Alamethicin (ALM) is an antimicrobial peptide that is frequently employed in studies of the mechanism of action of pore-forming molecules. Advanced techniques of solid-state NMR spectroscopy (SSNMR) are important in these studies, as they are capable of describing the alignment of helical peptides, such as ALM, in lipid bilayers. Here, it is demonstrated how an analysis of the SSNMR measurements can benefit from fully periodic calculations, which employ the plane-wave density-functional theory (PW DFT) of the solid-phase geometry and related spectral parameters of ALM. The PW DFT calculations are used to obtain the structure of desolvated crystalline ALM and predict the NMR chemical shift tensors (CSTs) of its nuclei. A variation in the CSTs of the amidic nitrogens and carbonyl carbons along the ALM backbone is evaluated and included in simulations of the orientation-dependent anisotropic 15N and 13C chemical shift components. In this way, the influence of the site-specific structural effects on the experimentally determined orientation of ALM is shown in models of cell membranes.

Institute of Macromolecular Chemistry Czech Academy of Sciences 16206 Prague Czech Republic

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Hwon J.H., Powderly W.G. The post anti-biotic era is here. Nature. 2021;373:471. doi: 10.1126/science.abl5997. PubMed DOI

Antimicrobial Resistance. [(accessed on 13 September 2021)]. Available online: https://www.who.int/health-topics/antimicrobial-resistance.

Hanna C.C., Hermant Y.O., Harris P.W.R., Brimble M.A. Discovery, Synthesis, and Optimization of Peptide-Based Antibiotics. Acc. Chem. Res. 2021;54:1878–1890. doi: 10.1021/acs.accounts.0c00841. PubMed DOI

Yan Y., Zhang Z., Wang X., Niu Y., Zhang S., Xu W., Ren C. Advances of peptides for antibacterial applications. Colloids Surf. B. 2021;202:11682. doi: 10.1016/j.colsurfb.2021.111682. PubMed DOI

Kabelka I., Vácha R. Advances in Molecular Understanding of α-Helical Membrane-Active Peptides. Acc. Chem. Res. 2021;54:2196–2204. doi: 10.1021/acs.accounts.1c00047. PubMed DOI

Marquette A., Bechinger B. Biophysical Investigations Elucidating the Mechanisms of Action of Antimicrobial Peptides and Their Synergism. Biomolecules. 2018;8:18. doi: 10.3390/biom8020018. PubMed DOI PMC

Malanovic N., Marx L., Blondelle S.E., Pabst G., Semeraro E.F. Experimental concepts for linking the biological activities of antimicrobial peptides to their molecular modes of action. BBA Biomembr. 2020;1862:183275. doi: 10.1016/j.bbamem.2020.183275. PubMed DOI

Bechinger B. The SMART model: Soft Membranes Adapt and Respond, also Transiently, in the presence of antimicrobial peptides. J. Pept. Sci. 2015;21:346–355. doi: 10.1002/psc.2729. PubMed DOI

Simcock P.W., Bublitz M., Cipcigan F., Ryadnov M.G., Crain J., Stansfeld P.J., Sansom M.S.P. Membrane Binding of Antimicrobial Peptides Is Modulated by Lipid Charge Modification. J. Chem. Theory Comput. 2021;17:1218–1228. doi: 10.1021/acs.jctc.0c01025. PubMed DOI

Aronica P.G.A., Reid L.M., Desai N., Li J., Fox S.J., Yadahalli S., Essex J.W., Verma C.S. Computational Methods and Tools in Antimicrobial Peptide Research. J. Chem. Inf. Model. 2021;61:3172–3196. doi: 10.1021/acs.jcim.1c00175. PubMed DOI

Kirschbaum J., Krause C., Winzheimer R.K., Brückner H. Sequences of alamethicins F30 and F50 reconsidered and reconciled. J. Pept. Sci. 2003;9:799–809. doi: 10.1002/psc.535. PubMed DOI

Pieta P., Mirza J., Lipkowski J. Direct visualization of the alamethicin pore formed in a planar phospholipid matrix. Proc. Natl. Acad. Sci. USA. 2012;109:21223–21227. doi: 10.1073/pnas.1201559110. PubMed DOI PMC

McClintic W.T., Taylor G.J., Simpson M.L., Collier C.P. Macromolecular Crowding Affects Voltage-Dependent Alamethicin Pore Formation in Lipid Bilayer Membranes. J. Phys. Chem. B. 2020;124:5095–5102. doi: 10.1021/acs.jpcb.0c01650. PubMed DOI

