Luke A. Clifton

Binding of pentagalloyl glucose to two globular proteins occurs via multiple surface sites

The interaction between pentagalloyl glucose (PGG) and two globular proteins, bovine serum albumin (BSA) and ribulose-1,5-bisphosphate carboxylase oxygenase (rubisco), was investigated by isothermal titration calorimetry (ITC). ITC data fit to a binding model consisting of two sets of multiple binding sites, which reveal similarities in the mode of binding of PGG to BSA and rubisco. In both cases, the interaction is characterized by a high number of binding sites, which suggests that binding occurs by a surface adsorption mechanism that leads to coating of the protein surface, which promotes aggregation and precipitation of the PGG-protein complex. This model was confirmed by turbidimetry analysis of the PGG-BSA interaction. Analysis of tryptophan fluorescence quenching during the interaction of PGG with BSA suggests that binding of PGG leads to some conformational changes that are energetically closer to the unfolded state of the BSA structure, because small red shifts in the resulting emission spectra were observed.

Interaction of Amphotericin B with Lipid Monolayers

Langmuir isotherm, neutron reflectivity, and Brewster angle microscopy experiments have been performed to study the interaction of amphotericin B (AmB) with monolayers prepared from 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and mixtures of this lipid with cholesterol or ergosterol to mimic mammalian and fungal cell membranes, respectively. Isotherm data show that AmB causes a more pronounced change in surface pressure in the POPC/ergosterol system than in the POPC and POPC/cholesterol systems, and its interaction with the POPC/ergosterol monolayer is also more rapid than with the POPC and POPC/cholesterol monolayers. Brewster angle microscopy shows that, in interaction with POPC monolayers, AmB causes the formation of small domains which shrink and disappear within a few minutes. The drug also causes domain formation in the POPC/cholesterol and POPC/ergosterol monolayers; in the former case, these are formed more slowly than is seen with the POPC monolayers and are ultimately much smaller; in the latter case, they are formed rather more quickly and are more heterogeneous in size. Neutron reflectivity data show that the changes in monolayer structure following interaction with AmB are the same for all three systems studied: the data are consistent with the drug inserting into the monolayers with its macrocyclic ring intercalated among the lipid acyl chains and sterol ring systems, with its mycosamine moiety colocalizing with the sterol hydroxyl and POPC head groups. On the basis of these studies, it is concluded that AmB inserts in a similar manner into POPC, POPC/cholesterol, and POPC/ergosterol monolayers but does so with differing kinetics and with the formation of quite different in-plane structures. The more rapid time scale for interaction of the drug with the POPC/ergosterol monolayer, its more pronounced effect on monolayer surface pressure, and its more marked changes as regards domain formation are all consistent with the drugs selectivity for fungal vs mammalian cell membranes.

Examining Protein–Lipid Complexes Using Neutron Scattering

Studying the structure of protein-lipid complexes, be they in vesicles, planar bilayers, monolayers, or nanodiscs, poses two particular challenges. Firstly such complexes are often dynamic. Secondly we need to resolve the lipid and protein structures within the complex. Neutron scattering is well placed to help in both respects since it deals with molecules in large, complex, dynamic structures and can easily differentiate between different molecular species. This comes from the great penetrating power of neutrons and their sensitivity to the difference between hydrogen (H) and deuterium (D). Both membrane proteins and lipids can be produced with varying degrees of deuteration, thus allowing us to dissect complexes with great accuracy. Two main scattering techniques are immediately applicable to the study of protein-lipid interactions. Neutron reflection exploits the constructive interference, which occurs when neutrons are reflected from different points in a layer. An everyday example is the rainbow of colors reflected from an oil film on water, which result from varying film thickness and the angle of reflection. Neutrons because of their short wavelengths (4-15 Å) and H/D sensitivity can, in reflectometry mode, provide accurate cross sections of lipid monolayers and bilayers. Small-angle neutron scattering (SANS) can resolve the structures of protein-lipid complexes if they are present as homogeneous dispersions. This is easiest with detergent micelles, but increasingly methods are being developed whereby vesicles, nanodiscs, etc., can be resolved. Again the ability to deuterate proteins and lipids enables SANS to resolve the inner structure of big, dynamic, lipid-protein complexes. The recent introduction of advanced neutron beam lines means that the technique is now within the grasp of a broad cross section of researchers.

