sheet structures. This β-sheet formation did not occur for the signal peptide when being part of the full length precursor. In this case it adopted an α-helical structure upon membrane insertion. The importance of the signal peptide and the mature domain for the membrane interaction is discussed in terms of current ideas of Tat substrate−membrane interactions.



INTRODUCTION The Tat system is a transport machinery that transports folded and often cofactor-containing proteins across energy-transducing membranes in prokaryotes and plant plastids (not to be confused with the lentiviral transcriptional transactivator protein Tat).1 Tat-dependently translocated proteins (Tat substrates) are synthesized as precursor proteins that possess unstructured N-terminal signal peptides that are followed by a folded mature domain (Figure 1).2 The signal peptide usually consists of three regions: An N-terminal hydrophilic n-region with a positive net charge is followed by a hydrophobic h-region andin the case of cleavable signal peptidesa hydrophilic short c-region that contains the signal peptidase cleavage site.3 The signal peptide is recognized by Tat system components during the translocation process and thus is an essential determinant for the translocation process.4 Most important for the specific recognition of the signal peptide is a conserved amino acid pattern at the interface between n- and h-regions, the eponymous twinarginine motif (Figure 1).1 In some cases, Tat signal peptides serve as N-terminal membrane anchors after transport. Their N-terminus inside topology indicates that signal peptide N-termini remain on the cytoplasmic (cis) face of the membrane while C-terminal globular domains are translocated.1 However, in most cases signal peptides are cleaved off after transport, and the translocated mature part of the protein © 2012 American Chemical Society

Figure 1. Schematic diagram of the HiPIP precursor. The mature domain has been adopted from its solved high-resolution oxidized structure (1NEH),33 and the position of the unfolded signal peptide has been schematically indicated, highlighting the twin arginines (underlined) that are part of the twin-arginine motif (bold).

is released from the membrane. The “N-terminus-cis” topology is also experimentally supported for cleavable signal peptides.5 Received: November 14, 2011 Revised: January 20, 2012 Published: January 20, 2012 3534

dx.doi.org/10.1021/la204473t | Langmuir 2012, 28, 3534−3541

Langmuir

Article

Boc (lysine, tryptophan). The Fmoc protecting group was cleaved with a mixture of 2% piperidine, 2% 1,8-diazabicyclo[5.4.0]undec7-ene (DBU) in DMF for 1 × 5 min and 1 × 15 min. After synthesis, peptides were cleaved from the resin and completely deprotected with a cleavage cocktail 5% phenol, 5% H2O, 5% thioanisol, 2.5% 1,2ethanedithiol, 82.5% trifluoroacetic acid (TFA) and purified with a preparative HPLC (Abimed, Langenfeld, Germany) using a SP250/21 Nucleosil 100-7 C8 column (Macherey-Nagel, Düren, Germany). The peptide purity was controlled with an ESQUIRE-LC ion-trap mass spectrometer equipped with an ESI source (Bruker Daltonics, Bremen, Germany) or a MALDI-ToF REFLEX mass spectrometer (Bruker Daltonics) and an analytic HPLC (Sykam, Fürstenfeldbrück, Germany) containing 125/4 LiChrospher RP-8 column (Merck, Darmstadt, Germany). The purity of the signal peptide was about 95% as estimated by HPLC analysis. A stock solution with a concentration of 150 μM in 20 mM potassium phosphate buffer pH 7.0 was used for the experiments. Preparation of Protein Samples. Purified recombinant precursor HiPIP and mature HiPIP (high potential iron−sulfur protein) from Allochromatium vinosum were generated as described previously.27 HiPIP preparations were dialyzed against 50 mM phosphate buffer pH 7.0 and concentrated to 180 μM by ultrafiltration, using the VivaspinTM ultrafiltration system (Sartorius). The purity of the samples was above 98% as estimated by SDS-PAGE and by the ratio of the absorptions 283 nm/388 nm, which are the absorption maxima of the peptide and the assembled iron−sulfur cofactor, respectively.28 Lipids. The lipids, E. coli polar lipid extract and E. coli phosphatidylglycerol, were purchased from Avanti Polar Lipids. The lipid extract was composed of phosphatidylethanolamine PE (67%), phosphatidylglycerol PG (23%), and cardiolipin CL (10%). The lipid purity was specified to be >99%. The lipids were therefore used without further purification and were dissolved in chloroform/methanol (20:1) to make a stock solution of about 1 mM. Chloroform and methanol (HPLC grade), potassium dihydrogen phosphate, and dipotassium hydrogen phosphate were purchased from Carl Roth GmbH (Karlsruhe, Germany). Ultrapure water with a resistivity of 18.2 MΩ cm was used (Milli-Q Advantage A10, Millipore GmbH, Schwalbach, Germany). Film Balance Experiments. All monolayer experiments were performed with film balances using a filter paper as Wilhelmy plate (Riegler & Kirstein, Berlin, Germany). Before each experiment, the trough was cleaned with Hellmanex solution (Carl Roth GmbH, Karlsruhe, Germany) and rinsed thoroughly with ultrapure water. The trough was then filled with a buffer solution (20 mM potassium phosphate, pH 7.0), and the surface was cleaned with a suction device. The temperature of the subphase was maintained at 20 ± 0.5 °C. In all experiments the amount of peptide/protein injected resulted in a subphase concentration of 100 nM. The adsorption experiments at the air/water interface in the absence of a lipid monolayer were performed at constant surface area. The respective surface pressure changes were recorded with time after the injection of the respective protein/peptide stock solution. These measurements were carried out in the smaller compartment of the IRRAS trough system in combination with IR spectroscopy (see below). For the protein/peptide adsorption at the lipid/water interface lipid films were spread at the air/water interface from chloroform/ methanol. After deposition of the lipid solution onto the buffer (20 mM potassium phosphate, pH 7.0) surface and an equilibration period of 15 min, the films were compressed to a surface pressure of 20 mN m−1 with a velocity of 0.02 nm2 per lipid molecule per minute. The pressure was kept constant, and the surface area was adjusted automatically by feedback regulation (π = constant). After a subsequent waiting period of 60 min, the peptide solution was injected into the subphase underneath the lipid film. The resulting change in area was recorded as a function of time. Infrared Reflection−Absorption Spectroscopy (IRRAS) of Monolayers. The IRRAS Teflon trough system (Riegler & Kirstein, Berlin, Germany) consists of a larger compartment (300 × 60 × 3 mm3) with two computer-controlled symmetrically moveable Teflon barriers and a smaller compartment (60 × 60 × 3 mm3). A constant

