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Determination of the structure of lipid vesicle-bound angiotensin II and angiotensin I
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ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 374 (2008) 346–357 www.elsevier.com/locate/yabio
Determination of the structure of lipid vesicle-bound angiotensin II and angiotensin I Pegah R. Jalili, Chhabil Dass * Department of Chemistry, University of Memphis, Memphis, TN 38152, USA Received 21 September 2007 Available online 4 December 2007
Abstract A mass spectrometry (MS)-based strategy was developed to determine the structure of lipid vesicle-bound angiotensin II (AII) and angiotensin I (AI). It involves hydrogen–deuterium exchange (HDX), chemical modifications (e.g., nitration of tyrosine, acetylation of free amino group), and ladder sequencing. HDX is also combined with tandem mass spectrometry (MS/MS) to provide structural details at individual amino acid residues. It was observed that a major portion of both of these peptide hormones interacts with the phospholipid head groups on the surface of the vesicles and that Tyr residue is embedded in the vesicles. Both peptides have a U-shaped structure in the lipid environment. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Hydrogen–deuterium exchange; Mass spectrometry; Angiotensin II; Angiotensin I; Conformation; Peptides
Angiotensin II (AII1, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) and angiotensin I (AI, Asp-Arg-Val-Tyr-Ile-His-Pro-PheHis-Leu) are endocrine hormone peptides that are synthesized locally in the pituitary gland and brain to form the renin–angiotensin system. AI, an inactive peptide, is produced when the liver protein, angiotensinogen, is cleaved with renin protease. AI is further converted by angiotensin-converting enzyme (ACE) to the bioactive octapeptide AII. These two peptides have identical N-terminal sequences but differ at the C terminal. AII is vital to regu-
*
Corresponding author. Fax: +1 901 678 3447. E-mail address: [email protected] (C. Dass). 1 Abbreviations used: AII, angiotensin II; AI, angiotensin I; ACE, angiotensin-converting enzyme; CNS, central nervous system; AT1 receptor, guanosine nucleotide protein-coupled receptor; CD, circular dichroism; NMR, nuclear magnetic resonance; HDX, hydrogen–deuterium exchange; MS, mass spectrometry; ESI, electrospray ionization; MALDI, matrix-assisted laser desorption/ionization; MS/MS, tandem mass spectrometry; DMPC, dimyristoyl phosphatidylcholine; OG, n-octyl-b-Dglycopyranoside; a-CHCA, a-cyano-4-hydroxycinnamic acid; TFA, trifluoroacetic acid; TOF, time-of-flight; TNM, tetranitromethane; DE, delayed extraction; m/z, mass-to-charge; IT, ion trap; HDX, hydrogen– deuterium exchange. 0003-2697/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.11.038
lating cardiovascular functions, fluid homeostasis, adrenal aldosterone secretion, and the release of pituitary hormones. It also acts on the central nervous system (CNS) receptors, such as the guanosine nucleotide protein-coupled (AT1) receptor, to regulate the blood pressure [1,2]. Excessive production of AII causes inadequate function of the renin–angiotensin system, leading to hypertension, heart failure, and renal diseases. AII receptor blockers and ACE inhibitors are used for treatment of these diseases. To design effective drugs such as the AII receptor blockers, it is imperative to have knowledge of the higher order structure of AII in the receptor-binding domain. Although the conformation that AII adopts at the receptor-binding site is decided by the geometry of this domain, determination of the conformation of the AT1 receptor at high resolution is difficult. Therefore, a practical alternative is to study the active structure of the peptide in a medium that mimics the natural environment of the receptor. Because of its biological activity and pharmaceutical interest, the structure of AII has been studied by a variety of analytical approaches, such as circular dichroism (CD) [3,4], nuclear magnetic resonance (NMR) [4–8], UV resonance Raman spectroscopy techniques [9,10], X-ray crys-
Structure of angiotensin II and angiotensin I / P.R. Jalili, C. Dass / Anal. Biochem. 374 (2008) 346–357
tallography [11,12], and conformational calculations [12,13], yet no consensus has emerged. Various AII structures that have been proposed include a-helix [7], b-sheet [4], b-turn [9], c-turn [12], and random coil [14]. Several structure–activity studies have revealed the topological contribution of the Tyr4, His6, and Phe8 aromatic residues and the C-terminal carboxyl group on the biological activity of AII [12,15–17]. It has also been documented that pstacking in Tyr4 and His6 allows a charge transfer process between the two peptide termini of AII and plays an important role in its biological activity [18,19]. A recent study by Spyroulias and coworkers revealed that both AI and AII assume an S-shaped structure in organic solvents and a U-shaped structure in the receptor-binding site [20]. In the current study, we report the conformational states of AII and AI in lipid vesicles (liposomes). This medium mimics the receptor environment more accurately than do the other commonly used membrane-mimetic media such as detergent micelles and organic solvents (e.g., methanol, 1-chloroethanol, trifluoroethanol). We present here, for the first time, a multipronged strategy, shown in Fig. 1, to determine the membrane topology of peptides in lipid vesicles. In this strategy, hydrogen–deuterium exchange (HDX), chemical modifications (nitration of tyrosine and acetylation of free amino group), and ladder sequencing are combined with mass spectrometry (MS) to provide detailed structural information about these peptide hormones. HDX is a well-known method for studying the conformational states and folding–unfolding mechanisms of peptides and proteins in solution [21–29]. HDX relies on the fact that a region of the molecule that is involved in noncovalent interactions and has low accessibility to the solvent exhibits slow HDX rates, whereas a highly exposed region undergoes rapid exchange. The mass of the target peptide increases by 1 u for each hydrogen exchanged with deuterium, and the resulting mass changes can be measured conveniently with MS. Electrospray ionization (ESI)–MS and matrix-assisted laser desorption/ionization (MALDI)–MS
347
are commonly used for this purpose. The combination of HDX with tandem mass spectrometry (MS/MS) yields even more structure-specific information about the conformation of peptides. The product ions in MS/MS spectra can provide knowledge of the amino acid sequence of the target peptide. The deuterium-labeled amino acid residues will shift to higher masses in the mass spectrum than will the nondeuterated residues, thereby providing information about individual amino acids that are involved in interaction with the membrane or are shielded by the solvent. Nitration of the peptide–vesicle complex is proposed to determine the topology of Tyr residues. Although tyrosine has a significant nonpolar character owing to the aromatic ring, its phenolic hydroxyl group (pKa = 10.1) acts as a charged polar entity at high pH. Because of these special characteristics, in most cases tyrosine is the site of interaction in enzyme–receptor reactions. Many structure–activity studies support tyrosine as being important for activity of AII [18–20]. Tyrosine can interact with the hydrophobic core of the receptor membrane via hydrogen bonding and/or hydrophobic interaction. In the current study, nitration was performed to determine whether tyrosine is buried in phospholipid vesicles. When the lipid-bound peptide is reacted with tetranitromethane (TNM) under moderate alkaline conditions, only the free tyrosine will be converted to 3-nitrotyrosine, increasing its mass by 45 u (Scheme 1) [30]. Acetylation will be performed by reacting the peptide-vesicle complex with acetic anhydride. This reaction converts the free amino group to the acetylated form, increasing the mass by 42 u (Scheme 2) [31]. We also propose using ladder sequencing to determine whether the C- and N-terminal residues of the peptide are buried in the membrane’s hydrophobic core or are outside. Traditionally, this method has played an important role in determining the amino acid sequence of peptides. The N- and C-terminal ladders are generated by reacting the target peptide with leucine aminopeptidase and carboxypeptidase Y, respectively [32,33]. Sequential cleavage of the peptide with each of these exopeptidases produces a group of fragments, the molecular mass of which differs from one another by the mass of the cleaved amino acid residue. The amino acid sequence of the peptide can be determined from the measured mass differences between a series of molecular ion peaks in the spectrum [32,33]. In the peptide–vesicle complex, the terminal amino acids that are outside of the vesicle will be cleaved sequentially to produce smaller fragments. Cleavage will stop at the site of an amino acid that is involved in any kind of interaction. The knowledge gained in the current study will facilitate understanding of biological processes, hormone–receptor interactions, and receptor-binding mechanisms. Materials and methods
Fig. 1. Experimental protocol for the study of conformation of bioactive peptides in phospholipid vesicles.
AI, AII, dimyristoyl phosphatidylcholine (DMPC), noctyl-b-d-glycopyranoside (OG), a-cyano-4-hydroxycinnamic acid (a-CHCA), anhydrous acetonitrile, tetrani-
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Scheme 1. Nitration of tyrosine.
with 2 ll of 20% acetic anhydride in acetonitrile. The resulting solution was centrifuged, and the pellet was dissolved in water and analyzed with MALDI–TOF MS.
