Thermal analysis of the NQ

Thermal analysis of the NQ21 peptide was performed with the Solar CM2203 spectrofluorometer. We prepared three 1 μg/mL peptide solutions with different desipramine hcl levels (5.0, 7.4, 8.5) in 0.01 M phosphate buffers. Each solution was slowly heated up from 28 °C to 54 °C with a step of 1 °C. Spectra were collected in the range from 300 to 400 nm (excitation wavelength was 270 nm). For the secondary structure prediction we used original PentaFOLD algorithm [22] based on probability scales for the most stable alpha helices, beta strands and random coils. The algorithm predicts secondary structure elements using alpha-helical (in which just the most stable beta-strands are predicted) and beta-structural patterns (in which just the most stable alpha-helices are predicted). For the tertiary structure prediction of the peptide we used both homology based methods (SWISS-model [23] and I-TASSER [24]) and ab-initio methods (PepFOLD 3.1 [25] and QUARK [26]). For the prediction of quaternary structure of peptide oligomers we used Hex 8.0.0 method [27] that arranges all the models according to the value of ΔU of the complex formation. To predict dimer structure the best model of the NQ21 peptide created by PepFOLD 3.1 was used. Then we took the best model of a dimer as an input for the prediction of the tetramer structure. Similarly the best tetramer model was an input for the octamer structure prediction. Circular dichroism (CD) analysis of the NQ21 water solution in far UV has been performed at pH = 6.8, at pH = 7.4, and at pH = 8.0 in 0.01 M phosphate buffer on a Chiroscan CD spectrometer. Scans were obtained from 195 to 300 nm with a step size of 1 nm in 300 μl quartz cuvette with a path length of 1 mm, at a rate of 2 nm in second. Lyophilized NQ21 peptide has been dissolved at the temperature of 4 °C in each buffer in the concentration of 0.2–0.4 mg/mL. Each probe has been heated up from 5° to 80 °C at the rate of 1° in a minute. The recording of each new spectrum has been started after the increase in temperature equal to 5°. Thermal analysis in near UV has been performed in the same manner, at pH = 7.4, but in a cuvette with a path length of 10 mm. Obtained spectra have been smoothed with the help of the GraphWork program. Then milliabsorbance units have been converted into molar ellipticity (in deg cm2/dmol) using actual concentrations, the path length of a cuvette, the number of amino acid residues and the molar weight of a peptide. Spectra were analyzed with the CAPITO web server [10] that performed a search of the most similar spectra from PCDDB [9]. Differential spectra have been calculated between each pair of spectra with a difference in 30°.
Results and discussion
Conclusions
Introduction The CD4-binding site (CD4-BS) of gp120 in HIV-1 has been recognized to be the most vulnerable site for antibody targeting and viral neutralization [1], [2], [3], [4], [5], [6], [7], [8]. Recently, identification of several potent neutralizing antibodies through the screening of HIV-1 patient sera, such as VRC01 [9] and PGV04 [5], have been known to be elicited by the CD4-BS of gp120. Further analysis of these antibodies has found that the binding sites for some CD4-BS antibodies primarily depend on the outer domain of gp120, which are unlike CD4 binding wherein the binding requires all the three domains: outer domain, inner domain and the bridging sheet [10]. It is known that the outer domain is relatively more stable than the inner domain, and the outer-domain core structure, which does not include the V3 loop, is even more stable [11], [12]. An optimized outer domain from a HIV-1 subtype A strain can bind to the CD4-BS antibodies VRC01 and PG04. The outer domain structure in complex with PG04 has been also solved [13]. The outer domain used as an immunogen has been tested for its immunogenicity previously, and it shows a comparable immune response to gp120, but a weaker response than the gp140 envelope trimer. However, it is an un-stabilized wildtype outer domain in which the V3-loop and the glycan site (antibody 2G12) were focused, not the CD4-BS [14]. A membrane anchored-outer domain is recognized by antibody b12 and is also found to have elicited CD4-BS antibodies [15]. All of these previous studies are based on the wildtype outer-domains, but the outcomes have provided the feasibility for developing outer-domain based immunogens.