Recently, it has been shown in vitro that Tat substrates can interact with membranes prior to their interaction with the translocation machinery.6,7 The N-terminal signal peptide has been found to be essential for membrane binding and proteaseprotected upon membrane interaction, and thus is believed to directly interact with the membranes.8−11 Peptides may interact with phospholipid membranes mainly via electrostatic or hydrophobic interactions.12,13 Such interactions can influence the structure of a peptide anddepending on the peptide sequence they may even result in a membrane insertion of the peptide.14 Membrane integration of peptides has been intensively studied with cytotoxic peptides that affect membrane integrity.12,15 However, the mode of membrane interaction of Tat substrates is not understood so far and is therefore the focus of this study. Here we report on the interaction of the bacterial model Tat substrate HiPIP (high potential iron−sulfur protein) with model membrane surfaces. HiPIP is a monomeric 9 kDa periplasmic protein containing one [4Fe−4S]2+/3+ cluster bound via four cysteines. The protein was used as a model because it is structurally well characterized and has a high stability.16,17 The signal peptide has 37 amino acids with the sequence MSDKPISKSRRDAVKVMLGTAAAIPMINLVGFGTARA, containing the characteristic twin-arginine motif needed for Tat-dependent translocation. In the precursor protein, the signal peptide is unstructured in solution and its N-teminal methionine is cleaved off in the cytoplasm, which is why the membrane-interacting signal peptide actually has a length of only 36 residues in vivo.2 Langmuir monolayers are convenient model systems to mimic half of a biological membrane.18 Monolayers as model membrane surfaces have the advantage that (i) lipid compositions can be varied as well as the lipid phase state, (ii) lipid lateral packing density (control of the surface pressure π, the area per film molecule A) and aqueous subphase composition (pH, ionic strength) can be regulated, and (iii) the lipid headgroup area exposed to the aqueous phase is known. This technique can be used directly for lipid/protein interaction studies.19−21 We combined the monolayer technique with infrared reflection−absorption spectroscopy (IRRAS) to analyze the conformation of the lipid-interacting peptides.22 The usefulness of this technique was demonstrated before with various proteins and lipid monolayers.23−25 Our data show that the globular mature domain of HiPIP strongly influences the mode of interaction of the signal peptide with the lipid monolayer. We could show that the signal peptide is essential for membrane binding of the precursor protein as the mature protein alone does not interact with lipid monolayers.



EXPERIMENTAL SECTION

Peptide Synthesis. The HiPIP signal peptide with the sequence SDKPISKSRRDAVKVMLGTAAAIPMINLVGFGTARA was chemically synthesized as described below. The N-terminal Met is cleaved off in vivo and therefore was not included in the synthesis. All amino acid derivatives, resins, and coupling reagents were obtained from Calbiochem-Novabiochem. Other solvents and chemicals were purchased from Sigma-Aldrich. Peptides were synthesized in a SYRO II MULTISYNTECH peptide synthesizer on a Fmoc-Ala-Wang resin using Fmoc chemistry.26 In each cycle, Fmoc-protected amino acids were preactivated with 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3tetramethylaminium hexafluorophosphate (HCTU) and N,Ndiisopropylethylamine (DIPEA) in N,N-dimethylformamide (DMF) and coupled for 2 h. The side chains of the amino acids were protected as follows: Pbf (arginine), Trt (asparagine, glutamine, histidine, cysteine), tBu (threonine, tyrosine, serine), OtBu (aspartic and glutamic acid), 3535