Scheme 2. Acetylation of free amino group.
tromethane, leucine aminopeptidase, and carboxypeptidase Y were obtained from Sigma (St. Louis, MO, USA). D2O (99.98%) was obtained from Aldrich (Milwaukee, WI, USA).
Nitration The pellet obtained after centrifuging 20 ll of the peptide–vesicle complex was dissolved in Tris buffer at pH 8.0 and treated with 0.5 ll of the nitration reagent (0.1 M tetranitromethane in ethanol) for 3 h. The resulting solution was centrifuged, and the pellet was dissolved in water and analyzed with MALDI–TOF MS.
The unilamellar DMPC peptide–vesicle complex was prepared by dissolving 50 ll of 1 nmol of each peptide in 0.5 ml of a solution that contained 5% (w/v) OG and 1.25% (w/v) DMPC. Solubilization of the peptides was aided by sonication in a water bath for 15 min at 38 °C. A small amount of water (0.5 ml) was added to this mixture, and the contents were incubated for 5 min at 38 °C. Dialysis (cellulose ester DispoDialyzer, 1 kDa) for 24 h at 38 °C against water removed OG detergent to produce large-size unilamellar vesicles. The resulting mixture was dialyzed again at 38 °C (cellulose ester DispoDialyzer, 15 kDa) to remove free peptides present in the solution.
Ladder sequencing A 2-ll portion of carboxypeptidase Y or leucine aminopeptidase was added to a 10-ll solution of the peptide–vesicle complex. The final concentration of endopeptidases was 10 pmol/ll in each solution. Several 1-ll aliquots were removed from the reaction vial at different reaction times for analysis by MALDI–TOF MS. For MALDI–TOF MS analysis, the solutions from the nitration, acetylation, and ladder sequencing experiments were mixed 1:1 with the matrix (10 mg/ml a-CHCA in 50% acetonitrile in 0.1% TFA). As controls, solutions of free peptides were reacted with acetylation, nitration, and ladder sequencing reagents and were analyzed in the same way.
Hydrogen–deuterium exchange
MS analysis
A 20-ll portion of the peptide–vesicle solution was centrifuged in a benchtop centrifuge at 10,000 rpm. HDX was initiated by dissolving the resulting pellet that contained the peptide–vesicle complex in 20 ll of D2O. The exchange was quenched after defined time points with 2% formic acid in D2O at pH 2.5 and was monitored within 1 min using nanoESI–MS and nanoESI–MS/MS. For MALDI–MS analysis, the pellet was dissolved in 20 ll of D2O. After 1 min, the exchange was quenched by adding in a 1:1 ratio the MALDI matrix solution (10 mg a-CHCA in 1 ml of 50% acetonitrile in 0.1% trifluoroacetic acid [TFA]/D2O) at pH 2.5 and 0 °C. The resulting solution was promptly dried in a desiccator and analyzed using MALDI–timeof-flight (TOF) MS.
The MALDI mass spectra were obtained on a Voyager DE-RP MALDI–TOF mass spectrometer (PerSeptive Biosystems, Foster City, CA, USA) operated in the delayed extraction (DE) reflectron mode at an accelerating voltage of 20 kV, a grid voltage of 57%, a mirror voltage ratio of 1.07, and an extraction delay time of 150 ns. The sample was irradiated with the nitrogen laser (k = 337 nm, pulse width = 4 ns, laser intensity = 2211–2600 W/cm2). The instrument was scanned in the mass-to-charge (m/z) range of 200 to 2000. The mass scale was calibrated via an external calibration procedure using the masses of the matrix dimer, AII, and AI (m/z 379.09, 1046.53, and 1296.68, respectively). The final spectra are averages of typically 200 laser shots. ESI–MS and ESI–MS/MS analyses were performed on a Finnigan LCQDeca (ThermoQuest, San Jose, CA, USA) ion trap (IT) mass analyzer that was equipped with a nanoESI source. The samples were injected via direct infusion at a rate of 0.5 ll/min. The spray voltage was maintained at 2 kV, and the capillary temperature was set at 150 °C. The ESI–MS spectra were acquired in the centroid mode by
Preparation of phospholipid–vesicle complexes
Chemical modification and ladder sequencing Acetylation An aliquot (20 ll) of the peptide–vesicle complex was centrifuged. The resulting pellet was dissolved in 20 ll of ammonium bicarbonate at pH 8.0 and treated overnight
Structure of angiotensin II and angiotensin I / P.R. Jalili, C. Dass / Anal. Biochem. 374 (2008) 346–357
scanning the instrument in the m/z range of 200 to 2000. For the MS/MS experiments, the precursor ions were mass-selected using a 2-u mass window. The collision gas was helium, and the relative collision energy was set at 25 to 45% of the maximum. The MS/MS spectra were also obtained in the centroid mode in the m/z scan range of 200 to 1500. To minimize the back exchange, the deuterated solvent was flushed for 30 min prior to analysis. Also, the ion source was isolated from the ambient atmosphere, and the sheath nitrogen gas with a pressure of 10 psi was flushed through the ion source during the HDX experiments. A previous study in our laboratory demonstrated that the back exchange under these conditions is less than 6% [34]. The backbone amide hydrogens and the ones that are involved in secondary structural elements have a slow exchange rate. Therefore, prior flushing of the ion source with the deuterated solvent is less likely to enhance deuterium incorporation during the short analysis time (1–30 s) used in these experiments. Calculations of number of hydrogens exchanged The number of exchanged hydrogens (Hx) in the peptide at several time points can be directly calculated from the MS data by observing the m/z ratio of the most abundant isotopic peak of the nth charge state and using the following expression [23]: H x ¼ M D M ¼ n½ðm=zÞD Dþ M;
ð1Þ
where M and MD are the molecular mass of the nondeuterated species and the measured molecular mass of the partially deuterated species, respectively. The deuterium (D) content for each data point is defined as D ¼ H x =Ht:100%;
ð2Þ
where Ht is the total number of exchangeable hydrogens (i.e., the sum of the number of exchangeable hydrogens on the side chains, backbones, and N and C termini). The information about the number of exchanged hydrogens in the MS/MS spectra is derived from the m/z values of b and y ions and can be calculated using the following expressions [25]:
349
nþ nþ bnþ j : H x ¼ n½M D ðbj Þ Mðbj Þ
ð3Þ
y nþ j
ð4Þ
: Hx ¼
n½M D ðy nþ j Þ
Mðy nþ j Þ
1 1:
Results and discussion Conformational analysis of AII It has been hypothesized that the lipid phase of a target cell membrane may facilitate the accumulation of the hormone near the receptor-binding site and, even more important, may induce the correct folding of the peptide prior to its interaction with the receptor [35,36]. In addition, the AII receptor-binding site is located at or near the epithetical plasma membrane surface, and lipid-induced peptide folding is important in the peptide–receptor interaction [9,12,35,36]. Therefore, the structure of AII was explored in phospholipid vesicles, which are known to resemble more closely the physical properties of the receptor-binding site. Hydrogen–deuterium exchange at various time points was studied for free AII in D2O and phospholipid vesicle-bound AII using ESI–IT MS. The time-resolved data are summarized in Table 1 and plotted in Fig. 2. The upper curve is the HDX profile of AII in D2O, and the lower curve shows corresponding data for the vesicle-bound AII. Two distinct HDX rates are clearly discernible in the two media; AII has lower deuterium content in lipid vesicles. For example, 9 hydrogens were exchanged in 30 min in this medium, as compared with 16 hydrogens in D2O, suggesting that AII has different structures in these media. The extent of HDX in the phospholipid vesicle-bound AII was determined using both MALDI–TOF MS and ESI–IT MS. Fig. 3A contains the MALDI mass spectrum of the AII peptide–vesicle complex. The corresponding spectrum of the deuterated analog after 1 min of HDX is presented in Fig. 3B. The signals due to the monomer and dimer ions of DMPC and the [M + H]+ or [M + D]+ ions of AII are clearly visible. A shift in the m/z of [M + H]+ ions from 1046.52 (Fig. 3A) to 1056.92 (Fig. 3B) on deuteration shows that 9 hydrogens are
Table 1 Number of hydrogens exchanged and deuterium content of AII and AI at different time points Time (min)
0.16 0.3 0.5 1 2 10 20 30
AII
AI
Number of hydrogens exchanged
Deuterium content (%)
Number of hydrogens exchanged
Deuterium content (%)
Water
Vesicles
Water
Vesicles
Water
Vesicles
Water
Vesicles
8 10 10 15 15 15 15 16
4 6 8 9 9 9 9 9
50 62 62 93 93 93 93 100
25 37 50 56 56 56 56 56
5 5 7 16 16 16 16 16
4 4 5 10 10 10 10 10
26 26 36 84 84 84 84 84
21 21 26 52 52 52 52 52
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Fig. 2. Deuterium content in D2O (upper curve) and phospholipid vesicle-bound (A) AII (lower curve) and (B) AI as a function of incubation time.
