Communication pubs.acs.org/Biomac
Hemopressin Forms Self-Assembled Fibrillar Nanostructures under Physiologically Relevant Conditions Martha G. Bomar, Steven J. Samuelsson,† Patrick Kibler, Krishna Kodukula, and Amit K. Galande* Center for Advanced Drug Research (CADRE), SRI International, 140 Research Drive, Harrisonburg, Virginia 22802, United States S Supporting Information *
ABSTRACT: The nonapeptide hemopressin, which is derived from the α chain of hemoglobin, has been reported to exhibit inverse agonist activity against the CB1 receptor. Administration of this peptide in animal models led to decreased food intake and elicited hypotensive and antinociceptive effects. On the basis of hemopressin’s potential in therapeutic applications and the lack of a structure−activity relationship study in literature, we aimed to determine the conformational features of hemopressin under physiological conditions. We conducted transmission electron microscopy experiments of hemopressin, revealing that it self-assembles into fibrils under aqueous conditions at pH 7.4. Circular dichroism and nuclear magnetic resonance experiments indicate that the peptide adopts a mostly extended β-like structure, which may contribute to its self-assembly and fibril formation.
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INTRODUCTION The endocannabinoid system consists of two cannabinoid receptors CB1 and CB2, which are G-protein coupled receptors and are primarily activated by lipophilic compounds called cannabinoids. The endocannabinoid pathways have diverse roles in a variety of biological processes, including addiction, appetite, memory, pain sensation, and cognition. Therefore, the cannabinoid receptors are well-established targets for therapeutic intervention in addictive and metabolic disorders. Indeed, studies have demonstrated the efficacy of rimonabant, a CB1 selective inverse agonist, in treating obesity and addictive disorders.1 Unfortunately, severe central nervous system (CNS) side effects prevented rimonabant’s approval in the United States as an antiobesity drug and suspended its use elsewhere. Therefore, selective CB1 antagonists lacking adverse side effects while maintaining therapeutic benefits are highly desired. The bioactive peptide hemopressin, which is derived from the α chain of hemoglobin, was recently reported to exhibit similar inverse agonist activity and binding affinity (EC50 = 0.35 nM) for the CB1 receptor as rimonabant.2 Originally, isolated from rat brain homogenates as a substrate for metallopeptidases,3 in vivo studies revealed that hemopressin administration causes significant antinociceptive effects in rats.2 Moreover, hemopressin was reported to cause dose-dependent hypotension in rats, rabbits, and mice.3,4 More recently, hemopressin was shown to inhibit food intake in normal and obese animal models without causing obvious adverse side effects.5 Considering the significance of hemopressin’s pharmacological properties and its potential in therapeutic applications, we aimed to determine the conformational features of hemopressin under physiological conditions. Transmission electron microscopy (TEM) of hemopressin in solution revealed that the peptide self-assembles into fibrils at physiological pH. Investigation into the structural basis of hemopressin self-assembly using nuclear © 2012 American Chemical Society
magnetic resonance (NMR) and circular dichroism (CD) revealed the presence of a mostly extended β-like structure, which may contribute to its self-assembly.