dx.doi.org/10.1021/la204473t | Langmuir 2012, 28, 3534−3541

Langmuir

Article

water level was maintained in both troughs with a home-built laser reflection-measuring device, which controls a corresponding peristaltic pump for each trough. If the film was prepared on the larger compartment, this trough was called the “sample” trough (and the smaller trough therefore the “reference” trough). In case the film was spread on the smaller compartment the denominations change accordingly. For IRRAS measurements an XA 511 reflection attachment (Bruker, Karlsruhe, Germany) with the trough system described above and an external MCT detector was interfaced to an Equinox 55 FTIR spectrometer (Bruker). The IR beam is focused onto the water surface at a defined and computer-controlled angle of incidence. A rotatable KRS-5 polarizer (>98% degree of polarization) was used to generate parallel and perpendicularly polarized light, respectively. The trough system was positioned on a moveable platform in order to shuttle between the “sample” and the “reference” trough. This shuttle technique diminishes spectral interference from water vapor absorption in the light beam.29−31 IRRA spectra were recorded at a 40° angle of incidence with parallel polarized light and at a spectral resolution of 8 cm−1. For each spectrum, 2000 scans were coadded over a total acquisition time of 6 min, zerofilled by two levels, apodized by using a Blackman−Harris four-term function, and Fourier transformed. The single-beam reflectance spectrum of the reference trough surface was ratioed as background to the single-beam reflectance spectrum of the monolayer on the sample trough to calculate the reflection absorption spectrum as −log(R/R0). Difference spectra of adsorbed polypeptides at lipid monolayers were calculated by subtracting the plain lipid spectrum from the protein/lipid interaction spectrum. CD Spectroscopy. Circular dichroism (CD) spectra were recorded on a JASCO J-815 spectropolarimeter (sample temperature 20 °C, Peltier thermostat, data interval 1 nm, scanning speed 50 nm/ min, 1 nm bandwidth, 10 scans averaging, cell path length 0.1 cm) with sample concentrations of 4.0 μM (purified precursor HiPIP) or 5.6 μM (purified mature HiPIP) as determined by UV−vis spectroscopy, using the known extinction coefficient of HiPIP (ε283 nm = 41.3 mM−1 cm−1),32 which is identical for precursor and mature forms of HiPIP due to the lack of tyrosines or tryptophanes in the signal peptide.

Figure 2. Far-UV circular dichroism spectra of mature HiPIP (open circles), precursor HiPIP (open squares), and the difference spectrum (signal peptide, filled triangles).

techniques in order to reveal differences in surface activities and possible structures that could be already induced by lipidindependent surface interactions. The polypeptide-containing solution was injected into the subphase buffer, resulting in a final concentration of 100 nM, the change in surface pressure was measured, and IRRA spectra were recorded concomitantly. Figures 3A,B show the results of these adsorption experiments to the air/water interface. The mature HiPIP exhibited the weakest surface activity (Figure 3). The change in surface pressure was very small, but the IRRA spectra recorded after the injection showed a characteristic group of vibrational bands arising from the protein and from the aqueous subphase. The appearance of the OH stretching vibrational band ν(OH) at ∼3600 cm−1 indicates the formation of a protein film on the buffer surface. During the adsorption process the ν(OH) band increased, which indicates an increasing film thickness, assuming a constant film refractive index.35 The frequency and shape of the amide I and II bands of the protein contain information about the protein secondary structure at the air/water interface. Approximately 2 h after the injection, the amide I and II bands showed two distinct minima at 1661 and 1544 cm−1, respectively, which can be attributed to α-helical secondary structure elements. Two shoulders at ∼1634 and ∼1529 cm−1 indicate additional contributions from β-sheets.36,37 These assignments correspond to the secondary structure elements of the HiPIP structure in Figure 1.16 Because of additional adsorption of the mature HiPIP at the air/water interface, the intensities of all bands increased with time. However, the ratio of the intensity of the band components characteristic for α-helices and β-sheets changed, too (Figure 3B, cf. gray dashed and solid). After 5 h, the amide I band showed two distinct minima at 1661 and 1624 cm−1, the low-frequency component corresponding to β-sheet structures having higher intensity. The amide II band was very broad with only one minimum at 1528 cm−1, which also reflects a higher amount of β-sheets. This result is somewhat surprising as it is known that the mature HiPIP is very stably folded and resistant against proteases in bulk.27,38 The increasing content of β-sheet structure and the slight shift to lower wavenumbers of the β-sheet amide I band indicates a conformational transition with at least a partial unfolding and aggregation of the protein at the air/water interface. This is not uncommon, as the concentration



RESULTS AND DISCUSSION Protein Adsorption at the Air/Water Interface. The interaction of bacterial Tat substrates with lipid monolayers was studied using the high potential iron−sulfur protein (HiPIP) of Allochromatium vinosum. Like all soluble Tat substrates, HiPIP is synthesized as a precursor protein that consists of an N-terminal signal peptide, followed by the folded mature domain (Figure 1).33 We prepared the signal peptide-containing precursor of HiPIP, the signal peptide-lacking mature HiPIP, and the individual signal peptide and used these three (poly)peptides for lipid monolayer interaction studies. It has been shown by NMR and H/D exchange that the signal peptide of the HiPIP precursor is unfolded in solution.2 With our preparations, we could confirm this by circular dichroism spectroscopy (Figure 2). Mature HiPIP gave rise to a typical HiPIP spectrum with a characteristic minimum near 230 nm, which has been proposed to be caused by the residue W80 that is positioned closely to the [4Fe−4S]2+/3+ cofactor.34 The CD spectrum of the HiPIP precursor showed significant differences due to the presence of the signal peptide in the precursor protein. The difference spectrum confirmed the unfolded characteristics of the signal peptide, including a predominant minimum below 200 nm and the absence of any minimum in the 205−230 nm region, which would have been expected for α-helical of β-sheet structures. With these homogeneous and structurally characterized polypeptides in our hands, we analyzed the adsorption behavior at the air/water interface. We used film balance and IRRAS 3536