Fig. 3. MALDI mass spectra of AII (A,B) and AI (C,D) incorporated into phospholipid vesicles before deuteration (A,C) and after deuteration (B,D).
exchanged. A similar conclusion is also reached with the ESI–MS data. In the zoom scan ESI mass spectra acquired after 1 min of isotope exchange, the miz of the doubly charged AII ion shifts from 524.3 (in water [Fig. 4A]) to 529.8 (in vesicles [Fig. 4C]), indicating that a total of 9 hydrogens are exchanged.
The MS/MS spectra of the doubly charged ions of nondeuterated AII and AII bound to phospholipid vesicles after 1 min of HDX in D2O are shown in Figs. 4B and D, respectively. The b and y ions are the prominent sequence-specific ions in these spectra. However, not all b and y ions are clearly discernible. The y2 and y72+ C-termi-
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Fig. 4. Zoom scan ESI–MS (A) and MS/MS (B) spectra of AII in water as well as zoom scan ESI–MS (C) and MS/MS (D) spectra of AII in phospholipid vesicles after 1 min HDX.
nal ions and the b4, b5, b6, b62+, and b72+ N-terminal ions are abundant and well resolved. The exact numbers of hydrogens exchanged in different segments of the peptide derived from the MS/MS data are presented in Table 2. The m/z of the y72+ ion shifts from 466.5 to 471.3, indicating that 7 hydrogens are free to exchange in the -Arg2Val3-Tyr4-Ile5-His6-Pro7-Phe8-OH segment. Because the total number of exchanged hydrogens in the intact peptide is 9, the remaining 2 hydrogens are ascribed to H2N-Asp1-.
Of the total 11 labile hydrogens in the b4 segment (H2NAsp1-Arg2-Val3-Tyr4-), 6 are exchanged (shift of m/z from 534.2 to 540.2), 2 of which have already been assigned to H2N-Asp1-. Therefore, the remaining 4 must be part of the -Arg2-Val3-Tyr4- segment. The number of exchanged hydrogens in the b5 ion (H2N-Asp1-Arg2-Val3-Tyr4-Ile5) is 7, implying that the Ile5 amide hydrogen undergoes isotope exchange. In the b6 ion (H2N-Asp1-Arg2-Val3-Tyr4Ile5-His6-), 9 hydrogens are exchanged, suggesting that
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Table 2 HDX–MS/MS of AII bound to phospholipid vesicles Ion
Theoretical m/z
m/z in vesicles
Hx in vesicles
y2 y72+ b4 b5 b6 b62+ b72+ RVYIH
263.0 466.5 534.2 647.3 784.3 392.7 441.3 669.3
266.1 471.3 540.2 654.4 793.4 397.5 446.3 677.4
1.0 6.6 6.0 7.0 9.0 10.0 10.0 8.0
Note. Hx, number of exchanged hydrogens.
both labile His6 hydrogens are susceptible to HDX and that none of the labile hydrogens in the -Pro7-Phe8-OH segment is exchanged. Thus, of the 7 unexchanged hydrogens in AII, 4 are involved in hydrogen bonding in the Arg2-Val3-Tyr4- segment, 1 is involved in the H2N-Asp1residue, and 2 are involved in the Phe8-OH moiety (Fig. 5A). The similar HDX expriment with AII in D2O reveals that only 1 hydrogen in the -Arg2-Val3-Tyr4- segment remains unexchanged (data not shown); thus, the structures of the peptide in the two media are different. Fig. 6A shows the MALDI mass spectrum of the products of the nitration reaction of free AII. The signal of the nitrated AII is observed at m/z 1091.47 (i.e., a shift of 45 u from the mass of the unreacted AII). Several other peaks are also observed due to successive loss of oxygen (NO3 ? NO2 ? NO ? NH2) from the nitrated molecular ion of AII. The vesicle-bound AII does not exhibit a mass shift (i.e., remains at m/z 1046.52) on reaction with the TNM reagent (not shown), suggesting that the Tyr4 side chain is either connected to the vesicles or involved in intramolecular hydrogen bonding. To identify whether the N-terminal amino group is free or bound to the lipid, acetylation was performed. Fig. 7A is the MALDI mass spectrum of the acetylated AII. Contrary to the expected increase of 42 u, the mass of the acetylated AII increased by 84 u (m/z 1130.37), pointing to the addition of two acetyl groups. It is interesting to note which
Fig. 6. MALDI mass spectra of nitrated free AII (A) and AI (B). The MALDI mass spectra of the vesicle-bound AI and AII on treatment with TNM do not show any peak shifts, implying that tyrosine is embedded in vesicles.
other residue, apart from the N-terminal amino group, is involved in this reaction. A literature search revealed that because of the resonance between Tyr4 and His6 aromatic residues, one of them becomes the target of acetylation and, depending on the environment, the acetyl group can translocate from one residue to the other [19]. To corroborate this finding, the MS/MS spectrum of the acetylated AII was acquired (not shown). It revealed that one acetyl group is attached to the N-terminal amino group and that another is attached to the Tyr4 residue. In contrast, the spectrum of the acetylated lipid-bound AII shows that only one acetyl group is attached to the peptide (Fig. 7B). From this observation, it is concluded that the N terminal is out-
Fig. 5. Pictorial depiction of the number of exchanged hydrogens on b and y fragments of vesicle-bound AII (A) and AI (B) derived from ESI–MS/MS data. The total labile hydrogens are given in parentheses, and the unexchanged hydrogens (U EX) are shown under the bars.
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Fig. 7. MALDI mass spectra of acetylated AII (A), vesicle-bound AII after acetylation (B), acetylated AI (C), and vesicle-bound AI after acetylation (D).
side the membrane and that Tyr4 is either embedded within the vesicles or involved in hydrogen bonding with the vesicles. Treatment of the vesicle-bound AII with carboxypeptidase Y results in cleavage of the Phe8 residue, indicating that this residue resides outside the vesicles (see Fig. 8A for the MALDI spectrum). However, no mass shift was observed in the MALDI mass spectrum of the leucine aminopeptidase treated AII (not shown), indicating that the Asp1-Arg2 peptide bond is resistant to the leucine aminopeptidase activity, perhaps due to the connectivity between these two residues proposed by other researchers [5,7,8]. According to them, the carboxylic group of Asp1 and the amino group of Arg2 are involved in hydrogen bonding, imparting resistance to the leucine aminopeptidase activity. Comparing the ESI and MALDI MS information obtained here with the proposals of other researchers, we can arrive at an optimal structure of AII. The active conformation of AII has long been debated between the U shape and the twisted extended structures. Carpenter and coworkers suggested that AII has a well-defined hairpin structure when in contact with phospholipid vesicles [5]. In this structure, the C and N termini approach to within 0.76 nm of each other. These researchers also provided evidence of the presence of three stable hydrogen bonds. The first hydrogen bond between the Phe8 amide group and the His6 carbonyl group results in the formation of an inverse c-turn centered on the Pro7 residue [5]. The other two hydrogen bonds involve the Asp1 side chain carbonyl group and the Arg2 side chain amino group. The Asp1 residue is flexible and does not interact with the micelles, but the His6 side chain and the Ile5 amide group interact with
the phospholipid head groups. Matsoukas and coworkers also suggested that p-stacking between the two termini of the peptide has a significant impact on its biological activity [15,17,18]. Mavromoustakos and coworkers reported that AII interacts with the phospholipid head groups up to the level of interface and does not enter deeper into phospholipid vesicles [37]. Infrared spectroscopy has provided clear evidence that there is very little interaction between AII and the zwitterionic lipid phosphatidylcholine [35]. UV resonance Raman and absorption studies show that AII adopts a b-turn-like folded structure in dodecylphosphocholine and that the side chains of Tyr4 and Phe8 residues reside in the hydrophobic environment [36]. The HDX experiments in the current study are consistent with some of these proposals. Because exchanged hydrogens are distributed throughout the peptide (e.g., in the Asp1, -Arg2-Val3-Tyr4-, Ile5, and His6 segments), it is unlikely that AII is buried entirely in the vesicles, a conclusion that supports the observations of Mavromoustakos and coworkers [37] and Surewicz and Mantsch [35]. Interaction of AII with the hydrophilic group of phospholipid vesicles is a strong possibility because of the presence of several hydrophilic residues such as Asp, Arg, and His. The nitration reaction suggests that the aromatic side chain of Tyr4 is buried into vesicles, whereas ladder sequencing shows that the Phe8 aromatic ring interacts with the surface of vesicles, consistent with the UV resonance Raman and absorption study that proposed that Tyr4 and Phe8 side chains of AII reside in the hydrophobic core of vesicles and that the remaining part of the peptide interacts with the surface of the vesicles [36]. The presence of unexchanged hydrogens in the NH2-Asp1 (1 hydrogen), -
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Fig. 8. MALDI mass spectra of vesicle-bound AII (A) and AI (B) after treatment with carboxypeptidase Y. In panel A, the peak at 899.48 is due to loss of Phe from AII. In panel B, the peaks at 1183.67 and 1046.66 represent the loss of Leu5 and His, respectively.