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EXPERIMENTAL SECTION
Peptide Synthesis, Purification, and Characterization. Using standard Fmoc chemistry, hemopressin (PVNFKFLSH) was synthesized by an Advanced Chemtech automated synthesizer (APEX 496) on Wang resin (Peptides International). All amino acids were purchased from either Novabiochem or Peptides International. A diisopropylcarbodiimide (DIC)/1-hydroxybenzotriazole (HOBt) double coupling protocol was used for coupling reactions with a 1:1 ratio of DIC/HOBt to amino acids. A solution of 0.5 M HOBt was used to dissolve the amino acids. The peptides were cleaved from the resin using 95% trifluoroacetic acid (TFA), 3% triisopropylsilane, and 2% water. Following cleavage and precipitation, the peptides were dissolved in 5−10 mL of water and put on a VirTis (Sentry 2.0) freezedryer for 24−48 h. The peptides were then purified by reversed-phase high-performance liquid chromatography (RP-HPLC) on a Shimadzu instrument. The HPLC conditions were as follows: Vydac C18 column; solvent gradient: A, 0.01% TFA in water; B, 0.01% TFA in acetonitrile; flow rate: 5 mL/min; and UV detection: 254 and 220 nm. A 4800 Plus MALDI TOF/TOF high-resolution mass spectrometer (Applied Biosystems) was then used to confirm the molecular weights of the peptide (expected MW 1088.26 Da, observed MW 1088.64 Da). RVDhemopressin (RVDPVNFKFLSH) was synthesized following the same procedure (expected MW 1458.67 Da, observed MW 1458.88 Da). Angiotensin II (DRVYIHPF) was purchased from Anaspec. NMR Spectroscopy. The NMR experiments were recorded with a Bruker Avance 600 MHz NMR spectrometer. In all of the NMR experiments, DSS (sodium 4,4-dimethyl-4-silapentane-1-sulfonate) was used as an internal standard (Cambridge Isotope Laboratories). To assign the amide protons, 2D TOCSY and ROESY spectra were
Received: December 22, 2011 Revised: January 22, 2012 Published: February 3, 2012 579
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acquired for each peptide at 298 K. For these experiments, 1 mM hemopressin was dissolved in either 90% H2O and 10% D2O or 65% H2O, 25% TFE-d3 and 10% D2O (Cambridge Isotope Laboratories) both with a pH of 3. An additional ROESY spectrum in 50% TFE-d3 was also collected to help with the assignments of overlapping amide peaks. All NMR data were processed in Topspin 2.1 (Bruker) and analyzed in CARA.24 CD Spectroscopy. All CD spectra were recorded on a Jasco810 CD spectrometer. The sample concentration for all spectra was 100 μM. The samples collected at pH 7.4 were buffered in 25 mM phosphate, whereas those at pH 3.0 were buffered against residual TFA from the peptide purification. Nondeuterated 2,2,2-trifluorethanol (TFE) was obtained from Sigma-Aldrich. All CD spectra were acquired at room temperature with a 1 mm quartz cuvette and a scanning speed of 50 nm/min. A blank spectrum was subtracted from each spectrum. The data were plotted and smoothed in SigmaPlot Version 11 (Systat Software). Transmission Electron Microscopy. A solution of 1 mM hemopressin, 25 mM phosphate, 50 mM NaCl, pH 7.4 was prepared. The sample was gently triturated, and two 10 μL aliquots were put on a parafilm base. A 200-mesh copper grid with carbon-stabilized Formvar substrates was floated on each drop for ∼90 s, aspirated dry, or gently aspirated before floating on a drop (10 μL) of 0.5% uranyl acetate (aqueous; 0.22 μm filtered) for another 90 s and then aspirated to dryness. The grids were imaged by TEM (CM12, FEI) at a 120 kV accelerating voltage using a liquid-nitrogen coldfinger and a 200 μm diffraction lens aperture to improve the contrast. The preparation was surveyed in scan-mode for signs of optimal material density before increasing the magnification to medium and high resolution (∼100 000×). Fields of interest were captured using optimal under-focus and were corrected for condenser and objective lens astigmatisms onto film (KODAK electron microscope film 4489). The negatives were digitized (Artixscan M1 Dual Pro Scanner, Microtek) at 16 bits and 600 ppi (file size of 9.14 Mb). The digitized files were imported into Image ProPlus image analysis software (IPP, MediaCy), where each image was calibrated and stamped with an appropriate micrometer-marker. Micrograph features were measured using an IPP caliper tool in manual-measure mode with the file electronically zoomed to 200 or 400%. The snapshot tool created a duplicate of the original file with the micrometer marker and caliper measures overlaid as a meta-image. All images were saved as tiffs without alterations to gamma settings using Adobe Photoshop (CS5).