dx.doi.org/10.1021/la204473t | Langmuir 2012, 28, 3534−3541

Langmuir

Article

protein film at the air/water interface compared to the mature HiPIP. This could be caused by a higher amount of adsorbed molecules, a different aggregation behavior, or another orientation of the protein. As a consequence, the intensity of the amide bands was also higher. The amide I band showed a minimum at 1628 cm−1 which reflects β-sheet structures. The shoulder at 1658 cm−1 indicates also the presence of α-helical structural elements. The amide II band showed a broad minimum at about 1534 cm−1, confirming the previous conclusions. During the adsorption process the ratio of the band intensities of α-helices and β-sheets remained almost constant. Comparison of the spectra of the mature and the precursor HiPIP protein at short times, when aggregation effects can be excluded, reveals that the band intensity ratio of β-sheet to α-helical structure elements are nearly the same, despite the fact that the precursor HiPIP contains the additional signal peptide. The secondary structure of the additionally attached signal peptide remains therefore unclear. It is likely that it is mainly unfolded and thus contributing to the background intensity of the amide I band coming from unstructured stretches of HiPIP and those in β-turns. The functional HiPIP signal peptide has 36 amino acids and thus a molar mass of almost 40% compared to the mature HiPIP. The signal peptide itself showed a moderate surface activity. The surface pressure increased slowly and continuously after injection into the subphase to a value slightly higher than that for the mature protein but lower compared to the value of the precursor protein. Five hours after the injection the pressure had increased to ∼2.5 mN m−1. The intensity of the ν(OH) band (∼3600 cm−1) indicating the thickness of the film was comparable to the one of the adsorbed mature HiPIP. The amide I band shape of the signal peptide is distinctly different from those of the precursor or the mature HiPIP. The intensity of the sharp peak at 1620 cm−1 is much higher and smaller peaks at 1661 and 1685 cm−1, respectively, are visible. The frequency of 1620 cm−1 indicates definitely that aggregated β-sheets are present, and the shoulder at 1685 cm−1 points to an antiparallel arrangement of the β-strands. The band at 1661 cm−1 points to some α-helical elements. Thus, the IRRA spectra clearly indicate the presence of β-sheets that originate most likely from intermolecular aggregation of the signal peptide at the air/water surface. Comparing the spectra of the individual signal peptide and the precursor protein containing the signal peptide, one can conclude that the attachment of the signal peptide to the mature HiPIP prevents the intermolecular aggregation of the signal sequence, probably because of steric reasons. Interaction of the HiPIP Precursor and Its Signal Peptide with Escherichia coli Phospholipids. After having analyzed the surface activity and structure of the precursor and mature forms of HiPIP as well as of the individual signal peptide in the absence of lipids at the air−water interface, we addressed the interactions with lipid monolayers at the air/ water interface. In different experiments, the precursor HiPIP, the mature HiPIP, and the individual signal peptide were injected into the subphase underneath a lipid monolayer. For our first interaction studies with monolayers we used a lipid composition closely corresponding to that of the cytoplasmic membrane of E. coli (PE/PG/CL 67:23:10 mol %). The compression isotherm of this lipid mixture showed a liquidexpanded phase behavior over the whole compression range (data not shown).

Figure 3. (A) Surface pressure versus time course of mature HiPIP, precursor HiPIP, and HiPIP signal peptide adsorption at the air/water interface in the absence of a lipid monolayer with a trough concentration of 100 nM, starting with the injection of the respective volume of the stock solution into the subphase (20 mM potassium phosphate, pH 7.0 at 20 °C). (B) IRRA spectra of the respective films of mature HiPIP, precursor HiPIP, and HiPIP signal peptide at the air/water interface shown in (A) after ∼5 h. The red dashed line shows the spectrum of mature HiPIP ∼2 h after injection. All spectra were recorded with p-polarized light at an angle of incidence of 40°. Inset: detail enlargement of amide I and II bands.

at the air/water interface is high due to the adsorption process so that aggregation is easily induced. Comparable phenomena have been observed before in many cases with small peptides at the air/water interface in a surface pressure-, time-, or concentrationdependent manner.13,39,40 However, also a reorientation of the molecule at the air/water surface with increasing surface concentration could be the cause of the time-dependent shift of the amide bands. HiPIP itself has a very low amount of secondary structure elements. Only 15−20% are in α-helical segments and even less in β-sheet structures. Therefore, slight changes in the reorientation of β-sheet segments can lead to an increase in the corresponding band intensity. The precursor of HiPIP containing the signal peptide turned out to have the highest surface activity of the three polypeptides. About 1 h after injection of the protein the surface pressure increased rapidly up to ∼7 mN m−1 followed by a slower surface pressure increase due to additional protein adsorption. The high surface activity and concentration were also detectable in the IRRA spectra by more intensive bands. The IRRA spectrum recorded 5 h after injection indicated, based on the intensity of the ν(OH) band, the formation of a thicker 3537

