Monitoring of peptide induced disruption of artificial lipid membrane constructed on boron-doped nanocrystalline diamond by electrochemical impedance spectroscopy

With the rise of antibiotic resistance of pathogenic bacteria there is an increased demand for monitoring of the functionality of bacteria membranes, whose disruption can be induced by peptide-lipid interactions. In this work we attempt to monitor formation and subsequent peptide induced disruption of supported lipid membranes (SLBs) on boron-doped nanocrystalline diamond (B-NCD). Electrochemical Impedance Spectroscopy (EIS) was used to study in situ changes related to lipid membrane formation and disruption by peptide-induced interactions. The observed impedance changes were minimal for oxidized B-NCD samples. The sensitivity for the detection of membrane formation and disruption was significantly higher for hydrogenated B-NCD surfaces. Data modelling indicates large differences in the structure of electrical double layer at the B-NCD/SLB interface for hydrogen and oxygen terminated B-NCD surfaces. For oxidized B-NCD surfaces, EIS changes are negligible. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.status: publishe

The Position of His-Tag in Recombinant OspC and Application of Various Adjuvants Affects the Intensity and Quality of Specific Antibody Response after Immunization of Experimental Mice.

Lyme disease, Borrelia burgdorferi-caused infection, if not recognized and appropriately treated by antibiotics, may lead to chronic complications, thus stressing the need for protective vaccine development. The immune protection is mediated by phagocytic cells and by Borrelia-specific complement-activating antibodies, associated with the Th1 immune response. Surface antigen OspC is involved in Borrelia spreading through the host body. Previously we reported that recombinant histidine tagged (His-tag) OspC (rOspC) could be attached onto liposome surfaces by metallochelation. Here we report that levels of OspC-specific antibodies vary substantially depending upon whether rOspC possesses an N' or C' terminal His-tag. This is the case in mice immunized: (a) with rOspC proteoliposomes containing adjuvants MPLA or non-pyrogenic MDP analogue MT06; (b) with free rOspC and Montanide PET GEL A; (c) with free rOspC and alum; or (d) with adjuvant-free rOspC. Stronger responses are noted with all N'-terminal His-tag rOspC formulations. OspC-specific Th1-type antibodies predominate post-immunization with rOspC proteoliposomes formulated with MPLA or MT06 adjuvants. Further analyses confirmed that the structural features of soluble N' and C' terminal His-tag rOspC and respective rOspC proteoliposomes are similar including their thermal stabilities at physiological temperatures. On the other hand, a change in the position of the rOspC His-tag from N' to C' terminal appears to affect substantially the immunogenicity of rOspC arguably due to steric hindrance of OspC epitopes by the C' terminal His-tag itself and not due to differences in overall conformations induced by changes in the His-tag position in rOspC variants

CD spectra of rOspC proteins.

<p>Secondary structure of N' and C' terminal His-tag rOspC proteins determined by CD and FTIR. <b>A</b>) CD and FTIR spectra were obtained at room temperature (22°C) using a Chirascan CD spectrometer and Tensor 27 FTIR, respectively. <b>B)</b> Comparison of secondary structures of rOspC proteins obtained by calculations based on FTIR and CD measurements.</p

Synthetic molecular adjuvants MT06 and MPLA.

<p>Left panel shows MT06, a non-pyrogenic lipophilic derivative of muramyl dipeptide (MDP) analogue—Nor-AbuMDP. The right panel shows monophosphoryl lipid A, a derivative of lipopolysaccharide from <i>S</i>. <i>minnesota</i>.</p

Characterization of metallochelating N' and C´ terminal His-tag rOspC proteoliposomes by immuno EM and DLS.

<p>The EM picture of <b>A)</b> plain nanoliposome, <b>B</b>) metallochelation nanoliposome with surface bound N´ terminal His-tag rOspC proteins. Metallochelation liposomes were incubated with N' and C' terminal His-tag rOspC proteins followed by incubation with pooled sera from rOspC-immunized mice (1:50 dilution) followed by addition of protein A-labeled 10-nm colloidal gold particles. After 12-h, the proteoliposomes were negatively stained by ammonium molybdate and observed using Philips Morgagni transmission EM. Black dots represent immunogold particles on rOspC proteins bound to the liposome surface (black arrows). rOspC protein molecules (white dots) forms chains on proteoliposome surfaces (white arrows). <b>C)</b> The increase of hydrodynamic radius after binding of rOspC proteins was measured by DLS. <b>D)</b> Tabular data characterizing the size of plane and rOspC proteoliposomes in detail.</p

SDS-PAGE and MS analyses of recombinant N' and C' terminal His-tag rOspC.

<p><b>A)</b> Recombinant N' and C' terminal His-tag rOspC proteins were separated using SDS-PAGE and stained by CBB G-250. The differences in mobility of both rOspC variants are caused by the length of labeling tags and spacers and correspond to theoretical prediction based on cDNA sequences: 23.96 kDa for C' and 27.12 for N' terminal His-tag rOspC. <b>B, C</b>) MALDI-TOF peptide mass fingerprinting of rOspC proteins. All spectra were acquired using CHCA matrix on Microflex LRF20 MALDI-TOF mass spectrometer. Panel <b>B</b>) refers to C' terminal His-tag rOspC and panel <b>C</b>) N' terminal His-tag rOspC.</p