Figure 1. Rat hemoglobin (PDB: 3DHT) showing where hemopressin (red) is located in the context of the intact protein. The heme groups are shown in blue. The N- and C-termini are denoted for clarity and the peptide sequence (with numbering used for discussion) is listed below. The Figure was made with Pymol.7
adopts a helical conformation in the protein crystal structure, the structural behavior of the synthetic nonapeptide by itself has not been thoroughly investigated. A recent NMR investigation of hemopressin reported a random coil conformation in water and a series of turn-like structures in mixed micelles.6 The lack of a well-defined ensemble of conformers in water prompted us to investigate the possibility that hemopressin might be forming self-assembled nanostructures under aqueous conditions. Indeed, we observed immediate formation of an aggregated or self-assembled matter upon preparation of a 1 mM sample of hemopressin in 25 mM phosphate, 50 mM NaCl, pH 7.4. Accordingly, a primary objective of this study was to characterize the self-assembly and nanostructural properties of hemopressin in water at physiological pH using TEM along with NMR and CD spectroscopy. TEM of a negative-stained preparation (1 mM hemopressin in 25 mM phosphate, 50 mM NaCl, pH 7.4) provided sufficient structural details for general characterization. After calibration, the filamentous structures were measured and analyzed for morphological information including shape, size, and assembly patterns. Hemopressin was found to be organized as semiregular bundles of fibrillar tangles (Figure 2). The fibrils were curvilinear
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RESULTS AND DISCUSSION The bioactive nonapeptide hemopressin is generated by proteolytic cleavage of hemoglobin. In the context of the α chain of hemoglobin, the hemopressin fragment (Figure 1, red) forms the first half of the 18-residue helix G, which lines the heme-binding pocket. Whereas the hemopressin sequence
Figure 2. TEM images of hemopressin in 25 mM phosphate, 50 mM NaCl, pH 7.4. (A) Curvilinear fibrils organize into a macromolecular lattice varying from few to bundles of fibrils. (B) Higher magnification shows close, lateral association of individual fibrillar assemblies. (C) Region of interest revealing fibrils with regular densities with a periodicity of ∼3.5 nm (inset; arrows). 580
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and complexed with adjacent fibrils into higher order structures, which ranged from a few fibril-diameters to clusters 600 to 800 nm wide (Supporting Information, Figure S1A). Fibrils were rarely unassociated or found alone. Therefore, individual fibrils were difficult to measure, but a close approximation showed diameters in the 7 to 8 nm range (Supporting Information, Figure S1B). A few TEM micrographs also showed fields with periodic densities along individual fibrils (Figure 2C, inset), which were highly regular with periods of ∼3.5 nm. These periodic repeats (Figure 2C) are indicative of super assemblies of monomers. Similarly, globular actin (G-actin) has been reported to organize into microfilaments (F-actin) under physiological conditions, which exhibit a regular period of 5.5 nm and can transverse the length of a eukaryotic organism or cell.8,9 In addition to these fibrils, particulate and very thin sheets of amorphous materials were found over much of the substrate and were attributed to the nonpeptide content of the sample preparation. Buffers alone were generally free of negatively stained materials. To confirm that the observed fibrils are specific to hemopressin, we conducted TEM on two additional peptide samples, the sequence-related peptide RVD-hemopressin, which is an agonist of the CB1 receptor, and a sequenceunrelated octapeptide angiotensin II, which has about the same
molecular size as that of hemopressin. Neither of the two control peptide samples exhibited any evidence of self-assembly or fibril formation (Supporting Information, Figure S2). We also investigated fibril formation of hemopressin in 25% TFE, a cosolvent frequently used as a mimic of biological membranes because of its lower dielectric constant compared with that of water. The hemopressin sample with 25% TFE adopted fibrils with similar morphologies and in somewhat greater abundance than their counterpart without TFE (Supporting Information, Figure S3). We conducted NMR and CD experiments to elucidate the structural basis for the self-assembly of hemopressin. The NMR experiments were run at pH 3 to minimize exchange of the backbone amide protons with the solvent. However, CD spectra acquired at pH 3 and 7.4 revealed the same global conformational signature at both pH values (Supporting Information, Figure S4), suggesting that the NMR results at pH 3 are also reflective of pH 7.4. The nuclear Overhauser effect (NOE) pattern of hemopressin in water was indicative of a mostly unstructured peptide, because no NH-NH or other long-range crosspeaks were observed (Figure 3A). We used TFE to induce secondary structure formation in hemopressin and hoped that the addition of the cosolvent would populate