Molugu T.R., Lee S., Brown M.F. Concepts and Methods of Solid-State NMR Spectroscopy Applied to Biomembranes. Chem. Rev. 2017;117:12087–12132. doi: 10.1021/acs.chemrev.6b00619. PubMed DOI

Yeh V., Bonev B.B. Solid state NMR of membrane proteins: Methods and applications. Biochem. Soc. Trans. 2021;49:BST20200070. doi: 10.1042/BST20200070. PubMed DOI

Salnikov E.S., Friedrich H., Li X., Bertani P., Reissmann S., Hertweck C., O’Neil J.D.J., Raap J., Bechinger B. Structure and Alignment of the Membrane-Associated Peptaibols Ampullosporin A and Alamethicin by Oriented 15N and 31P Solid-State NMR Spectroscopy. Biophys. J. 2009;96:86–100. doi: 10.1529/biophysj.108.136242. PubMed DOI PMC

Bertelsen K., Paaske B., Thøgersen L., Tajkhorshid E., Schiøtt B., Skrydstrup T., Nielsen N.C., Vosegaard T. Residue-Specific Information about the Dynamics of Antimicrobial Peptides from 1H–15N Solid-State NMR Spectroscopy. J. Am. Chem. Soc. 2009;131:18335–18342. doi: 10.1021/ja908604u. PubMed DOI

Toraya S., Nishimura K., Naito A. Dynamic Structure of Vesicle-Bound Melittin in a Variety of Lipid Chain Lenghts by Solid-State NMR. Biophys. J. 2004;87:3323–3335. doi: 10.1529/biophysj.104.046102. PubMed DOI PMC

Nagao T., Mishima D., Jakhlantugs N., Wang J., Ishioka D., Yokota K., Norisada K., Kawamura I., Ueda K., Naito A. Structure and orientation of antibiotic peptide alamethicin in phospholipid bilayers as revealed by chemical shift oscillation analysis of solid state nuclear magnetic resonance and molecular dynamics simulation. BBA Biomembr. 2015;1848:2789–2798. doi: 10.1016/j.bbamem.2015.07.019. PubMed DOI

Salnikov E.S., Aisebrey C., Raya J., Bechinger B. Investigations of the Structure, Topology and Dynamics of Membrane-Associated Polypeptides by Solid-State NMR Spectroscopy. In: Separovic F., Naito A., editors. Advances in Biological Solid-State NMR: Proteins and Membrane-Active Peptides. 1st ed. Royal Society of Chemistry; London, UK: 2014. pp. 214–234. DOI

Hansen S.K., Bertelsen K., Paaske B., Nielsen N.C., Vosegaard T. Solid-state NMR methods for oriented membrane proteins. Prog. Nucl. Mag. Res. Sp. 2015;88:48–85. doi: 10.1016/j.pnmrs.2015.05.001. PubMed DOI

Naito A., Matsumori N., Ramamoorthy A. Dynamic membrane interactions of antibacterial and antifungal biomolecules, and amyloid peptides, revealed by solid-state NMR spectroscopy. Biochim. Biophys. Acta Gen. Subj. 2018;1862:307–323. doi: 10.1016/j.bbagen.2017.06.004. PubMed DOI PMC

Hodgkinson P. NMR Crystallography of Molecular Organics. Prog. Nucl. Mag. Res. Sp. 2020;118:10–53. doi: 10.1016/j.pnmrs.2020.03.001. PubMed DOI

Czernek J., Brus J. Monitoring the Site-Specific Solid-State NMR Data in Oligopeptides. Int. J. Mol. Sci. 2020;21:2700. doi: 10.3390/ijms21082700. PubMed DOI PMC

Czernek J., Brus J. Polymorphic Forms of Valinomycin Investigated by NMR Crystallography. Int. J. Mol. Sci. 2020;21:4907. doi: 10.3390/ijms21144907. PubMed DOI PMC

Fox R.O., Richard F.M. A voltage-gated ion-channel model inferred from the crystal structure of alamethicin at 1.5-Å resolution. Nature. 1982;300:325–330. doi: 10.1038/300325a0. PubMed DOI

Chugh J.K., Wallace B.A. Peptaibols: Models for ion channels. Biochem. Soc. Trans. 2001;29:565–570. doi: 10.1042/bst0290565. PubMed DOI

Miura Y. NMR studies of the conformation, stability, and dynamics of alamethicin in methanol. Eur. Biophys. J. 2020;49:113–124. doi: 10.1007/s00249-019-01418-8. PubMed DOI

