Structural analysis of the neuropeptide substance P by using vibrational spectroscopy

Abstract

Substance P (SP) is one of the most studied peptide hormones and knowing the relationship between its structure and function may have important therapeutic applications in the treatment of a variety of stress-related illnesses. In order to obtain a deeper insight into its folding, the effects of different factors, such as pH changes, the presence of Ca2+ ions, and the substitution of the Met-NH2 moiety in the SP structure, was studied by Raman and infrared spectroscopies. SP has a pH-dependent structure. Under acidic–neutral conditions, SP possesses a prevalent β-sheet structure although also other secondary structure elements are present. By increasing pH, a higher orderliness in the SP secondary structure is induced, as well as the formation of strongly bound intermolecular β-strands with a parallel alignment, which favour the self-assembly of SP in β-aggregates. The substitution of the Met-NH2 moiety with the acidic functional group in the SP sequence, giving rise to a not biologically active SP analogue, results in a more disordered folding, where the predominant contribution comes from a random coil. Conversely, the presence of Ca2+ ions affects slightly but sensitively the folding of the polypeptide chain, by favouring the α-helical content and a different alignment of β-strands; these are structural elements, which may favour the SP biological activity. In addition, the capability of SERS spectroscopy to detect SP in its biologically active form was also tested by using different metal nanoparticles. Thanks to the use of silver NPs prepared by reduction of silver nitrate with hydroxylamine hydrochloride, SP can be detected at very low peptide concentration (~ 90 nM). However, the SERS spectra cannot be obtained under alkaline conditions since both the formation of SP aggregates and the lack of ion pairs do not allow a strong enough interaction of SP with silver NPs.

Graphical abstract

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Abbreviations

(Ag/Au) NPs:

(Silver/gold) Nanoparticles

FWHM:

Full width at half maximum

LOD:

Limit of detection

SP:

Substance P in amidated form

SP-OH:

Substance P in acidic form

SERS:

Surface-enhanced Raman scattering

Arg:

Arginine

Gly:

Glycine

Ile:

Isoleucine

Leu:

Leucine

Lys:

Lysine

Met:

Methionine

Phe:

Phenylalanine

Pro:

Proline

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Funding

This work was supported by the Scientific Grant Agency of the Ministry of the Education of Slovak Republic (APVV-15-0485) and the Ministerio de Economía y Competitividad from Spain under the grant FIS2017-84314-R.

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Correspondence to Zuzana Jurasekova or Armida Torreggiani.

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Jurasekova, Z., Garcia-Leis, A., Sanchez-Cortes, S. et al. Structural analysis of the neuropeptide substance P by using vibrational spectroscopy. Anal Bioanal Chem 411, 7419–7430 (2019). https://doi.org/10.1007/s00216-019-02097-2

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Keywords

  • Substance P
  • Vibrational (Raman and infrared) spectroscopy
  • Surface-enhanced Raman spectroscopy (SERS)