Article pubs.acs.org/ac
An Experimental Study of the Solvent-Dependent Self-Assembly/ Disassembly and Conformer Preferences of Gramicidin A Liuxi Chen, Shu-Hua Chen, and David H. Russell* Laboratory for Biological Mass Spectrometry Department of Chemistry Texas A&M University College Station, Texas 77843, United States S Supporting Information *
ABSTRACT: The solvent dependence of self-assembly/disassembly kinetics and conformer preferences of the gramicidin A (GA) dimer is investigated using a combination of techniques, viz., electrospray ionization−ion mobility−mass spectrometry (IM-MS), collision-induced dissociation (CID), and hydrogen/deuterium exchange (HDX)-MS. IM-MS measurements reveal that there are possibly three distinct GA dimeric species, detected as sodium ion adduct ions [2GA + 2Na]2+, and these are assigned as the parallel βhelix, antiparallel β-helix, and head-to-head dimer. The monomerization kinetics and equilibrium abundances of the dimer ions depend upon solvent polarity. The antiparallel β-helix was the thermodynamically preferred species in less polar solvents. HDX measurements and collision-induced dissociation (CID) of the intermediate complex confirm the well-protected dimer geometry with strong intermolecular hydrogen bonds. This combined IM-HDX-CID methodology provides a comprehensive view of GA self-assembly/disassembly in low dielectric solutions, showing its potential utility in solving solution-phase protein self-assembly/disassembly kinetics and providing structural information of the multimers at the same time.
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Nuclear magnetic resonance (NMR) spectroscopy data suggest that head-to-head helical dimers exist in artificial lipid bilayers and micelles.14 On the other hand, GA exists in a monomer− dimer equilibrium as well as an equilibrium between different dimer conformations in nonpolar solvents. The distribution of species depends on solvent polarity, concentration, solution ionic strength, and temperature.15 For example, in low dielectric solvents, including short chain alcohols, significant amounts of dimer are present;16 however, small amounts of water significantly increase the equilibrium monomer population.17 More interestingly, the self-assembled behavior of GA is known to be highly sensitive to the environment.15,16,18−21 The environment-dependent conformations of dimeric GA have been examined using various techniques, including circular dichroism (CD),15,16,19,21,22 NMR,14,23−25 liquid chromatography,26−29 X-ray crystallography,24,30−33 and mass spectrometry (MS).34−36 Although the aforementioned techniques provide evidence for the detailed structural elements, interpretation of the data is often ambiguous owing to an equilibrium distribution of conformations as both monomer and dimer, which complicates the monitoring of variations in the abundance of the different species and the determination of kinetics of self-assembly. Electrospray ionization (ESI) MS has been established to be a
eptide/protein self-assembly and aggregation are important biological processes involved in essential cell functions that include the formation of ion channels that can result in cell destruction/lysis.1,2 Peptide/protein misfolding and self-assembly are also associated with diseases such as Alzheimer’s, Parkinson’s, bovine spongiform encephalopathy (mad cow disease), and type II diabetes.3 Understanding how folding dynamics and electrostatic interactions, especially intermolecular hydrogen bonding, affect the self-assembly of peptides in a low dielectric environment, i.e., lipid vesicles,4 lipid bilayers,5−8 and as native complexes,9,10 remains a major challenge to structural biology. Equally challenging is the need to directly monitor the kinetics of self-assembly/disassembly processes under native conditions. Such studies may potentially provide molecular level understanding of the mechanism of selfassembly of a range of bioactive peptides (e.g., gramicidins,11 protegrins,12 and cecropins13) that possess a broad spectrum of bioactivity, ranging from antibacterial and anti-inflammatory to potential antitumor/anticancer activity. The gramicidin peptides are unique because of their small size and specificity of biological function, i.e., transporting monovalent cations across membranes.11 Gramicidin A (GA), a linear pentadecapeptide isolated from the soil bacterium Bacillus brevis, has been extensively investigated using a variety of experimental and computational tools; however, numerous questions concerning the environment-dependent conformer preferences remain unanswered. Equally important is the limited understanding of pathways by which different conformations interconvert and the kinetics of such processes. © 2013 American Chemical Society
Received: May 6, 2013 Accepted: July 15, 2013 Published: July 15, 2013 7826
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Figure 1. (A) Zoom-in mass spectra of GA incubated in propanol as a function of time showing the m/z 1905 region of [GA + Na]+/[2GA + 2Na]2+. (B) Ion mobility arrival time distributions (ATD) of the mass envelope of m/z 1905. (C) The kinetic plots of the monomerization of GA at a concentration of 5 μM in ethanol, propanol, and isobutanol, expressed as the intensity ratio of monomers as a function of incubation time. The apparent rate constants are (4.9 ± 0.3) × 10−4, (8.6 ± 0.8) × 10−5, and (3.2 ± 0.4) × 10−5 s−1 for ethanol, propanol, and isobutanol, respectively. (D) The kinetic plots of three dimer conformers (D1, D2, D3) in ethanol, propanol, and isobutanol. The monomerization kinetic plot is also shown for comparison (open circle).