dx.doi.org/10.1021/la204473t | Langmuir 2012, 28, 3534−3541

Langmuir

Article

For studies of the interaction of peptides and proteins with lipid monolayers it is important to select the correct monolayer pressure. Many experiments have shown that the so-called monolayer−bilayer equivalence pressure, where the lipid packing behavior is similar in the bilayer and the monolayer, is in the range between 30 and 35 mN m−1.41 In many cases, however, the amount of bound proteins/peptides to the monolayer is very low at this film pressure due to the fact that the bulk concentration of the peptides or proteins cannot be increased. This was similar in our system as we used a bulk concentration of only 100 nM. To increase the degree of binding, we therefore slightly reduced the lateral pressure to 20 mN m−1 and kept the film pressure constant. (Our own data and also those published by other groups always show a linear correlation between increasing protein/peptide binding and decreasing initial pressure of the lipid monolayer as, for instance, shown for melittin and δ-lysin by Maget-Dana.14) The adsorption of the protein was then followed by observing changes in area per lipid molecule. As described above, IRRA spectra were recorded during the adsorption process. Injection of mature HiPIP underneath the lipid monolayer did not result in changes of the area per lipid molecule, and thus it could be concluded that mature HiPIP does not interact with the monolayer (Figure 4A). We recorded almost no changes to the E. coli lipid IRRA spectrum after the injection of HiPIP. Figure 4B shows that the spectra of the pure lipid film and the spectrum after injection of the mature HiPIP are identical. The largest bands were again the OH stretching vibrational band ν(OH) at ∼3600 cm−1 and the H2O deformation vibrational band δ(H2O) at ∼1660 cm−1. In addition, specific lipid bands like the antisymmetric and symmetric CH2 stretching vibrational bands of the lipid alkyl chains are seen (νas(CH2) at ∼2925 cm−1 and νs(CH2) at ∼2855 cm−1). As was expected from the surface pressure area isotherm, the frequency of these bands indicates that the E. coli polar lipid mixture is in a liquid-expanded phase. The carbonyl stretching vibrational band ν(CO) of the fatty acid ester bonds is seen at ∼1735 cm−1. In contrast to mature HiPIP, the addition of the signal peptide-containing precursor of HiPIP resulted in an increase of the area occupied per lipid molecule. After 10 h of incubation, the lipid area was increased by almost 11% (Figure 4A). This is a clear indication for a binding of the precursor HiPIP into the lipid monolayer. The IRRA spectra of the film after injection of the precursor HiPIP showed a very small increase of the ν(OH) band reflecting a small increase of the film thickness due to the interaction of the protein. In the region of the protein specific amide I and II bands small spectral changes were observed, and also a slight intensity decrease of the ν(CO) band of the lipid could be detected. To enhance the spectral changes occurring after injection, we analyzed the respective difference spectrum (Figure 4C). The difference spectrum clearly shows the presence of the protein in the film as amide I and amide II bands are visible. The position of the amide I band at ∼1655 cm−1 and the amide II band at ∼1545 cm−1 suggest almost exclusively α-helical secondary structure elements. Compared to the experiment at the air/ water interface, the amount of β-sheets was thus strongly reduced when the protein was bound to the monolayer. Most importantly, the difference spectrum indicates an α-helical conformation of the attached signal peptide as almost no β-sheet structures could be detected.

Figure 4. (A) Relative area change ΔA/A0 versus time after injection of HiPIP, precursor HiPIP, and HiPIP signal peptide underneath a monolayer of E. coli polar lipids(PE/PG/CL 67:23:10 mol %) at a constant surface pressure of 20 mN m −1.The final protein concentration in the buffer subphase (20 mM potassium phosphate, pH 7.0 at 20 °C) was 100 nM. (B) IRRA spectra of a monolayer of E. coli polar lipids at a surface pressure of 20 mN m−1 (black dashed) and of the respective films after adsorption of HiPIP, precursor HiPIP, and HiPIP signal peptide to the lipid monolayer shown in (A) after ∼5 h. All spectra were recorded with p-polarized light at an angle of incidence of 40°. Inset: detailed enlargement of amide I and II bands. (C) Difference spectra between the films ∼5 h after injection of HiPIP, precursor HiPIP, and signal peptide, respectively, and the pure lipid monolayer shown in (B).

The signal peptide of HiPIP interacted most intensively with the E. coli polar lipid monolayer. The change in area per lipid molecule amounted to 35%, and the corresponding IRRA 3538

