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However even if immunological tests with the NQ peptide
However, even if immunological tests with the NQ21 peptide are mostly positive [8], it doesn't mean that the structure of the NQ21 peptide is the same as that of the corresponding gp120 region characterized by a long alpha helix. Therefore, the aim of the current work was to check whether the NQ21 peptide has the same alpha-helical structure, as the corresponding fragment of gp120.
X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy are widely used to study the structure of proteins. However, it is possible that such methods will not provide an information on short N1-Methylpseudouridine if there are several rather different structural variants of the same peptide or even oligomers of the different order in the same sample. In our case the reproduction of the required structure can be confirmed by checking the content of secondary structure in the peptide. Circular dichroism (CD) spectroscopy may be used for this purpose, but the number of short peptides in PCDDB (Protein Circular Dichroism Data Base) is limited [9,10]. So, there is a risk that the most similar spectrum will poorly correspond to that of the given short peptide to speculate about its secondary structure content. Moreover, in peptides with Trp residues making aromatic-aromatic interactions with each other CD signal from secondary structure may be completely obscured by a signal from interacting Trp side chains [11,12]. The heating of the peptide solution under the control of CD may help to confirm the existence of interacting Trp residues, and even to check the secondary structure in the differential spectra, but anyway one needs a confirmation from an alternative method in such cases.
To overcome the abovementioned limitations Raman spectroscopy can be considered as an alternative technique because successful interpretation of its results does not require a set of reference spectra. It is known that the wavelength of amide I band (-C=O) of Raman spectrum is a sensible indicator of the secondary structure content of a protein or a peptide [[13], [14], [15]]. It is widely accepted that the position of amide I band between 1640 and 1658 cm−1 shows that the protein is alpha helical, if it is situated from 1665 to 1680 cm−1 the protein is beta structural, while the peak variation in the range 1660–1665 cm−1 shows that the protein is mixed or mostly consists of random coil [15].
It should be noted that to obtain informative Raman spectra of proteins or peptides rather high concentrations of these substances (nearly 1 mg per mL) must be used. However, such concentration is overabundant for the vaccine. Usually 1–30 μcg of an antigen is enough for successful immunization. Raman spectroscopy is not sensitive to small amounts of substances because only one Raman photon is scattered from the 106–108 photons of an incident light. To solve this problem we studied peptides by surface enhanced Raman scattering (SERS) spectroscopy using so-called SERS substrates based on noble metals' nanostructures [16]. The number of photons scattered from the molecules deposited on the SERS substrates greatly increases due to surface plasmon resonance (SPR) in metallic nanostructures [17] providing high sensitivity at extremely low concentration down to single molecule detection [18]. Since peptides may have different structure in dry form and in water solution, we recorded SERS spectra from solutions of peptides in phosphate buffers. It is known that with the help of SERS it is possible to observe the structure of molecules mainly from the first layer that is adsorbed on a surface. Since the surface in our case is quite hydrophilic and has a positive charge, there is a high probability that drastic changes in secondary structure will not occur during the adsorption: peptide molecules may still be hydrated and interact with silver cations from the SERS substrate by their carboxylic groups.
Material and methods
As the material for in vitro experiments we used lyophilized peptides NQ21 and biotin-NQ21 (with a purity level higher than 95%), commercially synthesized by Peptide 2.0 Inc. (USA). The synthesis has been performed on the automatic synthesizer Symphony, the quality control included HPLC and MS detection of peptides.