Arg2-Val3-Tyr4- (4 hydrogens), and Phe8 (2 hydrogens) moities is also consistant with this proposal. Thus, the proposal that AII has a U-shaped structure in the lipid environment is not unreasonable. Conformational analysis of AI A strategy similar to that discussed for AII was also employed to determine the structure of AI trapped in phospholipid vesicles. The MALDI mass spectrum shows that the m/z of the phospholipid-bound AI shifts from 1296.69 to 1307.68 (Figs. 3C and D), and the ESI mass spectrum of the lipid-bound AI indicates that the m/z of its doubly charged ion shifts from 648.8 to 654.9 on deuteration (Figs. 9A and C). AI has a total of 19 exchangeable hydrogens. Both of these ionization techniques reveal that a total of 10 hydrogens are exchanged. The MS/MS spectra of the [M + H]+ ions in AI and the corresponding deutrated ions are shown in Figs. 9B and D, respectively. The number of hydrogens exchanged in various segments of AI as deduced from these MS/MS spectra are summarized
in Table 3. In short, these results indicate that of the 9 unexchanged hydrogens in AI, 6 are involved in hydrogen bonding in the H2N-Asp1-Arg2-Val3-Tyr4- segment and 1 each is involved in the His6, His9, and Leu10 residues (see Fig. 5D). HDX of the free peptide in D2O shows that there are 3 unexchanged hydrogens (1 each in the H2N-Asp1Arg2-Val3-Tyr4- segment, His, and Phe). The MALDI mass spectrum (Fig. 6B) of the TNM-treated free AI indicates a mass shift of 45 u from 1296.68 to 1341.77 u, suggesting that the Tyr4 side chain is accessible to TNM in water. However, the lipid-bound peptide does not show a mass shift on reaction with TNM (spectrum not shown). Therefore, tyrosine is either bound to the vesicles or involved in intramolecular hydrogen bonding. Similar to AII, acetylation of the free AI (Fig. 7C) also shows a mass shift of 84 u in the MALDI mass spectrum, pointing to the addition of two acetyl groups; the MS/MS spectrum of the acetylated AI shows that acetylation occurs at the N-terminal amino group and at the Tyr4 residue (spectrum not shown). In contrast, only one residue can be acetylated in the lipid-bound AI (Fig. 7D). There-
Structure of angiotensin II and angiotensin I / P.R. Jalili, C. Dass / Anal. Biochem. 374 (2008) 346–357
355
Fig. 9. Zoom scan ESI–MS (A) and MS/MS (B) spectra of AI in water as well as zoom scan ESI–MS (C) and MS/MS (D) spectra of AI bound to vesicles after 1 min HDX.
fore, the N terminal of AI is outside of the membrane and Tyr4 is either embedded inside of the vesicles or involved in hydrogen bonding with the vesicles. Treatment of AI incorporated in DMPC vesicles with carboxypeptidase Y shows that both Leu10 (m/z 1183.67) and His9 (m/z 1046.66) can be cleaved, suggesting that these residues are outside of the vesicles (Fig. 8B). The cleavage stops at the Phe8 residue, implying that the aro-
matic ring of Phe8 is embedded in hydrophobic core of the membrane vesicles. Similar to AII, the N-terminal Asp1 remains unaffected on treatment of AI with aminopeptidase, confirming connectivity between Asp1 and Arg2 residues. From the HDX–MS/MS data, it can be stated that the structure of AI in phospholipid vesicles has some elements of a secondary structure. Both His residues in AI
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Table 3 Results of HDX–MS/MS of AI bound to phospholipid vesicles Ion
Theoretical m/z
m/z in vesicles
Hx in vesicles
y2 y4 b4 b5 b6 b7 b8 b92+
269.4 513.2 534.2 647.3 784.5 881.4 1028.5 583.7
272.2 517.4 539.4 653.3 791.4 888.5 1036.5 588.3
1.0 2.0 5.0 6.0 7.0 7.0 8.0 9.0
Note. Hx, number of exchanged hydrogens.
play an important role in interaction with the hydrophilic groups on the surface of the phospholipid vesicles. Similar to AII, the HDX data for AI fits with a U-shaped structure. The distribution of hydrogens exchanged on AI indicates that this peptide intracts with the surface of phospholipid vesicles and does not enter into the lipid environment completely. Also, the first four N-terminal residues H2N-Asp1-Arg2-Val3-Tyr4- of AI have a significant impact on stabilizing the conformation via hydrogen bonding. The nitration and ladder sequencing experiments suggest that the hydrophobic groups of Phe8 and Tyr4 are embedded in the hydrophobic core of the membrane. Conclusion An MS-based multipronged strategy that includes HDX, nitration of tyrosine, acetylation of free amino groups, ladder sequencing, and MS/MS can be applied successfully to determine the conformation of biologically active peptides in phospholipid vesicles. Because exchanged hydrogens are distributed throughout the peptide, AII interacts with the phospholipid head groups at the surface of the vesicles. The Tyr residue is embedded in the vesicles, whereas Phe is outside of the vesicles. AII has a U-shaped structure in the lipid environment. Similar conclusions were drawn for AI; that is, this peptide also interacts with the surface of phospholipid vesicles and does not enter into the lipid environment completely. Acknowledgment The MS data were acquired using MS instrumentation of the Charles B. Stout Neuroscience Mass Spectrometry Laboratory, Health Science Center, University of Tennessee. References [1] A.H. Barnett, The role of angiotensin II receptor antagonists in the management of diabetes, Blood Pressure 1 (2001) 21–26. [2] Q. Wang, E. Hummler, M. Maillard, J. Nussberger, B.C. Rossier, H.R. Brunner, M. Burnier, Compensatory up-regulation of angiotensin II subtype 1 receptors in (ENaC knockout heterozygous mice, Blood Pressure 59 (2001) 2216–2221.
[3] D. Greff, S. Fermandjian, P. Fromageot, M.C. Khosla, R.R. Smeby, F.M. Bumpus, Circular-dichroism spectra of truncated and other analogs of angiotensin II, Eur. J. Biochem. 61 (1976) 297–305. [4] N. D’Amelio, E. Gaggelli, N. Gaggelli, L. Lozzi, P. Neri, D. Valensin, G. Valensin, Interaction of angiotensin II with the C-terminal 300-320 fragment of the rat angiotensin II receptor AT1 as monitored by NMR, Biopolymers 70 (2003) 134–144. [5] K.A. Carpenter, B.C. Wilkes, P. Schiller, The octapeptide angiotensin II adopts a well-defined structure in a phospholipid environment, Eur. J. Biochem. 251 (1998) 448–453. [6] S. Zimmer, W. Haar, W. Maurer, H. Rueterjans, S. Fermandjian, P. Fromageot, Investigation of the structure of angiotensin II using carbon-13 NMR spectra, Eur. J. Biochem. 29 (1972) 80–87. [7] N. Zhou, G.J. Moore, H.J. Vogel, Proton NMR studies of angiotensin II and its analogs in aqueous solution, J. Protein Chem. 10 (1991) 333–343. [8] G.A. Spyroulias, A.S. Galanis, G. Pairas, E. Manessi-Zoupa, P. Cordopatis, Structural features of angiotensin I-converting enzyme catalytic sites: Conformational studies in solution, homology models and comparison with other zinc metallopeptidases, Curr. Top. Med. Chem. 4 (2004) 403–429. [9] J.S. Holtz, I.K. Lednev, S.A. Asher, UV resonance Raman study of angiotensin II conformation in nonaqueous environments: Lipid micelles and acetonitrile, Biopolymers 57 (2000) 55–63. [10] S. Fermandjian, P. Fromageot, A.M. Tistchenko, J.P. Leiknam, M. Lutz, Angiotensin II conformations: Infrared and Raman studies, Eur. J. Biochem. 28 (1972) 174–182. [11] M.P. Printz, G. Nemethy, H. Bleich, Proposed models for angiotensin II in aqueous solution and conclusions about receptor topography, Nat. New Biol. 237 (1972) 135–140. [12] A.G. Tzakos, A.M. Bonvin, A. Troganis, P. Cordopatis, M.L. Amzel, I.P. Gerothanassis, N.A. van Nuland, On the molecular basis of the recognition of angiotensin II (AII): NMR structure of AII in solution compared with the X-ray structure of AII bound to the mAb Fab131, Eur. J. Biochem. 270 (2003) 849–860. [13] O. Collet, S. Premilat, Monte-Carlo calculations of the solvent effects on the conformation of angiotensin II, Intl. J. Pept. Protein Res. 47 (1996) 239–244. [14] M.P. Printz, H.P. William, L.C. Craig, Evidence for the presence of hydrogen-bonded secondary structure in angiotensin II in aqueous solution, Proc. Natl. Acad. Sci. USA 69 (1972) 378–382. [15] J.M. Matsoukas, J. Hondrelis, M. Keramida, T. Mavromoustakos, A. Makriyannis, R. Yamdagni, Q. Wu, G.J. Moore, Role of the NH2 -terminal domain of angiotensin II (ANG II) and [Sar1]angiotensin II on conformation and activity NMR evidence for aromatic ring clustering and peptide backbone folding compared with [des1,2,3]angiotensin II, J. Biol. Chem. 269 (1994) 5303–5312. [16] H.E. Bleich, R.J. Freer, S.S. Stafford, R.E. Galardy, Correlation of the biological activity and solution conformation of [Asp1,Ile5] and [Phe4,Tyr8] angiotensin II, Proc. Natl. Acad. Sci. USA 75 (1978) 3630–3634. [17] J.M. Matsoukas, G.J. Moore, NMR studies on angiotensin II: Histidine and phenylalanine ring stacking and biological activity, Biochem. Biophys. Res. Commun. 122 (1984) 419–423. [18] J.M. Matsoukas, G. Bigam, N. Zhou, G.J. Moore, 1 H-NMR studies of [Sar1]angiotensin II conformation by nuclear Overhauser effect spectroscopy in the rotating frame (ROESY): Clustering of the aromatic rings in dimethylsulfoxide, Peptides 11 (1990) 359–366. [19] G.J. Moore, Kinetics of acetylation–deacetylation of angiotensin II: Intramolecular interactions of the tyrosine and histidine side-chains, Intl. J. Pept. Protein Res. 26 (1985) 469–481. [20] G.A. Spyroulias, P. Nikolakopoulou, A. Tzakos, I.P. Gerothanassis, V. Magafa, E. Manessi-Zoupa, P. Cordopatis, Comparison of the solution structures of angiotensin I and II: Implication for structure– function relationship, Eur. J. Biochem. 270 (2003) 2163–2173. [21] V. Katta, B.T. Chait, Conformational changes in proteins probed by hydrogen-exchange electrospray-ionization mass spectrometry, Rapid Commun. Mass Spectrom. 5 (1991) 214–217.
