Neutron Reflectometry for Studying Proteins/Peptides in Biomimetic Membranes

The development of biomimetic surfaces for protein and peptide adsorptions is continuously expanding. Their biological functions can be influenced by the properties of the underlying artificial environment but the detailed mechanism is still not clear. In the past 30 years, neutron reflectometry has been widely applied to characterise the molecular structure of proteins or multi-protein complexes and their interactions with fluid artificial membrane that mimics the cellular environment. The specific interactions, bindings or structural changes between proteins and membranes play a crucial role in cellular responses and have promising potential in diagnostics and other biosensor applications. This chapter presents the progression of surface design for protein adsorption/interactions on membranes in detail, ranging from a simple phospholipid monolayer setup to more complicated artificial lipid bilayer systems. Furthermore, a new development of designed surfaces for studying the integral membrane protein system is also discussed in this chapter. A brief overview of various membrane mimetic surfaces is first outlined, followed by presenting specific examples of protein-membrane interactions studied by neutron reflectometry. The author demonstrates how to use neutron reflectometry as an advanced technique to provide step-by-step structural details for biomolecular applications in a well-controlled manner

Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1).

Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions

Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1)

<div><p>Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive <i>in silico</i>, <i>in vivo</i> and <i>in vitro</i> analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. <i>In silico</i> modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions.</p></div

Cyclic voltammetric analysis of P450c17, wt cyt b5, E48G/E49G cyt b5 and sequential additions.

<p><b>a, c,</b> d.c. voltammograms and <b>b, d,</b> the 6^th harmonic of the a.c. voltammograms for the bare (<i>grey</i>; left axis) and modified CNT electrodes. <b>a</b>, <b>b,</b> Adsorption of hemin (black, right axes) produced a much larger faradaic current and an <math><msubsup><mrow><mi>E</mi></mrow><mrow><mi>a</mi><mi>c</mi></mrow><mrow><mn>0</mn></mrow></msubsup></math> value (dashed lines) that were at least 0.02 V more negative than those derived from adsorption of proteins (left axes): wt cyt b5 (green), E48G/E49G cyt b5 (red) or P450c17 (blue). <b>c,</b> Adsorption of E48G/E49G cyt b5 on P450c17/CNT (blue) produced a substantial increase in the surface concentration of hemin (orange), but negligible changes in <i>Γ</i> were derived from adsorption of wt cyt b5 (magenta) on P450c17/CNT. <b>d,</b> The faradaic current in the a.c. components for P450c17 (blue) was suppressed upon interaction with wt cyt b5 (magenta); the a.c. data for wt cyt b5 (green) is shown as a control. (<b>e</b>) Schematic representation of the modes of interaction of the wt cyt b5 and the E48G/E49G cyt b5 with the P450c17/CNT electrode. All currents were normalized to the geometric electrode surface area (a-c) or to the amount of electroactive heme (d); The electrolyte solution in each case was a deoxygenated aqueous 0.20 M NaCl+0.02 M (K₂HPO₄+KH₂PO₄), pH = 7.0.</p

Reversible potentials (Eac0) of the surface confined Fe^3+/2+ redox couple derived from the a.c. voltammograms (<i>f</i> = 219 Hz)<i>^{^a}</i> obtained from CNT electrodes modified with hemin, wt cyt b5, E48G/E49G cyt b5, P450c17, and their combinations.

<p>^<i>a</i> Average of the potentials of the central minimum in the 6^th a.c. harmonic (envelope presentation, in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141252#pone.0141252.g003" target="_blank">Fig 3</a>) from the forward and backward d.c. potential sweep directions.</p><p>^<i>b</i> Values are reproducible to ±0.001 V.</p><p>^<i>c</i> Hemin adsorbed on the electrode pre-modified with wt cyt b5.</p><p>^<i>d</i> either wt or E48G/E49G cyt b5 adsorbed on the electrode pre-modified with P450c17.</p><p>Reversible potentials (<math><msubsup><mrow><mi>E</mi></mrow><mrow><mi>a</mi><mi>c</mi></mrow><mrow><mn>0</mn></mrow></msubsup></math>) of the surface confined Fe^3+/2+ redox couple derived from the a.c. voltammograms (<i>f</i> = 219 Hz)<i>^{^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141252#t001fn001" target="_blank">a</a>}}</i> obtained from CNT electrodes modified with hemin, wt cyt b5, E48G/E49G cyt b5, P450c17, and their combinations.</p

Quartz Crystal Microbalance data for proteins binding to a membrane layer.

<p><b>a,</b> QCM data showing changes in frequency (lines, left axes) and dissipation (symbols, right axes) (7^th harmonic; Δ<i>f</i>_QCM data are normalized to the overtone number) derived from deposition of proteins onto a DMPC-cholesterol membrane pre-deposited on a mpa-layer adhered to a Au-coated quartz crystal. QCM profiles obtained with protein mixtures 20 nM P450c17+20 nM CPR+20 nM wt cyt b5 (blue) and 20 nM P450c17 + 20 nM CPR + 100 nM wt cyt b5 (black) were similar to each other and also to that for 20 nM P450c17+20 nM CPR (Figure H in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141252#pone.0141252.s001" target="_blank">S1 File</a>). In contrast, the E48G/E49G cyt b5 was deposited non-specifically and competitively with P450c17+CPR, as determined with 20 nM P450c17+20 nM CPR+20 nM E48G/E49G cyt b5 (green) and especially 20 nM P450c17+20 nM CPR+100 nM E48G/E49G cyt b5 (red) mixtures. <b>b,</b> Analysis of the QCM data using Δ<i>f</i>_QCM<i>vs</i>. Δ<i>D</i> ‘fingerprint’ plots of the temporally resolved data from panel (a). The traces were divided into characteristic deposition stages labeled with roman numerals (I–III). DMPC = 1,2-dimyristoyl-<i>sn</i>-glycero-3-phosphocholine; mpa = mercaptopropionic acid.</p