Figure 3. NMR spectra of 1 mM hemopressin in 25 mM phosphate, 50 mM NaCl, pH 3 with and without 25% TFE-d3. (A) ROESY spectrum of hemopressin without 25% TFE-d3 indicates the lack of any NHi-NHi+1 crosspeaks. (B) ROESY spectrum of hemopressin with 25% TFE-d3 showing NHi-NHi+1 crosspeaks between HNK5 and HNF6 (red) and between HNL7 and HNS8 (green). (C) Long-range crosspeaks between Hα of F4 and Hβ and Hδ protons of L7 were observed in 25% TFE-d3. (D) 1D spectra of hemopressin with and without 25% TFE-d3. The greater dispersity of peaks with 25% TFE-d3 suggests a more structured peptide in the more hydrophobic environment. 581
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secondary structural elements that may contribute to its selfassembly. TFE can promote secondary structure formation of peptides by either preferentially solvating the folded state of the peptide or by solvating the peptide and displacing water from the peptide surface, thus favoring intrapeptide hydrogen bond formation.10 Because of its lower dielectric constant than water, TFE also has the advantage of serving as a membrane mimic. The rotating frame Overhauser effect spectroscopy (ROESY) spectrum of hemopressin in 25% TFE-d3 revealed weak NHNH crosspeaks between K5 and F6 and between L7 and S8 (Figure 3B). Spectral overlap of the amide protons of F4 and K5 and of F6 and L7 prevented observation of additional NH-NH crosspeaks, if present. Strong Hαi-HNi+1 crosspeaks were also observed for the entire peptide chain in 25% TFE-d3. In addition, long-range crosspeaks were observed between Hα of F4 and the Hβ and Hδ protons of L7 (Figure 3C), suggesting the formation of a turn-like structure within the peptide. However, the lack of long-range HN-HN crosspeaks and the presence of weak sequential HNi-HNi+1 crosspeaks and strong Hαi-HNi+1 crosspeaks suggest that the peptide mostly adopts an extended or β-like conformation.11 Accordingly, the greater dispersity of peaks in the 1D spectrum of hemopressin in the presence of 25% TFE-d3 indicates a more compact structure in the hydrophobic environment than in water alone (Figure 3D).12 CD was also used to assess the structure of hemopressin in aqueous solution at pH 7.4 in both the absence and presence of TFE. Without TFE, the spectrum of hemopressin exhibited a slight minimum or shoulder at 188 nm and a maximum at 216 nm, which corresponds to a mainly unstructured state. However, a single negative peak around 195 nm, indicative of a completely unstructured peptide, was lacking, thereby suggesting the presence of some structure within the sample. A maximum at ∼195 nm appeared with TFE and increased with increasing percentages of TFE. The signal of the maximum at 216 nm decreased with increasing TFE, and a broad shoulder appeared around 220 nm. The enhanced signal at 195 nm indicates a more ordered state in the more hydrophobic environment, which may be attributed to an increase in β-like or extended structures in accordance with the NMR data. Indeed, similar CD profiles have also been observed for other fibril-forming peptides13−16 and were attributed to β-sheet formation. An isodichroic point at 203 nm in the CD spectra of hemopressin suggests that the peptide adopts mainly two conformational states, which are likely an unordered and ordered state. In other fibril-forming peptides, the transition from an unordered to ordered state plays a significant part in fibrillization and may also contribute to fibril formation by hemopressin (Figure 4).17 Scrima et al.6 recently reported CD and NMR data for hemopressin in water and dodecylphosphocholine/sodium dodecyl sulfate (DPC/SDS) micelles at pH 5.4. Notably, the CD spectra of hemopressin in DPC/SDS micelles and in TFE appeared very different. Whereas a minimum at 200 nm was observed in the micelles, we observed a maximum at 195 nm, which increased with increasing TFE. These differences may be attributed to the distinct properties of micelles and TFE, which are both frequently used as membrane-mimicking media for structural studies. The structure of hemopressin in the DPC/SDS micelles reflects a micelle-associated peptide state; aromatic and hydrophobic residues within peptides preferentially interact with the hydrophobic lipid tails of micelles, facilitating structure formation by the peptide. By contrast, peptides remain in solution with TFE albeit with a different solvation state and dielectric constant than in water alone; the cosolvent TFE
Figure 4. CD spectra of hemopressin in 25 mM phosphate, pH 7.4 with increasing TFE concentrations.