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ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 374 (2008) 346–357 www.elsevier.com/locate/yabio
Determination of the structure of lipid vesicle-bound angiotensin II and angiotensin I Pegah R. Jalili, Chhabil Dass * Department of Chemistry, University of Memphis, Memphis, TN 38152, USA Received 21 September 2007 Available online 4 December 2007
Abstract A mass spectrometry (MS)-based strategy was developed to determine the structure of lipid vesicle-bound angiotensin II (AII) and angiotensin I (AI). It involves hydrogen–deuterium exchange (HDX), chemical modifications (e.g., nitration of tyrosine, acetylation of free amino group), and ladder sequencing. HDX is also combined with tandem mass spectrometry (MS/MS) to provide structural details at individual amino acid residues. It was observed that a major portion of both of these peptide hormones interacts with the phospholipid head groups on the surface of the vesicles and that Tyr residue is embedded in the vesicles. Both peptides have a U-shaped structure in the lipid environment. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Hydrogen–deuterium exchange; Mass spectrometry; Angiotensin II; Angiotensin I; Conformation; Peptides
Angiotensin II (AII1, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) and angiotensin I (AI, Asp-Arg-Val-Tyr-Ile-His-Pro-PheHis-Leu) are endocrine hormone peptides that are synthesized locally in the pituitary gland and brain to form the renin–angiotensin system. AI, an inactive peptide, is produced when the liver protein, angiotensinogen, is cleaved with renin protease. AI is further converted by angiotensin-converting enzyme (ACE) to the bioactive octapeptide AII. These two peptides have identical N-terminal sequences but differ at the C terminal. AII is vital to regu-
*
Corresponding author. Fax: +1 901 678 3447. E-mail address: [email protected] (C. Dass). 1 Abbreviations used: AII, angiotensin II; AI, angiotensin I; ACE, angiotensin-converting enzyme; CNS, central nervous system; AT1 receptor, guanosine nucleotide protein-coupled receptor; CD, circular dichroism; NMR, nuclear magnetic resonance; HDX, hydrogen–deuterium exchange; MS, mass spectrometry; ESI, electrospray ionization; MALDI, matrix-assisted laser desorption/ionization; MS/MS, tandem mass spectrometry; DMPC, dimyristoyl phosphatidylcholine; OG, n-octyl-b-Dglycopyranoside; a-CHCA, a-cyano-4-hydroxycinnamic acid; TFA, trifluoroacetic acid; TOF, time-of-flight; TNM, tetranitromethane; DE, delayed extraction; m/z, mass-to-charge; IT, ion trap; HDX, hydrogen– deuterium exchange. 0003-2697/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2007.11.038
lating cardiovascular functions, fluid homeostasis, adrenal aldosterone secretion, and the release of pituitary hormones. It also acts on the central nervous system (CNS) receptors, such as the guanosine nucleotide protein-coupled (AT1) receptor, to regulate the blood pressure [1,2]. Excessive production of AII causes inadequate function of the renin–angiotensin system, leading to hypertension, heart failure, and renal diseases. AII receptor blockers and ACE inhibitors are used for treatment of these diseases. To design effective drugs such as the AII receptor blockers, it is imperative to have knowledge of the higher order structure of AII in the receptor-binding domain. Although the conformation that AII adopts at the receptor-binding site is decided by the geometry of this domain, determination of the conformation of the AT1 receptor at high resolution is difficult. Therefore, a practical alternative is to study the active structure of the peptide in a medium that mimics the natural environment of the receptor. Because of its biological activity and pharmaceutical interest, the structure of AII has been studied by a variety of analytical approaches, such as circular dichroism (CD) [3,4], nuclear magnetic resonance (NMR) [4–8], UV resonance Raman spectroscopy techniques [9,10], X-ray crys-
Structure of angiotensin II and angiotensin I / P.R. Jalili, C. Dass / Anal. Biochem. 374 (2008) 346–357
tallography [11,12], and conformational calculations [12,13], yet no consensus has emerged. Various AII structures that have been proposed include a-helix [7], b-sheet [4], b-turn [9], c-turn [12], and random coil [14]. Several structure–activity studies have revealed the topological contribution of the Tyr4, His6, and Phe8 aromatic residues and the C-terminal carboxyl group on the biological activity of AII [12,15–17]. It has also been documented that pstacking in Tyr4 and His6 allows a charge transfer process between the two peptide termini of AII and plays an important role in its biological activity [18,19]. A recent study by Spyroulias and coworkers revealed that both AI and AII assume an S-shaped structure in organic solvents and a U-shaped structure in the receptor-binding site [20]. In the current study, we report the conformational states of AII and AI in lipid vesicles (liposomes). This medium mimics the receptor environment more accurately than do the other commonly used membrane-mimetic media such as detergent micelles and organic solvents (e.g., methanol, 1-chloroethanol, trifluoroethanol). We present here, for the first time, a multipronged strategy, shown in Fig. 1, to determine the membrane topology of peptides in lipid vesicles. In this strategy, hydrogen–deuterium exchange (HDX), chemical modifications (nitration of tyrosine and acetylation of free amino group), and ladder sequencing are combined with mass spectrometry (MS) to provide detailed structural information about these peptide hormones. HDX is a well-known method for studying the conformational states and folding–unfolding mechanisms of peptides and proteins in solution [21–29]. HDX relies on the fact that a region of the molecule that is involved in noncovalent interactions and has low accessibility to the solvent exhibits slow HDX rates, whereas a highly exposed region undergoes rapid exchange. The mass of the target peptide increases by 1 u for each hydrogen exchanged with deuterium, and the resulting mass changes can be measured conveniently with MS. Electrospray ionization (ESI)–MS and matrix-assisted laser desorption/ionization (MALDI)–MS
347
are commonly used for this purpose. The combination of HDX with tandem mass spectrometry (MS/MS) yields even more structure-specific information about the conformation of peptides. The product ions in MS/MS spectra can provide knowledge of the amino acid sequence of the target peptide. The deuterium-labeled amino acid residues will shift to higher masses in the mass spectrum than will the nondeuterated residues, thereby providing information about individual amino acids that are involved in interaction with the membrane or are shielded by the solvent. Nitration of the peptide–vesicle complex is proposed to determine the topology of Tyr residues. Although tyrosine has a significant nonpolar character owing to the aromatic ring, its phenolic hydroxyl group (pKa = 10.1) acts as a charged polar entity at high pH. Because of these special characteristics, in most cases tyrosine is the site of interaction in enzyme–receptor reactions. Many structure–activity studies support tyrosine as being important for activity of AII [18–20]. Tyrosine can interact with the hydrophobic core of the receptor membrane via hydrogen bonding and/or hydrophobic interaction. In the current study, nitration was performed to determine whether tyrosine is buried in phospholipid vesicles. When the lipid-bound peptide is reacted with tetranitromethane (TNM) under moderate alkaline conditions, only the free tyrosine will be converted to 3-nitrotyrosine, increasing its mass by 45 u (Scheme 1) [30]. Acetylation will be performed by reacting the peptide-vesicle complex with acetic anhydride. This reaction converts the free amino group to the acetylated form, increasing the mass by 42 u (Scheme 2) [31]. We also propose using ladder sequencing to determine whether the C- and N-terminal residues of the peptide are buried in the membrane’s hydrophobic core or are outside. Traditionally, this method has played an important role in determining the amino acid sequence of peptides. The N- and C-terminal ladders are generated by reacting the target peptide with leucine aminopeptidase and carboxypeptidase Y, respectively [32,33]. Sequential cleavage of the peptide with each of these exopeptidases produces a group of fragments, the molecular mass of which differs from one another by the mass of the cleaved amino acid residue. The amino acid sequence of the peptide can be determined from the measured mass differences between a series of molecular ion peaks in the spectrum [32,33]. In the peptide–vesicle complex, the terminal amino acids that are outside of the vesicle will be cleaved sequentially to produce smaller fragments. Cleavage will stop at the site of an amino acid that is involved in any kind of interaction. The knowledge gained in the current study will facilitate understanding of biological processes, hormone–receptor interactions, and receptor-binding mechanisms. Materials and methods
Fig. 1. Experimental protocol for the study of conformation of bioactive peptides in phospholipid vesicles.