Lee T.-H., Hall K.N., Aguilar M.-I. Antimicrobial Peptide Structure and Mechanism of Action: A Focus on the Role of Membrane Structure. Curr. Top. Med. Chem. 2016;16:25–39. doi: 10.2174/1568026615666150703121700. PubMed DOI

Kumar P., Kizhakkedathu J.N., Straus S.K. Antimicrobial Peptides: Diversity, Mechanism of Action, and Strategies to Improve the Activity and Biocompatibility In Vivo. Biomolecules. 2018;8:4. doi: 10.3390/biom8010004. PubMed DOI PMC

Birdsall E.R., Petti M.K., Saraswat V., Ostrander J.S., Arnold M.S., Zanni M.T. Structure Changes of a Membrane Polypeptide under an Applied Voltage Observed with Surface-Enhanced 2D IR Spectroscopy. J. Phys. Chem. Lett. 2021;12:1786–1792. doi: 10.1021/acs.jpclett.0c03706. PubMed DOI PMC

Esteban-Martín S., Strandberg E., Fuertes G., Ulrich A.S., Salgado J. Influence of Whole-Body Dynamics on 15N PISEMA NMR Spectra of Membrane Proteins: A Theoretical Analysis. Biophys. J. 2009;96:3233–3241. doi: 10.1016/j.bpj.2008.12.3950. PubMed DOI PMC

Salnikov E., Bertani P., Raap J., Bechinger B. Analysis of the amide 15N chemical shift tensor of the Ca tetrasubstituted constituent of membrane-active peptaibols, the a-aminoisobutyric acid residue, compared to those of di- and tri-substituted proteinogenic amino acid residues. J. Biomol. NMR. 2009;45:373–387. doi: 10.1007/s10858-009-9380-5. PubMed DOI

Czernek J., Brus J. Theoretical predictions of the two-dimensional solid-state NMR spectra: A case study of the 13C—1H correlations in metergoline. Chem. Phys. Lett. 2013;586:56–60. doi: 10.1016/j.cplett.2013.09.015. DOI

Czernek J., Brus J. The covariance of the differences between experimental and theoretical chemical shifts as an aid for assigning two-dimensional heteronuclear correlation solid-state NMR spectra. Chem. Phys. Lett. 2014;608:334–339. doi: 10.1016/j.cplett.2014.05.099. DOI

Harris R.K., Becker E.D., De Menezes S.M.C., Granger P., Hoffman R.E., Zilm K.W. Further conventions for NMR shielding and chemical shifts (IUPAC Recommendations 2008) Pure Appl. Chem. 2008;82:59–84. doi: 10.1351/pac200880010059. PubMed DOI

Czernek J., Brus J. Theoretical Investigations into the Variability of the N-15 Solid-State NMR Parameters Within an Antimicrobial Peptide Ampullosporin A. Phys. Res. 2018;67:S349–S356. doi: 10.33549/physiolres.933976. PubMed DOI

Quine J.R., Achuthan S., Asbury T., Bertram R., Chapman M.S., Hu J., Cross T.A. Intensity and mosaic spread analysis from PISEMA tensors in solid-state NMR. J. Magn. Reson. 2006;179:190–198. doi: 10.1016/j.jmr.2005.12.002. PubMed DOI

Opella S.J. Structure Determination of Membrane Proteins in Their Native Phospholipid Bilayer Environment by Rotationally Aligned Solid-State NMR Spectroscopy. Acc. Chem. Res. 2013;49:2145–2153. doi: 10.1021/ar400067z. PubMed DOI PMC

Takeda N., Kuroki S., Kurosu H., Ando S. 13C-NMR Chemical Shift Tensor and Hydrogen-Bonded Structure of Glycine-Containing Peptides in a Single Crystal. Biopolymers. 1999;50:61–69. doi: 10.1002/(SICI)1097-0282(199907)50:1<61::AID-BIP6>3.0.CO;2-9. DOI

Saito H., Ando I., Ramamoorthy A. Chemical shift tensor—The heart of NMR: Insights into biological aspects of proteins. Prog. Nucl. Mag. Res. Sp. 2010;57:181–228. doi: 10.1016/j.pnmrs.2010.04.005. PubMed DOI PMC

Asakawa N., Kuroki S., Kurosu H., Ando I., Shoji A., Ozaki T. Hydrogen-bonding effect on 13C NMR chemical shifts of L-alanine residue carbonyl carbons of peptides in the solid state. J. Am. Chem. Soc. 1992;114:3261–3265. doi: 10.1021/ja00035a016. DOI

Kresse G., Joubert D. From ultrasoft pseudopotentials to the projector augmented-wave method. Phys. Rev. B. 1999;59:1758–1775. doi: 10.1103/PhysRevB.59.1758. DOI