dx.doi.org/10.1021/la204473t | Langmuir 2012, 28, 3534−3541

Langmuir

Article

spectra clearly showed specific peptide bands such as the amide A band at ∼3290 cm−1 and the amide I and II bands at 1621 and 1530 cm−1, respectively (Figures 4A,B). This frequency of the amide I band indicates an aggregated β-sheet secondary structure of the signal peptide. The difference spectrum (Figure 4C) showed this more clearly as in addition to the broad asymmetric amide I band with a minimum at 1623 cm−1 an additional band at 1684 cm−1 was visible which is characteristic for antiparallel β-sheets. The shoulder visible at higher frequency (1658 cm−1) indicates some contributions from α-helical segments. In Figure 4B, a small intensity decrease of the ν(OH) band is evident which is due to a reduction of the average film thickness. This is to be expected as the peptide film (Figure 3A) had a smaller thickness than the lipid film (Figure 4A). Insertion of the signal peptide into the lipid monolayer led also to an intensity decrease of the specific lipid bands like the antisymmetric and symmetric methylene stretching vibrational bands ν(CH2) and the carbonyl stretching vibrational band ν(CO) at 2924, 2854, and 1736 cm−1, respectively. This is due to a dilution of the lipids by the peptide insertion as the monolayer area increases. Theoretically, the decrease should amount to ca. 35%, i.e., the same amount as the area per lipid is increased. The actual reduction of intensity was, however, stronger. The cause for this was probably a not completely homogeneous insertion of the peptide into the lipid monolayer which could be analyzed by measuring spectra at different locations in the monolayer, but with the used equipment those studies are not possible. Interaction of the HiPIP Precursor and Its Signal Peptide with Phosphatidylglycerol Monolayers. To analyze whether the interaction of the HiPIP precursor and the signal peptide with phospholipids was influenced by electrostatic attractions, we investigated their interaction with pure phosphatidylglycerol (PG) monolayers at a surface pressure of 20 mN m−1 (Figure 5A). At this surface pressure the E. coli PG is also in a liquid-expanded state as shown by its compression isotherm (data not shown). As described before, we recorded IRRA spectra during the adsorption process. The spectrum of the pure lipid monolayer in Figure 5B is showing the usual bands originating from the lipids and the subphase as described for Figure 4B (see above). As in the case of the interactions with the mixed E. coli polar lipids, the mature HiPIP showed hardly any interaction with the PG monolayer (Figure 5A). In agreement with this, no changes in the corresponding IRRA spectra were detectable. Indeed, the spectra after the injection of the protein overlap the spectrum of the pure lipid (Figure 5B). The HiPIP precursor protein showed a much higher affinity to the negatively charged PG monolayer compared to the E. coli polar lipid mixture described above. The increase in area per lipid molecule 10 h after the injection of the protein amounted to 30% in the presence of the negatively charged PG (Figure 5A) compared to 11% in the presence of the E. coli polar lipid mixture (Figure 4A). Moreover, significant changes in the IRRA spectra indicated the enhanced adsorption of the protein to the PG monolayer. This was evident from the intensity increase of the ν(OH) as well as from the higher intensities of the amide I and II bands at 1658 and 1547 cm−1, respectively (cf. Figures 4B and 5B). The analysis of the respective difference spectra confirms the α-helical conformation of the membrane-interacting portion of the precursor (Figure 5C). Again, an intensity decrease of the asymmetric and symmetric ν(CH2) bands (2924 and 2854 cm−1) and also of the ν(CO) band (1733 cm−1)

Figure 5. (A) Relative area change ΔA/A0 versus time course of HiPIP, precursor HiPIP, and HiPIP signal peptide adsorption at an E. coli PG monolayer at a constant surface pressure of 20 mN m−1 with a trough concentration of 100 nM, starting with the injection of the respective volume of the stock solution into the subphase (20 mM potassium phosphate, pH 7.0 at 20 °C). (B) IRRA spectra of an E. coli PG lipid monolayer at a surface pressure of 20 mN m−1 (black dashed) and of the respective films of HiPIP, precursor HiPIP, and HiPIP signal peptide adsorbed at the lipid monolayer shown in (A) after ∼5 h. All spectra were recorded with p-polarized light at an angle of incidence of 40°. Inset: detailed enlargement of amide I and II bands. (C) Difference spectra between the films ∼5 h after injection of HiPIP, precursor HiPIP, and signal peptide, respectively, and the E. coli PG monolayer shown in (B).

could be observed due to lipid dilution upon protein binding. It is therefore clear that the HiPIP precursor protein inserts into the PG monolayer to a larger extent than into the E. coli lipid 3539

dx.doi.org/10.1021/la204473t | Langmuir 2012, 28, 3534−3541

Langmuir

Article

As an example, we analyzed the 12 known iron−sulfur cofactorcontaining signal-peptide-containing Tat substrates in E. coli.1 The first cysteine ligand was never closer than 14 residues to the signal peptide, and in most cases this distance was between 14 and 22 residues. We thus cannot exclude a requirement of some unfolding very close to the N-terminus of the mature domain for transport, but the distance of cofactor ligands to the N-termini may also have other reasons.

mixture, as the overall area increase and the intensity decrease of the lipid specific bands as well as the intensity increase of the amide I band are more pronounced. The signal peptide interacts even more strongly with the negatively charged PG monolayer, also in comparison to the interaction with the E. coli polar lipid mixture. The increase of the area per lipid molecule amounted to 86% (Figure 5A). The band positions of the amide I and II bands seen in the difference spectrum are at 1621 and 1530 cm−1, respectively (Figure 5C), and indicate again mainly aggregated β-sheet secondary structure as observed before. The decrease in the vibrational band intensities of the lipids indicates again their displacement due to the incorporation of the peptide into the PG monolayer. This is also more pronounced than observed before with the E. coli lipid monolayer. Apparently electrostatic interactions are highly important for the interaction of the signal peptide or the whole precursor protein, which is in agreement with other studies.42 The signal peptide of HiPIP contains 5 positive and 2 negative charges within the n-region and one further positive charge in the c-region at position −2 relative to the cleavage site. Our data suggest that the positive net charge of the signal peptide especially in the n-regionmost likely initiates the contact with the lipid surface. An importance of negatively charged phospholipids for Tat transport has been documented in earlier studies,43 and our data suggest that the signal peptide−membrane interaction contributes to this importance. In addition, an essential domain of E. coli TatA, the amphipathic helix, requires negatively charged phospholipids for efficient membrane interaction.44 Different mechanisms could account for the suppression of a signal peptide β-sheet formation by the folded mature domain of the HiPIP precursor. The folded globular domain may sterically inhibit a lateral association of signal peptides to β-sheets, or it may influence the depth to which regions of the signal peptide can intercalate into the membrane. Especially, the C-terminal part of the signal peptide is likely to be directly influenced by the globular mature domain, which fixes this region to the lipid surface. There also exists the possibility that regions of the mature domain are directly involved in the membrane interaction. Such a direct involvement of a mature region in a membrane interaction has so far only been described recently for a thylakoidal Tat substrate.11 In the case of the thylakoid system, it has been shown that the region directly following the signal peptide is membrane-interacting in a way that protects this region from proteolytic sensitivity. It has been proposed that a partial unfolding of the N-terminal region occurs with this plant Tat substrate, and a portion of the mature domain traverses the membrane.11 Similar to this case, HiPIP could in principle unfold in the N-terminal region of the mature domain. It has been shown that this region can unfold under the influence of chaotropic salts without losing the [4Fe−4S]2+/3+ cofactor that is bound to the C-terminal region of HiPIP.38 Such a partial unfolding of the N-terminal part of the mature domain without loss of the tightly bound cofactor may be the basis for the observed β-sheet formation of mature HiPIP at the air/water interface. However, an extensive N-terminal unfolding of the mature domain is unlikely to occur when the full-length precursor interacts with lipid membranes, as β-sheet aggregates are not formed in this case. Also, the analysis of other Tat substrates argues against a generally occurring extensive N-terminal unfolding during Tat transport, as cofactors are often assembled near the N-terminus and an unfolding event during transport would result in the loss of the assembled cofactors.