AI, AII, dimyristoyl phosphatidylcholine (DMPC), noctyl-b-d-glycopyranoside (OG), a-cyano-4-hydroxycinnamic acid (a-CHCA), anhydrous acetonitrile, tetrani-
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Structure of angiotensin II and angiotensin I / P.R. Jalili, C. Dass / Anal. Biochem. 374 (2008) 346–357
Scheme 1. Nitration of tyrosine.
with 2 ll of 20% acetic anhydride in acetonitrile. The resulting solution was centrifuged, and the pellet was dissolved in water and analyzed with MALDI–TOF MS.
Scheme 2. Acetylation of free amino group.
tromethane, leucine aminopeptidase, and carboxypeptidase Y were obtained from Sigma (St. Louis, MO, USA). D2O (99.98%) was obtained from Aldrich (Milwaukee, WI, USA).
Nitration The pellet obtained after centrifuging 20 ll of the peptide–vesicle complex was dissolved in Tris buffer at pH 8.0 and treated with 0.5 ll of the nitration reagent (0.1 M tetranitromethane in ethanol) for 3 h. The resulting solution was centrifuged, and the pellet was dissolved in water and analyzed with MALDI–TOF MS.
The unilamellar DMPC peptide–vesicle complex was prepared by dissolving 50 ll of 1 nmol of each peptide in 0.5 ml of a solution that contained 5% (w/v) OG and 1.25% (w/v) DMPC. Solubilization of the peptides was aided by sonication in a water bath for 15 min at 38 °C. A small amount of water (0.5 ml) was added to this mixture, and the contents were incubated for 5 min at 38 °C. Dialysis (cellulose ester DispoDialyzer, 1 kDa) for 24 h at 38 °C against water removed OG detergent to produce large-size unilamellar vesicles. The resulting mixture was dialyzed again at 38 °C (cellulose ester DispoDialyzer, 15 kDa) to remove free peptides present in the solution.
Ladder sequencing A 2-ll portion of carboxypeptidase Y or leucine aminopeptidase was added to a 10-ll solution of the peptide–vesicle complex. The final concentration of endopeptidases was 10 pmol/ll in each solution. Several 1-ll aliquots were removed from the reaction vial at different reaction times for analysis by MALDI–TOF MS. For MALDI–TOF MS analysis, the solutions from the nitration, acetylation, and ladder sequencing experiments were mixed 1:1 with the matrix (10 mg/ml a-CHCA in 50% acetonitrile in 0.1% TFA). As controls, solutions of free peptides were reacted with acetylation, nitration, and ladder sequencing reagents and were analyzed in the same way.
Hydrogen–deuterium exchange
MS analysis
A 20-ll portion of the peptide–vesicle solution was centrifuged in a benchtop centrifuge at 10,000 rpm. HDX was initiated by dissolving the resulting pellet that contained the peptide–vesicle complex in 20 ll of D2O. The exchange was quenched after defined time points with 2% formic acid in D2O at pH 2.5 and was monitored within 1 min using nanoESI–MS and nanoESI–MS/MS. For MALDI–MS analysis, the pellet was dissolved in 20 ll of D2O. After 1 min, the exchange was quenched by adding in a 1:1 ratio the MALDI matrix solution (10 mg a-CHCA in 1 ml of 50% acetonitrile in 0.1% trifluoroacetic acid [TFA]/D2O) at pH 2.5 and 0 °C. The resulting solution was promptly dried in a desiccator and analyzed using MALDI–timeof-flight (TOF) MS.
The MALDI mass spectra were obtained on a Voyager DE-RP MALDI–TOF mass spectrometer (PerSeptive Biosystems, Foster City, CA, USA) operated in the delayed extraction (DE) reflectron mode at an accelerating voltage of 20 kV, a grid voltage of 57%, a mirror voltage ratio of 1.07, and an extraction delay time of 150 ns. The sample was irradiated with the nitrogen laser (k = 337 nm, pulse width = 4 ns, laser intensity = 2211–2600 W/cm2). The instrument was scanned in the mass-to-charge (m/z) range of 200 to 2000. The mass scale was calibrated via an external calibration procedure using the masses of the matrix dimer, AII, and AI (m/z 379.09, 1046.53, and 1296.68, respectively). The final spectra are averages of typically 200 laser shots. ESI–MS and ESI–MS/MS analyses were performed on a Finnigan LCQDeca (ThermoQuest, San Jose, CA, USA) ion trap (IT) mass analyzer that was equipped with a nanoESI source. The samples were injected via direct infusion at a rate of 0.5 ll/min. The spray voltage was maintained at 2 kV, and the capillary temperature was set at 150 °C. The ESI–MS spectra were acquired in the centroid mode by
Preparation of phospholipid–vesicle complexes
Chemical modification and ladder sequencing Acetylation An aliquot (20 ll) of the peptide–vesicle complex was centrifuged. The resulting pellet was dissolved in 20 ll of ammonium bicarbonate at pH 8.0 and treated overnight
Structure of angiotensin II and angiotensin I / P.R. Jalili, C. Dass / Anal. Biochem. 374 (2008) 346–357
scanning the instrument in the m/z range of 200 to 2000. For the MS/MS experiments, the precursor ions were mass-selected using a 2-u mass window. The collision gas was helium, and the relative collision energy was set at 25 to 45% of the maximum. The MS/MS spectra were also obtained in the centroid mode in the m/z scan range of 200 to 1500. To minimize the back exchange, the deuterated solvent was flushed for 30 min prior to analysis. Also, the ion source was isolated from the ambient atmosphere, and the sheath nitrogen gas with a pressure of 10 psi was flushed through the ion source during the HDX experiments. A previous study in our laboratory demonstrated that the back exchange under these conditions is less than 6% [34]. The backbone amide hydrogens and the ones that are involved in secondary structural elements have a slow exchange rate. Therefore, prior flushing of the ion source with the deuterated solvent is less likely to enhance deuterium incorporation during the short analysis time (1–30 s) used in these experiments. Calculations of number of hydrogens exchanged The number of exchanged hydrogens (Hx) in the peptide at several time points can be directly calculated from the MS data by observing the m/z ratio of the most abundant isotopic peak of the nth charge state and using the following expression [23]: H x ¼ M D M ¼ n½ðm=zÞD Dþ M;
ð1Þ
where M and MD are the molecular mass of the nondeuterated species and the measured molecular mass of the partially deuterated species, respectively. The deuterium (D) content for each data point is defined as D ¼ H x =Ht:100%;
ð2Þ
where Ht is the total number of exchangeable hydrogens (i.e., the sum of the number of exchangeable hydrogens on the side chains, backbones, and N and C termini). The information about the number of exchanged hydrogens in the MS/MS spectra is derived from the m/z values of b and y ions and can be calculated using the following expressions [25]:
349
nþ nþ bnþ j : H x ¼ n½M D ðbj Þ Mðbj Þ
ð3Þ
y nþ j
ð4Þ
: Hx ¼
n½M D ðy nþ j Þ
Mðy nþ j Þ
1 1:
Results and discussion Conformational analysis of AII It has been hypothesized that the lipid phase of a target cell membrane may facilitate the accumulation of the hormone near the receptor-binding site and, even more important, may induce the correct folding of the peptide prior to its interaction with the receptor [35,36]. In addition, the AII receptor-binding site is located at or near the epithetical plasma membrane surface, and lipid-induced peptide folding is important in the peptide–receptor interaction [9,12,35,36]. Therefore, the structure of AII was explored in phospholipid vesicles, which are known to resemble more closely the physical properties of the receptor-binding site. Hydrogen–deuterium exchange at various time points was studied for free AII in D2O and phospholipid vesicle-bound AII using ESI–IT MS. The time-resolved data are summarized in Table 1 and plotted in Fig. 2. The upper curve is the HDX profile of AII in D2O, and the lower curve shows corresponding data for the vesicle-bound AII. Two distinct HDX rates are clearly discernible in the two media; AII has lower deuterium content in lipid vesicles. For example, 9 hydrogens were exchanged in 30 min in this medium, as compared with 16 hydrogens in D2O, suggesting that AII has different structures in these media. The extent of HDX in the phospholipid vesicle-bound AII was determined using both MALDI–TOF MS and ESI–IT MS. Fig. 3A contains the MALDI mass spectrum of the AII peptide–vesicle complex. The corresponding spectrum of the deuterated analog after 1 min of HDX is presented in Fig. 3B. The signals due to the monomer and dimer ions of DMPC and the [M + H]+ or [M + D]+ ions of AII are clearly visible. A shift in the m/z of [M + H]+ ions from 1046.52 (Fig. 3A) to 1056.92 (Fig. 3B) on deuteration shows that 9 hydrogens are
Table 1 Number of hydrogens exchanged and deuterium content of AII and AI at different time points Time (min)
0.16 0.3 0.5 1 2 10 20 30
AII
AI
Number of hydrogens exchanged
Deuterium content (%)
Number of hydrogens exchanged
Deuterium content (%)
Water
Vesicles
Water
Vesicles
Water
Vesicles
Water
Vesicles
8 10 10 15 15 15 15 16
4 6 8 9 9 9 9 9
50 62 62 93 93 93 93 100
25 37 50 56 56 56 56 56
5 5 7 16 16 16 16 16
4 4 5 10 10 10 10 10
26 26 36 84 84 84 84 84
21 21 26 52 52 52 52 52
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Fig. 2. Deuterium content in D2O (upper curve) and phospholipid vesicle-bound (A) AII (lower curve) and (B) AI as a function of incubation time.
Fig. 3. MALDI mass spectra of AII (A,B) and AI (C,D) incorporated into phospholipid vesicles before deuteration (A,C) and after deuteration (B,D).
exchanged. A similar conclusion is also reached with the ESI–MS data. In the zoom scan ESI mass spectra acquired after 1 min of isotope exchange, the miz of the doubly charged AII ion shifts from 524.3 (in water [Fig. 4A]) to 529.8 (in vesicles [Fig. 4C]), indicating that a total of 9 hydrogens are exchanged.
The MS/MS spectra of the doubly charged ions of nondeuterated AII and AII bound to phospholipid vesicles after 1 min of HDX in D2O are shown in Figs. 4B and D, respectively. The b and y ions are the prominent sequence-specific ions in these spectra. However, not all b and y ions are clearly discernible. The y2 and y72+ C-termi-
Structure of angiotensin II and angiotensin I / P.R. Jalili, C. Dass / Anal. Biochem. 374 (2008) 346–357
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Fig. 4. Zoom scan ESI–MS (A) and MS/MS (B) spectra of AII in water as well as zoom scan ESI–MS (C) and MS/MS (D) spectra of AII in phospholipid vesicles after 1 min HDX.
nal ions and the b4, b5, b6, b62+, and b72+ N-terminal ions are abundant and well resolved. The exact numbers of hydrogens exchanged in different segments of the peptide derived from the MS/MS data are presented in Table 2. The m/z of the y72+ ion shifts from 466.5 to 471.3, indicating that 7 hydrogens are free to exchange in the -Arg2Val3-Tyr4-Ile5-His6-Pro7-Phe8-OH segment. Because the total number of exchanged hydrogens in the intact peptide is 9, the remaining 2 hydrogens are ascribed to H2N-Asp1-.
Of the total 11 labile hydrogens in the b4 segment (H2NAsp1-Arg2-Val3-Tyr4-), 6 are exchanged (shift of m/z from 534.2 to 540.2), 2 of which have already been assigned to H2N-Asp1-. Therefore, the remaining 4 must be part of the -Arg2-Val3-Tyr4- segment. The number of exchanged hydrogens in the b5 ion (H2N-Asp1-Arg2-Val3-Tyr4-Ile5) is 7, implying that the Ile5 amide hydrogen undergoes isotope exchange. In the b6 ion (H2N-Asp1-Arg2-Val3-Tyr4Ile5-His6-), 9 hydrogens are exchanged, suggesting that
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Table 2 HDX–MS/MS of AII bound to phospholipid vesicles Ion
Theoretical m/z
m/z in vesicles
Hx in vesicles
y2 y72+ b4 b5 b6 b62+ b72+ RVYIH
263.0 466.5 534.2 647.3 784.3 392.7 441.3 669.3
266.1 471.3 540.2 654.4 793.4 397.5 446.3 677.4
1.0 6.6 6.0 7.0 9.0 10.0 10.0 8.0
Note. Hx, number of exchanged hydrogens.
both labile His6 hydrogens are susceptible to HDX and that none of the labile hydrogens in the -Pro7-Phe8-OH segment is exchanged. Thus, of the 7 unexchanged hydrogens in AII, 4 are involved in hydrogen bonding in the Arg2-Val3-Tyr4- segment, 1 is involved in the H2N-Asp1residue, and 2 are involved in the Phe8-OH moiety (Fig. 5A). The similar HDX expriment with AII in D2O reveals that only 1 hydrogen in the -Arg2-Val3-Tyr4- segment remains unexchanged (data not shown); thus, the structures of the peptide in the two media are different. Fig. 6A shows the MALDI mass spectrum of the products of the nitration reaction of free AII. The signal of the nitrated AII is observed at m/z 1091.47 (i.e., a shift of 45 u from the mass of the unreacted AII). Several other peaks are also observed due to successive loss of oxygen (NO3 ? NO2 ? NO ? NH2) from the nitrated molecular ion of AII. The vesicle-bound AII does not exhibit a mass shift (i.e., remains at m/z 1046.52) on reaction with the TNM reagent (not shown), suggesting that the Tyr4 side chain is either connected to the vesicles or involved in intramolecular hydrogen bonding. To identify whether the N-terminal amino group is free or bound to the lipid, acetylation was performed. Fig. 7A is the MALDI mass spectrum of the acetylated AII. Contrary to the expected increase of 42 u, the mass of the acetylated AII increased by 84 u (m/z 1130.37), pointing to the addition of two acetyl groups. It is interesting to note which
Fig. 6. MALDI mass spectra of nitrated free AII (A) and AI (B). The MALDI mass spectra of the vesicle-bound AI and AII on treatment with TNM do not show any peak shifts, implying that tyrosine is embedded in vesicles.
other residue, apart from the N-terminal amino group, is involved in this reaction. A literature search revealed that because of the resonance between Tyr4 and His6 aromatic residues, one of them becomes the target of acetylation and, depending on the environment, the acetyl group can translocate from one residue to the other [19]. To corroborate this finding, the MS/MS spectrum of the acetylated AII was acquired (not shown). It revealed that one acetyl group is attached to the N-terminal amino group and that another is attached to the Tyr4 residue. In contrast, the spectrum of the acetylated lipid-bound AII shows that only one acetyl group is attached to the peptide (Fig. 7B). From this observation, it is concluded that the N terminal is out-
Fig. 5. Pictorial depiction of the number of exchanged hydrogens on b and y fragments of vesicle-bound AII (A) and AI (B) derived from ESI–MS/MS data. The total labile hydrogens are given in parentheses, and the unexchanged hydrogens (U EX) are shown under the bars.
