Název: Školitel: Spectroscopic and electrochemical characterization of the protein GP12 Mgr. Natalia Cernei PhD Datum: 22.2.214
Virus HIV HIV (Human Immunodeficiency Virus) is an enveloped RNA virus belonging to retroviruses, a group of viruses having the ability to produce by their RNA and DNA sequence inserted into the host cell genome. HIV is not resistant. Outside the organism survives poorly and only for short periods. This virus is very particularly sensitive to heat, the temperature of 6 C will not survive. The virus could infect an organism must to penetrate and come into contact with the blood of susceptible individuals. The spread of the AIDS pandemic is one of the main problems of many developing countries. The world today is around 4 million infected, three quarters of them live in sub-saharan Africa. In some African countries is infected even a quarter of the population. Is also strongly affected by the South Asia, especially India. In Europe, the countries of the former Soviet Union. The largest number of HIV infected live in South Africa, Nigeria and India.
Protein GP 12 Glycoprotein GP12 is a glycoprotein exposed on the surface of the HIV envelope The 12 in its name comes from its molecular weight of 12 Gp12 is essential for virus entry into cells as it plays a vital role in attachment to specific cell surface receptors.
Scheme of HIV-1 virion Transmembrane envelope protein (gp41) V3 loop Matrix (p17) Surface envelope protein (gp12) Nucleocapsid (p7) RNA genome CD4+ T-lymphocyte CD4 receptor Lipid Bilayer Integrase (p31) Proteases (p11) Reverse transcriptase (p66) Capsid (p24) gp12 ID gp12 OD 3 2 1 CD4 V3 loop Bridging sheet Overall scheme of HIV-1 virion, with expression of interaction between HIV-1 envelope glycoproteins with CD4 receptor binding site. One of the subunit of gp41 is depicted. The gp12 OD stays for outer domain of glycoprotein 12, gp12 ID stays for inner domain of glycoprotein 12, 1; 2; 3 stays for loops that form three topological layers. Binding with CD4 results in the apposition of layer 1 and layer 2, the formation of the bridging sheet and the projection of the V3 loop away from the gp12 core. This rearrangement of gp12 allows the gp41 ectodomain to undergo additional conformational changes, necessary for HIV-1 entry.
Synthesis of CD4 Binding loop SSGGDPEIVTH by peptide synthesizer Liberty Blue. Peptide with the amino acid sequence SSGGDPEIVTH with molecular weight 198 Da was prepared on Liberty Blue peptide synthesizer by standard F-moc solid-phase peptide synthesis.
Absorbance (AU) Aspartic Acid RT:24.22 Serine RT:29.3 Glutamic Acid RT:33.5 Glycine RT:37.73 Proline RT:41.42 Methionine RT:45.52 Valine RT: 53.28 Isoleucine RT: 63.3 Histidine RT: 8.2 Res His Met Val Ile Glu Pro Asp Gly Gly Ser Ser Absorbance (AU) Absorbance (AU) Absorbance (AU) CD4 binding loop RT 75.12..1 Characterization of CD4 binding loop of GP 12 A B 2, C D 2.4 2.1 1.8 1.5 1.2.9.6.3..7.6.5.4.3.2 Amino acids used for synthesis 1,6 1,2,8,4 3 4 5 6 7 8 Retention time (min), 244 344 444 Wavelength (nm) E Intensity x1 4 Intens. [a.u.] 4. 3. 2. 1. x14 5 4 3 2 1. 19 199.14 199.14.7.6.5.4.3.2.1. 1 2 34 5 6 7 8 91 [M+H] + Retention time (min) 1121.166 [M+Na] + 1137.159 1137.196 11 111 112 113 114 115 m/z 11 111 112 113 114 m/z [M+K] + (A) Record obtained from program Liberty Blue, confirming succesfull synthesis of CD4 binding loop (SSGGD PEIVMH), contained in region 366 376 of gp12 of HIV-1. (B) Absorption spectra of synthesized CD4 binding loop with absorption maximum at 272 nm. (C) Chromatogram obtained on ion-exchange chromatography (IEC) showing retention time (75.12 min) of CD4 binding loop peptide. (D) Expression of amino acid content in CD4 binding loop after hydrolysis in microwave reactor MW 3. (E) MALDI- TOF/TOF mass spectra of CD4 binding loop peptide with calculated mass of 198.13 Da. DHB was used as a matrix. Measurements were carried out in reflector positive mode, with laser power of 7 %. One spectrum is made as an average from 25 subspectra. M stays for molecule of analyte; H stays for atom of hydrogen.
Peak height (µa) Peak height (µa) Peak height (µa) Peak height (µa) A C 4 35 3 25 2 15 1 4 3 2 1 Electrochemical detection of CD4 Binding Loop of gp 12 5 MeOH ACN 5 7 9 11 Potentinal (mv) 5 B D Peek height (µa) B-R 1 B-R 9 B-R 8 7 8 9 1 11 5 4 3 2 1 µg.ml -1 6 B-R 3 B-R 4 1 5 7 9 11 Potentinal (mv) Flow injection analysis with electrochemical detection (FIA-ED) system consists of two chromatographic pumps Model 582 ESA (ESA Inc., Chelmsford, USA) (working range.1-9.999 ml/min) and CoulArray electrochemical detector (Model 56A, ESA, USA). Detector consists of flow analytical chamber (Model 621, ESA, USA). Chamber contains four analytical cells. One analytical cell contains two referent (hydrogen-palladium), and two counter electrodes and one porous graphite working electrode. Electrochemical detector is situated in control module, which is thermostated. Sample (2 µl) was injected by manual valve (Rheodyne, USA). Flow rate of mobile phase was 1 ml/min 4 2 B-R 7 E 5 25 B-R 6 B-R 5 y =,4421x -,1513 R² =,9987 25 5 75 1 Concentration (µg.ml -1 ) 5 µg.ml -1 25 µg.ml -1 12.5 µg.ml -1
Conclusion Was performed successfully synthesis of CD4 binding loop (SSGGD PEIVMH), contained in region 366 376 of gp12 of HIV-1. CD4 binding loop (SSGGD PEIVMH was characterization by spectroscopic, IELC and MALDI/TOF methods. Was optimized the optimum conditions (Britton-Robinson ph 8 with addition 3 % ACN in the 11 mv) of the electrochemical detection of CD4 binding loop (SSGGD PEIVMH), of gp12 of HIV-1.
Prospects for the future Interaction studies CD4 binding loop of GP 12 with peptides Interaction studies CD4 binding loop of GP 12 with QDOTs
Acknowledgment NAZV QJ1311 and SIX CZ.1.5/2.1./3.72.
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