CONCLUSIONS In this study, we applied film balance and IRRAS techniques to characterize the adsorption behavior of the Tat substrates at lipid monolayers. Monolayers are valuable model systems for the very initial step of the membrane interaction, and our results indicate that important information could be obtained that helps to understand the membrane association process of Tat substrates. We used a natural Tat substrate, the high potential iron−sulfur protein (HiPIP) for our analyses, which has several advantages: (i) natively folded precursor or mature forms of HiPIP can be prepared in sufficient quantities for this type of experiments;27 (ii) the signal peptide of HiPIP has experimentally been shown to be unstructured in solution when it is connected to a fully folded mature domain, and thus structures that can be attributed to the membrane-interacting signal peptide are induced by the lipid interaction;2 and (iii) HiPIP is a natural Tat substrate, and all observations result from interactions that in principle can occur naturally. The mature HiPIP did not interact with lipids, indicating that the signal peptide is essential for the membrane interaction. The signal peptide of HiPIP as well as the signal peptidecontaining precursor of HiPIP both interacted with phospholipid monolayers. It has been shown that also signal peptides of the Sec pathway have a high affinity for membrane lipids.14 In both cases the interaction was significantly enhanced when the monolayer was only composed of the negatively charged PG. Although the signal peptide in solution has been shown to be unstructured when it is part of the precursor protein2 (Figure 2), it forms β-sheet structures as an “unconnected” individual 36 residues peptide. Importantly, this β-sheet formation of the signal peptide is suppressed by the folded mature domain of the precursor protein, and an α-helix can be induced at the membrane surface. Previously, Briggs et al. showed that the Sec signal peptide of LamB formed β-sheets when it interacted only with the head groups of lipids, whereas an insertion into the monolayer induced α-helices.14 In the case of the Tat signal peptide studied here, the observed α-helix formation would thus be in agreement with an insertion into the hydrophobic regions of the membrane. In summary, the signal peptide and negatively charged phospholipids play key roles in the membrane interaction, and it is interesting to see that the mature domain prevents a β-sheet formation of the signal peptide and thus is highly important for the membrane interaction. The herein demonstrated α-helical structure of the membrane-interacting signal peptide as part of the precursor protein is likely to be highly important for substrate binding to the Tat system, as the helix may help to position the side chains of the twin-arginine motif to orientations that are recognized by the Tat system substrate binding site.



AUTHOR INFORMATION

Corresponding Author

*Tel +49-345-5525850; Fax +49-345-5527157; e-mail alfred. [email protected]. 3540

dx.doi.org/10.1021/la204473t | Langmuir 2012, 28, 3534−3541

Langmuir

Article

Notes

(32) Dus, K.; De Klerk, H.; Sletten, K.; Bartsch, R. G. Biochim. Biophys. Acta 1967, 140, 291. (33) Bertini, I.; Dikiy, A.; Kastrau, D. H. W.; Luchinat, C.; Sompornpisut, P. Biochemistry 1995, 34, 9851. (34) Przysiecki, C. T.; Meyer, T. E.; Cusanovich, M. A. Biochemistry 1985, 24, 2542. (35) Hussain, H.; Kerth, A.; Blume, A.; Kressler, J. J. Phys. Chem. B 2004, 108, 9962. (36) Goormaghtigh, E.; Cabiaux, V.; Ruysschaert, J.-M. Determination of Soluble and Membrane Protein Structure by Fourier Transform Infrared Spectroscopy. III. Secondary Structures. In Physicochemical Methods in the Study of Biomembranes, 23rd ed.; Hilderson, H. J., Ralston, G. B., Eds.; Plenum Press: New York, 1994; p 405. (37) Tamm, L. K.; Tatulian, S. A. Q. Rev. Biophys. 1997, 30, 365. (38) Bentrop, D.; Bertini, I.; Iacoviello, R.; Luchinat, C.; Niikura, Y.; Piccioli, M.; Presenti, C.; Rosato, A. Biochemistry 1999, 38, 4669. (39) Kerth, A.; Erbe, A.; Dathe, M.; Blume, A. Biophys. J. 2004, 86, 3750. (40) Lopes, D. H. J.; Meister, A.; Gohlke, A.; Hauser, A.; Blume, A.; Winter, R. Biophys. J. 2007, 93, 3132. (41) Blume, A. Biochim. Biophys. Acta 1979, 557, 32. (42) Batenburg, A. M.; Demel, R. A.; Verkleij, A. J.; De Kruijff, B. Biochemistry 1988, 27, 5678. (43) Mikhaleva, N. I.; Santini, C.-L.; Giordano, G.; Nesmeyanova, M. A.; Wu, L.-F. FEBS Lett. 1999, 463, 331. (44) Chan, C. S.; Haney, E. F.; Vogel, H. J.; Turner, R. J. Biochim. Biophys. Acta 2011, 1808, 2289.