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Fig. 7. MALDI mass spectra of acetylated AII (A), vesicle-bound AII after acetylation (B), acetylated AI (C), and vesicle-bound AI after acetylation (D).
side the membrane and that Tyr4 is either embedded within the vesicles or involved in hydrogen bonding with the vesicles. Treatment of the vesicle-bound AII with carboxypeptidase Y results in cleavage of the Phe8 residue, indicating that this residue resides outside the vesicles (see Fig. 8A for the MALDI spectrum). However, no mass shift was observed in the MALDI mass spectrum of the leucine aminopeptidase treated AII (not shown), indicating that the Asp1-Arg2 peptide bond is resistant to the leucine aminopeptidase activity, perhaps due to the connectivity between these two residues proposed by other researchers [5,7,8]. According to them, the carboxylic group of Asp1 and the amino group of Arg2 are involved in hydrogen bonding, imparting resistance to the leucine aminopeptidase activity. Comparing the ESI and MALDI MS information obtained here with the proposals of other researchers, we can arrive at an optimal structure of AII. The active conformation of AII has long been debated between the U shape and the twisted extended structures. Carpenter and coworkers suggested that AII has a well-defined hairpin structure when in contact with phospholipid vesicles [5]. In this structure, the C and N termini approach to within 0.76 nm of each other. These researchers also provided evidence of the presence of three stable hydrogen bonds. The first hydrogen bond between the Phe8 amide group and the His6 carbonyl group results in the formation of an inverse c-turn centered on the Pro7 residue [5]. The other two hydrogen bonds involve the Asp1 side chain carbonyl group and the Arg2 side chain amino group. The Asp1 residue is flexible and does not interact with the micelles, but the His6 side chain and the Ile5 amide group interact with
the phospholipid head groups. Matsoukas and coworkers also suggested that p-stacking between the two termini of the peptide has a significant impact on its biological activity [15,17,18]. Mavromoustakos and coworkers reported that AII interacts with the phospholipid head groups up to the level of interface and does not enter deeper into phospholipid vesicles [37]. Infrared spectroscopy has provided clear evidence that there is very little interaction between AII and the zwitterionic lipid phosphatidylcholine [35]. UV resonance Raman and absorption studies show that AII adopts a b-turn-like folded structure in dodecylphosphocholine and that the side chains of Tyr4 and Phe8 residues reside in the hydrophobic environment [36]. The HDX experiments in the current study are consistent with some of these proposals. Because exchanged hydrogens are distributed throughout the peptide (e.g., in the Asp1, -Arg2-Val3-Tyr4-, Ile5, and His6 segments), it is unlikely that AII is buried entirely in the vesicles, a conclusion that supports the observations of Mavromoustakos and coworkers [37] and Surewicz and Mantsch [35]. Interaction of AII with the hydrophilic group of phospholipid vesicles is a strong possibility because of the presence of several hydrophilic residues such as Asp, Arg, and His. The nitration reaction suggests that the aromatic side chain of Tyr4 is buried into vesicles, whereas ladder sequencing shows that the Phe8 aromatic ring interacts with the surface of vesicles, consistent with the UV resonance Raman and absorption study that proposed that Tyr4 and Phe8 side chains of AII reside in the hydrophobic core of vesicles and that the remaining part of the peptide interacts with the surface of the vesicles [36]. The presence of unexchanged hydrogens in the NH2-Asp1 (1 hydrogen), -
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Fig. 8. MALDI mass spectra of vesicle-bound AII (A) and AI (B) after treatment with carboxypeptidase Y. In panel A, the peak at 899.48 is due to loss of Phe from AII. In panel B, the peaks at 1183.67 and 1046.66 represent the loss of Leu5 and His, respectively.
Arg2-Val3-Tyr4- (4 hydrogens), and Phe8 (2 hydrogens) moities is also consistant with this proposal. Thus, the proposal that AII has a U-shaped structure in the lipid environment is not unreasonable. Conformational analysis of AI A strategy similar to that discussed for AII was also employed to determine the structure of AI trapped in phospholipid vesicles. The MALDI mass spectrum shows that the m/z of the phospholipid-bound AI shifts from 1296.69 to 1307.68 (Figs. 3C and D), and the ESI mass spectrum of the lipid-bound AI indicates that the m/z of its doubly charged ion shifts from 648.8 to 654.9 on deuteration (Figs. 9A and C). AI has a total of 19 exchangeable hydrogens. Both of these ionization techniques reveal that a total of 10 hydrogens are exchanged. The MS/MS spectra of the [M + H]+ ions in AI and the corresponding deutrated ions are shown in Figs. 9B and D, respectively. The number of hydrogens exchanged in various segments of AI as deduced from these MS/MS spectra are summarized
in Table 3. In short, these results indicate that of the 9 unexchanged hydrogens in AI, 6 are involved in hydrogen bonding in the H2N-Asp1-Arg2-Val3-Tyr4- segment and 1 each is involved in the His6, His9, and Leu10 residues (see Fig. 5D). HDX of the free peptide in D2O shows that there are 3 unexchanged hydrogens (1 each in the H2N-Asp1Arg2-Val3-Tyr4- segment, His, and Phe). The MALDI mass spectrum (Fig. 6B) of the TNM-treated free AI indicates a mass shift of 45 u from 1296.68 to 1341.77 u, suggesting that the Tyr4 side chain is accessible to TNM in water. However, the lipid-bound peptide does not show a mass shift on reaction with TNM (spectrum not shown). Therefore, tyrosine is either bound to the vesicles or involved in intramolecular hydrogen bonding. Similar to AII, acetylation of the free AI (Fig. 7C) also shows a mass shift of 84 u in the MALDI mass spectrum, pointing to the addition of two acetyl groups; the MS/MS spectrum of the acetylated AI shows that acetylation occurs at the N-terminal amino group and at the Tyr4 residue (spectrum not shown). In contrast, only one residue can be acetylated in the lipid-bound AI (Fig. 7D). There-
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Fig. 9. Zoom scan ESI–MS (A) and MS/MS (B) spectra of AI in water as well as zoom scan ESI–MS (C) and MS/MS (D) spectra of AI bound to vesicles after 1 min HDX.
fore, the N terminal of AI is outside of the membrane and Tyr4 is either embedded inside of the vesicles or involved in hydrogen bonding with the vesicles. Treatment of AI incorporated in DMPC vesicles with carboxypeptidase Y shows that both Leu10 (m/z 1183.67) and His9 (m/z 1046.66) can be cleaved, suggesting that these residues are outside of the vesicles (Fig. 8B). The cleavage stops at the Phe8 residue, implying that the aro-
matic ring of Phe8 is embedded in hydrophobic core of the membrane vesicles. Similar to AII, the N-terminal Asp1 remains unaffected on treatment of AI with aminopeptidase, confirming connectivity between Asp1 and Arg2 residues. From the HDX–MS/MS data, it can be stated that the structure of AI in phospholipid vesicles has some elements of a secondary structure. Both His residues in AI
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Table 3 Results of HDX–MS/MS of AI bound to phospholipid vesicles Ion
Theoretical m/z
m/z in vesicles
Hx in vesicles
y2 y4 b4 b5 b6 b7 b8 b92+
269.4 513.2 534.2 647.3 784.5 881.4 1028.5 583.7
272.2 517.4 539.4 653.3 791.4 888.5 1036.5 588.3
1.0 2.0 5.0 6.0 7.0 7.0 8.0 9.0
Note. Hx, number of exchanged hydrogens.
play an important role in interaction with the hydrophilic groups on the surface of the phospholipid vesicles. Similar to AII, the HDX data for AI fits with a U-shaped structure. The distribution of hydrogens exchanged on AI indicates that this peptide intracts with the surface of phospholipid vesicles and does not enter into the lipid environment completely. Also, the first four N-terminal residues H2N-Asp1-Arg2-Val3-Tyr4- of AI have a significant impact on stabilizing the conformation via hydrogen bonding. The nitration and ladder sequencing experiments suggest that the hydrophobic groups of Phe8 and Tyr4 are embedded in the hydrophobic core of the membrane. Conclusion An MS-based multipronged strategy that includes HDX, nitration of tyrosine, acetylation of free amino groups, ladder sequencing, and MS/MS can be applied successfully to determine the conformation of biologically active peptides in phospholipid vesicles. Because exchanged hydrogens are distributed throughout the peptide, AII interacts with the phospholipid head groups at the surface of the vesicles. The Tyr residue is embedded in the vesicles, whereas Phe is outside of the vesicles. AII has a U-shaped structure in the lipid environment. Similar conclusions were drawn for AI; that is, this peptide also interacts with the surface of phospholipid vesicles and does not enter into the lipid environment completely. Acknowledgment The MS data were acquired using MS instrumentation of the Charles B. Stout Neuroscience Mass Spectrometry Laboratory, Health Science Center, University of Tennessee. References [1] A.H. Barnett, The role of angiotensin II receptor antagonists in the management of diabetes, Blood Pressure 1 (2001) 21–26. [2] Q. Wang, E. Hummler, M. Maillard, J. Nussberger, B.C. Rossier, H.R. Brunner, M. Burnier, Compensatory up-regulation of angiotensin II subtype 1 receptors in (ENaC knockout heterozygous mice, Blood Pressure 59 (2001) 2216–2221.
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