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MATERIALS AND METHODS Materials. Gramicidin (Bacillus brevis), BioChemika, a mixture of gramicidins A, B, C, and D (percentages not specified by the manufacturer), was purchased from Fluka Chemicals (Sigma-Aldrich) and was used without further purification, at a concentration of 5 μM, for monitoring the monomerization process using ESI-IM-MS. The fully labeled mass spectrum of Fluka gramicidin is shown in the Supporting Information (Figure S1). High-purity (≥90%) gramicidin A purchased from Sigma-Aldrich (St. Louis, MO) was used for the HDX experiment. Ethanol, propanol, and isobutanol (ACS grade) were also purchased from Sigma-Aldrich (St. Louis, MO). Methanol-OD was purchased from Cambridge Isotope Laboratories (Andover, MA). Electrospray−Ion Mobility−Mass Spectrometry (ESIIM-MS). The ESI-IM-MS spectra in various solvents were
direct and powerful method for studying the protein multiplex in solution.37 Combined with ion mobility spectrometry (IMS), an ion size-based separation technique, provides a unique means to monitor microheterogeneity of the ion population.38 Furthermore, different conformations that comprise the ion population can be distinguished by using hydrogen/deuterium exchange (HDX),39 a powerful tool for studying protein folding/unfolding dynamics, with the added benefit of being able to explore the conformational space of biomolecules when combined with IMS.40 Here, we present a comprehensive study of the reaction pathway of the GA monomer/dimer as well as the conformer preferences of the dimer, utilizing native ESI,41−43 ion mobility-mass spectrometry (IM-MS), HDX, and collision-induced dissociation (CID). 7827
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the modified N- and C-termini (HCO-Val-Gly-Ala-DLeu-AlaDVal-Val-DVal-Trp-DLeu-Trp-DLeu-Trp-DLeu-TrpNHCH2CH2OH). In this study, IM-MS is used to measure the conformational microheterogeneity of GA ion population; i.e., IM-MS achieves separation on the basis of size-to-charge. Thus, conformers that have different shapes/sizes can be separated even if they have the same m/z. Furthermore, if the conformer preference is solvent-dependent, then IM-MS can be used to directly monitor the environment-dependent microheterogeneity. Here, we examine the self-assembly/disassembly of GA in short chain alcohols, viz., ethanol, proponal, and isobutanol. ESI of GA in these solvents yields abundant Na+ ion adducts, specifically [GA + Na]+ and [2GA + 2Na]2+ ions, owing to the presence of salts in the peptide sample and the solvents and the lack of basic/acidic amino acids in the GA sequence.47 Monomerization and Conformational Transitions of GA Dimer Monitored by ESI-IMS-MS. The net reaction that occurs upon dissolving GA lyophilized powder in alcohol is best described as a monomerization reaction in which the overall dimer population decreases as the monomer increases. The ESI mass spectrum of GA contains abundant ion signals between m/z 1905 and 1909 that correspond to [GA + Na]+ and [2GA + 2Na]2+ ions (see Figure 1A). These ions have the same m/z ratio, but one mass unit separates ions that carry a single charge, whereas 0.5 mass unit separates the ions that carry two charges. The ion abundance ratio of [GA + Na]+/[2GA + 2Na]2+ increases as a function of time following dissolving the lyophilized powder owing to dissociation of GA dimer in the alcohol solution (see Figure 1A). The signals for [GA + Na]+ and [2GA + 2Na]2+ ions can be clearly distinguished on the basis of ion mobility ATD (Figure 1B); the abundances of the corresponding ions change as a function of incubation time. Thus, it appears that GA is present in the powder as a dimer that slowly dissociates to a monomer in alcoholic solution. The apparent rate constant of the monomerization reaction was determined by using the relative abundances of the ion mobility separated monomer and dimer ions (Figure 1B). Figure 1C contains plots of the relative abundance of monomer ions as a function of incubation time. It is apparent that at low concentrations of GA, the equilibrium favors the monomer; however, the rates of monomerization and the relative abundances of the monomer and dimer at equilibrium are solvent-dependent. That is, the kinetics of monomerization is slower for the longer chain alcohols (kapp: ethanol > propanol > isobutanol), and the equilibrium abundance of the dimer also increases with increasing chain length (dimer abundances are ethanol < propanol < isobutanol). The apparent rate constants (4.9 ± 0.3) × 10−4, (8.6 ± 0.8) × 10−5, and (3.2 ± 0.4) × 10−5 s−1, respectively, for ethanol, propanol, and isobutanol agree very well with the literature values (5.0 ± 0.4) × 10−4, (8.2 ± 0.7) × 10−5, and (2.5 ± 0.4) × 10−5 s−1 obtained by HPLC and fluorescence measurement.28 It has been reported that GA exists as a dynamic mixture of monomer47 and several dimeric conformers in low dielectric solvents. In addition to gas phase separation of monomers and dimers, different conformers of the dimers can be clearly distinguished on the drift time scale, which allows simultaneous monitoring of the kinetics of the monomerization and the dimer conformer transitions. The IM ATD for the dimer ions is composed of three distinct (partially resolved) Gaussian distributions, labeled D1, D2, and D3, and the abundances of each species are time dependent (Figure 1B). It is worth noting that the ATD of the monomer is broader than that of ATDs of