promotes hydrogen bonding and hydrophobic interactions within peptides by forming a solvent matrix around the peptide.18 The results from both of these studies reveal a more ordered peptide under membrane-mimicking conditions, which may contribute to its fibrillization at the receptor. Our TEM experiments indicate that the CB1 inverse agonist hemopressin self-assembles at physiological pH values. The nonapeptide forms fibrils that feature higher order organization into a lattice of full, complex filaments, which may have implications for its pharmacology in vivo and may compromise in vitro experiments relying on specific soluble peptide concentrations. Indeed, in a recent review article, synthetic hemopressin was reported to exhibit variability in both in vitro and in vivo assays, and its oligomerization was tested using dialysis.19 The authors indicated that hemopressin forms aggregates that are retained in a dialysis cassette to a greater extent than a control peptide (angiotensin II, which we also used as a control for TEM studies; Supporting Information). Protein and peptide fibrillization contributes to numerous diseases, including Alzheimer’s and prion diseases.20 Few naturally occurring bioactive peptides including the larger insulin and amyloid-β have been shown to form fibrils, and smaller fibrilforming peptides have mainly been designed or derived from sequences of aggregating proteins, such as tau, transthyretin, and elastin. The significance of hemopressin self-assembly is underscored by the fact that the peptide is derived in vivo from the abundantly occurring hemoglobin and is found in the brain. Although a clear understanding of the aggregation process of peptides is lacking, the rate of self-assembly is significantly influenced by physicochemical properties including hydrophobicity, amino acid sequence, secondary structure, and charge.21 Moreover, various environmental factors can also affect peptide solubility, including temperature, solvent composition, agitation, and solvent pH. A transition from an unordered to ordered structure significantly contributes to fibrillization, and fibril formation is usually promoted by interactions between short β-strands, which often contain hydrophobic and aromatic residues.17,22 In our investigation of the structural basis for fibril formation, we determined that hemopressin adopts populations of both unordered and ordered conformations, which is likely an extended or β-like structure and may contribute to its selfassembly. Moreover, the hemopressin sequence contains several hydrophobic residues including two Phe and one Val, which both 582
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have high propensities to form β sheets and may also promote fibril formation.23 Furthermore, the high pI value of 9.2 for hemopressin might also contribute to its fibrillization and is especially relevant at physiological pH values. Indeed, a solvent pH near the pI of the peptide decreases the charge repulsion between different peptide molecules, thereby facilitating self-assembly. Although the pI of RVD-hemopressin and angiotensin II (8.75 and 6.74, respectively) are also near physiological pH, these peptides did not form fibrils under the same conditions as hemopressin. However, these peptides are slightly less hydrophobic than hemopressin, and the presence of the β-breaker proline within the RVD-hemopressin sequence (rather than at the N-terminus of hemopressin) may decrease its propensity to form fibrils. Whereas the mechanism of fibril formation in hemopressin remains unknown, its aromatic and hydrophobic nature, β-sheet propensity, and high pI value may contribute to its self-assembly at physiological pH, which may account for its reported variability in pharmacological activity and play a role in its in vivo activity.