Segall M.D., Lindan P.J.D., Probert M.J., Pickard C.J., Hasnip P.J., Clark S.J., Payne M.C. First principles simulation: Ideas, illustrations, and the CASTEP code. J. Phys. Condens. Matter. 2002;14:2717–2744. doi: 10.1088/0953-8984/14/11/301. DOI

Clark S.J., Segall M.D., Pickard C.J., Hasnip P.J., Probert M.J., Refson K., Payne M.C. First principles methods using CASTEP. Z. Kristallogr. 2005;220:567–570. doi: 10.1524/zkri.220.5.567.65075. DOI

BIOVIA Materials Studio . Dassault Systèmes. Vélizy-Villacoublay; Paris, France: [(accessed on 13 September 2021)]. Available online: https://www.3ds.com/products-services/biovia/products/molecular-modeling-simulation/biovia-materials-studio/

Biswas A.B., Hughes E.W., Sharma B.D., Wilson J.N. The crystal structure of α-glycylglycine. Acta Cryst. B. 1968;24:40–50. doi: 10.1107/S0567740868001688. PubMed DOI

Rao S.N., Parthasarathy R. Structure and conformational aspects of the nitrates of amino acids and peptides. I. Crystal structure of glycylglycine nitrate. Acta Cryst. B. 1973;29:2379–2388. doi: 10.1107/S0567740873006734. DOI

Koetzle T.F., Hamilton W.C. Precision neutron diffraction structure determination of protein and nucleic acid components. II. The crystal and molecular structure of the dipeptide glycylglycine monohydrochloride monohydrate. Acta Cryst. B. 1972;28:2083–2090. doi: 10.1107/S0567740872005576. DOI

Gao S.-P., Pickard C.J., Perlov A., Milman V. Core-Level Spectroscopy Calculation and the Plane Wave Pseudopotential Method. J. Phys. Condens. Matter. 2009;21:104203. doi: 10.1088/0953-8984/21/10/104203. PubMed DOI

Perdew J.P., Burke K., Ernzerhof M. Generalized gradient approximation made simple. Phys. Rev. Lett. 1996;77:3865–3868. doi: 10.1103/PhysRevLett.77.3865. PubMed DOI

Tkatchenko A., Scheffler M. Accurate Molecular Van Der Waals Interactions from Ground-State Electron Density and Free-Atom Reference Data. Phys. Rev. Lett. 2009;102:073005. doi: 10.1103/PhysRevLett.102.073005. PubMed DOI

Pickard C.J., Mauri F. All-electron magnetic response with pseudopotentials: NMR chemical shifts. Phys. Rev. B. 2001;63:245101. doi: 10.1103/PhysRevB.63.245101. DOI

Yates J.R., Pickard C.J., Mauri F. Calculations of NMR chemical shifts for extended systems using ultrasoft pseudopotentials. Phys. Rev. B. 2007;76:024401. doi: 10.1103/PhysRevB.76.024401. DOI

Czernek J. On the solid-state NMR spectra of naproxen. Chem. Phys. Lett. 2015;619:230–235. doi: 10.1016/j.cplett.2014.11.031. DOI

Bechinger B., Sizun C. Alignment and Structural Analysis of Membrane Polypeptides by 15N and 31P Solid-State NMR Spectroscopy. Concepts Magn. Reson. 2003;18A:130–145. doi: 10.1002/cmr.a.10070. DOI

Paulino J., Yi M., Hung I., Gan Z., Wang X.L., Chekmenev E.Y., Zhou H.X., Cross T.A. Functional stability of water wire–carbonyl interactions in an ion channel. Proc. Natl. Acad. Sci. USA. 2020;117:11908–11915. doi: 10.1073/pnas.2001083117. PubMed DOI PMC

Hung I., Gan Z., Wu G. Two- and Three-Dimensional 13C–17O Heteronuclear Correlation NMR Spectroscopy for Studying Organic and Biological Solid. J. Phys. Chem. Lett. 2021;12:8897–8902. doi: 10.1021/acs.jpclett.1c02465. PubMed DOI

Hauser K., He Y., Garcia-Diaz M., Simmerling C., Coutsias E. Characterization of Biomolecular Helices and Their Complementarity Using Geometric Analysis. J. Chem. Inf. Model. 2017;57:864–874. doi: 10.1021/acs.jcim.6b00721. PubMed DOI PMC

Czernek J., Brus J. On the predictions of the 11B solid state NMR parameters. Chem. Phys. Lett. 2016;655:66–70. doi: 10.1016/j.cplett.2016.05.027. DOI