acquired on a Waters Synapt HDMS G2 mass spectrometer (Waters UK Ltd., Manchester, U.K.) equipped with a travelingwave IM cell. Ions were formed by nano-ESI using a source temperature of ∼100 °C and a capillary voltage of 1.6−2.2 kV. The cone and extraction voltages were set to produce a high abundance of the dimer signals. Typical operating parameters for these experiments were a traveling wave IM cell pressure of 3 mbar of nitrogen, a wave velocity of 550 m/s, and a wave amplitude of 38 V. The data reported here were not sensitive to small changes in the TW ion cell parameters. ESI-IM-MS data for the GA system were acquired at different time points immediately after dissolving the lyophilized powder in solution. The monomeric and dimeric GA forms are separated by their different IM arrival time; their abundances from the ESI-IM-MS experiments are represented by the ratio of ion abundances calculated using the integrated areas of the IM arrival time distribution (ATD). The ATD of the GA dimer profile can be deconvoluted into three individual Gaussian distributions. To quantitatively determine the changes of monomeric and dimeric GA forms as a function of incubation time in different solvents, the total abundance of GA is defined as the loading amount of GA in its monomeric form, that is, the abundance of monomeric form and twice this abundance for the dimeric form. The relative abundances of the individual monomeric and dimeric species are the ratios of their abundance to the total abundance. Assuming the solid material is initially composed of dimers only, and at low concentration as in the ESI experiment, the monomerization process follows a simple first order exponential decay.15 Three independent measurements were made to determine the average and standard deviation of the apparent rate of the monomerization reaction. The ion-neutral collision cross section (CCS) values for the dimer conformers were derived using the method described previously by Ruotolo et al.44 Doubly protonated tryptic peptides obtained from cytochrome c and myoglobin were used as the CCS calibration standards. Literature values of CCS of the calibrant peptides were taken from the database generated by Clemmer and coworkers.45 Hydrogen−Deuterium Exchange Labeling. Fully deuterium-labeled GA samples were prepared by incubating the GA in methanol-OD at a concentration of 1 mM for over 24 h. The forward exchange profile of GA in methanol-OD is shown in the Supporting Information (Figure S2). The stock solution was then diluted to 1 μM in propanol to initiate the backexchange. The time between mixing the solution and initiating the MS measurement is ∼2 min. Mass spectra were taken as a function of time to monitor the back-exchange. Changes in deuterium content (in Da) were calculated by comparing the average mass-to-charge values of the isotope clusters of the partially deuterated peptides with that of nondeuterated peptides. CCS Calculations of Protein Data Bank GA Dimer Structures. Three GA dimer structures in the protein data bank (PDB) were selected for CCS calculation, which represent the three most commonly observed conformers of the dimer: parallel double helix (1MIC), antiparallel double helix (1ALZ), and head-to-head dimer (1NRU). CCS calculation was carried out using the MOBCAL46 trajectory method.
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RESULTS AND DISCUSSION GA is highly hydrophobic, sparingly soluble (only nM levels) in water, owing to its composition of nonpolar amino acids and 7828
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present in the solution without the need for prior isolation/ purification. GA Dimeric Conformers Probed by HDX and CID. Solution-phase HDX was performed as a means to further investigate structural elements of GA dimers. Figure 2 contains
the dimer. The broad profile of the monomer reported previously47 is contributed from four dynamic monomer conformers which varies in CCS, while the narrower ATDs of the dimers, especially D1 and D2, suggest much ordered assemblies, which is further illustrated in the following sections. Figure 1D contains plots of the relative abundances of D1, D2, and D3 in the three different solvents. Initially, the abundance of D2 is very low, but it increases and eventually becomes more abundant than both D1 and D3. Note that in each solvent the abundances of D1 and D2 greatly exceed that of D3, which is decreasing initially and remains constant (