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(7) DeLano, W. L. The PyMOL Molecular Graphics System; DeLano Scientific: San Carlos, CA, 2002. (8) Samuelsson, S. J.; Luther, P. W.; Pumplin, D. W.; Bloch, R. J. J. Cell. Biol. 1993, 122, 485−496. (9) Heuser, J. E.; Kirschner, M. W. J. Cell. Biol. 1980, 86, 212−234. (10) Roccatano, D.; Colombo, G.; Fioroni, M.; Mark, A. E. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 12179−12184. (11) Wuthrich, K. NMR of Proteins and Nucleic Acids; Wiley: New York, 1986. (12) Yao, J.; Dyson, H. J.; Wright, P. E. FEBS Lett. 1997, 419, 285− 289. (13) Goux, W. J.; Kopplin, L.; Nguyen, A. D.; Leak, K.; Rutkofsky, M.; Shanmuganandam, V. D.; Sharma, D.; Inouye, H.; Kirschner, D. A. J. Biol. Chem. 2004, 279, 26868−26875. (14) MacPhee, C. E.; Dobson, C. M. J. Mol. Biol. 2000, 297, 1203− 1215. (15) Satheeshkumar, K. S.; Jayakumar, R. Biophys. J. 2003, 85, 473− 483. (16) Bowerman, C. J.; Liyanage, W.; Federation, A. J.; Nilsson, B. L. Biomacromolecules 2011, 12, 2735−2745. (17) Chiti, F.; Dobson, C. M. Annu. Rev. Biochem. 2006, 75, 333− 366. (18) Reiersen, H.; Rees, A. R. Protein Eng. 2000, 13, 739−743. (19) Gomes, I.; Dale, C. S.; Casten, K.; Geigner, M. A.; Gozzo, F. C.; Ferro, E. S.; Heimann, A. S.; Devi, L. A. AAPS J. 2010, 12, 658−669. (20) Stefani, M.; Dobson, C. M. J. Mol. Med. (Heidelberg, Ger.) 2003, 81, 678−699. (21) Chiti, F.; Stefani, M.; Taddei, N.; Ramponi, G.; Dobson, C. M. Nature 2003, 424, 805−808. (22) Inouye, H.; Kirschner, D. A. Adv. Protein Chem. 2006, 73, 181− 215. (23) Street, A. G.; Mayo, S. L. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 9074−9076. (24) Keller R. The Computer-Aided Resonance Assignment Tutorial CARA; Cantina Verlag: Gouldau, Switzerland, 2004.
ASSOCIATED CONTENT
S Supporting Information *
Additional TEM images of hemopressin and peptide controls and CD spectra of hemopressin at different pH values. This material is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Present Address †
SRI International, 333 Ravenswood Avenue, Menlo Park, California 94025, United States Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS CADRE was established with support from the Commonwealth of Virginia. We thank Drs. Gina MacDonald, Richard Foust, and David Brakke at James Madison University for access to the NMR and CD spectrometers. We thank Bill Thomsen, Surendra Nayak, and Meghan Cupp at SRI International for helpful discussions. Dr. Walter Moos at SRI International is gratefully acknowledged.
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REFERENCES
(1) Lazary, J.; Juhasz, G.; Hunyady, L.; Bagdy, G. Trends Pharmacol. Sci. 2011, 32, 270−280. (2) Heimann, A. S.; Gomes, I.; Dale, C. S.; Pagano, R. L.; Gupta, A.; de Souza, L. L.; Luchessi, A. D.; Castro, L. M.; Giorgi, R.; Rioli, V.; Ferro, E. S.; Devi, L. A. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 20588− 20593. (3) Rioli, V.; Gozzo, F. C.; Heimann, A. S.; Linardi, A.; Krieger, J. E.; Shida, C. S.; Almeida, P. C.; Hyslop, S.; Eberlin, M. N.; Ferro, E. S. J. Biol. Chem. 2003, 278, 8547−8555. (4) Blais, P. A.; Cote, J.; Morin, J.; Larouche, A.; Gendron, G.; Fortier, A.; Regoli, D.; Neugebauer, W.; Gobeil, F. Peptides 2005, 26, 1317−1322. (5) Dodd, G. T.; Mancini, G.; Lutz, B.; Luckman, S. M. J. Neurosci. 2010, 30, 7369−7376. (6) Scrima, M.; Di Marino, S.; Grimaldi, M.; Mastrogiacomo, A.; Novellino, E.; Bifulco, M.; D’Ursi, A. Biochemistry 2010, 49, 10449− 10457. 583
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