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank Nebiyat Nigusie Woldeyohannis for providing protein samples. Financial support from the state Sachsen-Anhalt (Exzellenzcluster Biowissenschaften) and the DFG (BR2285/1-3) is gratefully acknowledged.



REFERENCES

(1) Berks, B. C.; Palmer, T.; Sargent, F. Curr. Opin. Microbiol. 2005, 8, 174. (2) Kipping, M.; Lilie, H.; Lindenstrauss, U.; Andreesen, J. R.; Griesinger, C.; Carlomagno, T.; Brüser, T. FEBS Lett. 2003, 550, 18. (3) Natale, P.; Brüser, T.; Driessen, A. J. Biochim. Biophys. Acta 2008, 1778, 1735. (4) Alami, M.; Luke, I.; Deitermann, S.; Eisner, G.; Koch, H. G.; Brunner, J.; Müller, M. Mol. Cell 2003, 12, 937. (5) Fincher, V.; McCaffery, M.; Cline, K. FEBS Lett. 1998, 423, 66. (6) Bageshwar, U. K.; Whitaker, N.; Liang, F. C.; Musser, S. M. Mol. Microbiol. 2009, 74, 209. (7) Hou, B.; Frielingsdorf, S.; Klösgen, R. B. J. Mol. Biol. 2006, 355, 957. (8) Brüser, T.; Yano, T.; Brune, D. C.; Daldal, F. Eur. J. Biochem. 2003, 270, 1211. (9) Shanmugham, A.; Wong Fong Sang, H. W.; Bollen, Y. J. M.; Lill, H. Biochemistry 2006, 45, 2243. (10) Musser, S. M.; Theg, S. M. Eur. J. Biochem. 2000, 267, 2588. (11) Schlesier, R.; Klösgen, R. B. Biol. Chem. 2010, 391, 1411. (12) Hong, J.; Oren, Z.; Shai, Y. Biochemistry 1999, 38, 16963. (13) Arouri, A.; Kerth, A.; Dathe, M.; Blume, A. Langmuir 2011, 27, 2811. (14) Briggs, M.; Cornell, D.; Dluhy, R.; Gierasch, L. Science 1986, 233, 206. (15) Bougis, P.; Rochat, H.; Pieroni, G.; Verger, R. Biochemistry 1981, 20, 4915. (16) Carter, C. W.; Kraut, J.; Freer, S. T.; Xuong, N.-h.; Alden, R. A.; Bartsch, R. G. J. Biol. Chem. 1974, 249, 4212. (17) Banci, L.; Bertini, I.; Dikiy, A.; Kastrau, D. H. W.; Luchinat, C.; Sompornpisut, P. Biochemistry 1995, 34, 206. (18) Maget-Dana, R. Biochim. Biophys. Acta 1999, 1462, 109. (19) Dennison, S. R.; Baker, R. D.; Nicholl, I. D.; Phoenix, D. A. Biochem. Biophys. Res. Commun. 2007, 363, 178. (20) Giehl, A.; Lemm, T.; Bartelsen, O.; Sandhoff, K.; Blume, A. Eur. J. Biochem. 1999, 261, 650. (21) Cornell, D. G.; Patterson, D. L. J. Agric. Food Chem. 1989, 37, 1455. (22) Mendelsohn, R.; Mao, G.; Flach, C. R. Biochim. Biophys. Acta 2010, 1798, 788. (23) Meister, A.; Nicolini, C.; Waldmann, H.; Kuhlmann, J.; Kerth, A.; Winter, R.; Blume, A. Biophys. J. 2006, 91, 1388. (24) Erbe, A.; Kerth, A.; Dathe, M.; Blume, A. ChemBioChem 2009, 10, 2884. (25) Hermelink, A.; Kirsch, C.; Klinger, R.; Reiter, G.; Brezesinski, G. Biophys. J. 2009, 96, 1016. (26) Sewald, N.; Jakubke, H. D. Peptides: Chemistry and Biology; Wiley-VCH: Weinheim, 2002; p 594. (27) Brüser, T.; Yano, T.; Brune, D. C.; Daldal, F. Eur. J. Biochem. 2003, 270, 1211. (28) Bartsch, R. G. Methods Enzymol. 1971, 23, 644. (29) Flach, C. R.; Brauner, J. W.; Taylor, J. W.; Baldwin, R. C.; Mendelsohn, R. Biophys. J. 1994, 67, 402. (30) Kerth, A.; Garidel, P.; Howe, J.; Alexander, C.; Mach, J. P.; Waelli, T.; Blume, A.; Th Rietschel, E.; Brandenburg, K. Med. Chem. 2009, 5, 535. (31) Maltseva, E.; Kerth, A.; Blume, A.; Mohwald, H.; Brezesinski, G. ChemBioChem 2005, 6, 1817. 3541

dx.doi.org/10.1021/la204473t | Langmuir 2012, 28, 3534−3541