Complete bilayer adsorption of C16TAB on the surface of mica using neutron reflection.

We present neutron reflection data from an alkylammonium surfactant (C16TAB) at the mica/water interface. The system is studied in situ in a noninvasive manner and indicates the formation of a complete adsorbed bilayer with little evidence of defects. A detailed analysis suggests that the data are not consistent with some other previously reported adsorbed structures, such as micelles or cylinders.

Specular neutron reflection at the mica/water interface – irreversible adsorption of a cationic dichain surfactant

Neutron reflection from the important mineral mica at the solid/liquid interface is presented here using a new approach – a very thin mica crystal supported on a silicon substrate. This approach avoids the problems of crystal defects and surface undulations that have hindered previous work. The use of mica as a reflectivity substrate is important as it is a model surface, which is atomically smooth with a high structural charge. In this work the mica/water interface is fully characterized. In particular, a characteristic double critical edge is observed, arising from the higher scattering length densities of the mica and D2O subphase relative to the silicon support. The experimental data are modelled using a combined approach: conventional amplitude summation (matrix method) for the thin layers and reflected intensity summation with attenuation terms for the thick layers of mica and hydrocarbon adhesive. Reflection data from the adsorption of the dichain cationic surfactant didodecyldimethylammonium bromide (DDAB) to the surface of muscovite mica from aqueous solution are also presented. It is found that, at twice the critical micelle concentration, a bilayer of DDAB with a thickness of 24 A is observed, containing essentially no water. Its partial removal by washing and ion exchange is also presented.

A comparison of didodecyldimethylammonium bromide adsorbed at mica/water and silica/water interfaces using neutron reflection.

The layer structure of the dichain alkyl ammonium surfactant, didodecyldimethylammonium bromide (DDAB), adsorbed from water on to silica and mica surfaces has been determined using neutron reflection. Although sometimes considered interchangeable surfaces for study, we present evidence of significant differences in the adsorbed layer structure below the critical micelle concentration. A complete DDAB bilayer was assembled at the water/mica interface at concentrations below the critical micelle concentration (CMC). In contrast it is not until the CMC was reached that the complete bilayer structure formed on the oxidised silicon crystal. Removal of the complete bilayer on both surfaces was attempted by both washing and ion exchange yet the adsorbed structure proved tenacious.

Lyophilised protein dynamics: more than just methyls?

Neutron spectroscopy has been used to probe picosecond to nanosecond dynamics in lyophilised apoferritin, insulin, superoxide dismutase and green fluorescent protein. These proteins have markedly different secondary structures yet similar CH3 compositions. Results suggest that while only CH3 activation is apparent in apoferritin, an enhanced dynamic environment presents itself in Ins, SOD and GfP. Our results hint at a structure dependent dynamic landscape.

Adsorption of Sodium Hexanoate on α-Alumina

Neutron reflection and adsorption isotherm measurements have been used to study the adsorption behaviour of hexanoic acid onto α-alumina surfaces. Importantly, the pH dependence of the behaviour has been characterised with a pronounced maximum in adsorption identified at a pH of approximately 5, close to the pKa of the acid. The adsorbed layer is identified as a bilayer, which is reasonable given the hydrophilic nature of both side of the layer, and has a thickness of 13 Å, suggesting significant extent of interdigitation. At pH 5, the layer has much lower extent of hydration relative to the higher pH of 7, consistent with the increased total adsorption at pH 5. A number of different mechanisms for the binding of the hexanoic acid to the surface are considered. The experimental data, combined with calculations using equilibrium/binding constants of the surface and ligands, indicates that a ligand exchange reaction may be the most significant mechanism.

Thermal motion in the multi-subunit protein, apoferritin, as probed by high energy resolution neutron spectroscopy

Insight into the dynamic landscape of the multi-subunit protein, apoferritin, using neutron spectroscopy is presented in this paper. We combine elastic and quasi-elastic neutron scattering data, collected using different neutron spectrometers, to probe length scales up to 10 A and timescales up to 2 ns. We show, for the first time without ambiguity, and via a thorough and systematic approach, that in its lyophilised form, apoferritin, above T ≈ 100 K and in the pico- to nanosecond time regime, exhibits a single dynamic response driven by methyl groups alone. No contribution is observed from protons associated with non-methyl species. A distribution of CH3 activation energies is obtained in line with the environmental heterogeneity that exists around the methyl species in this protein. In addition, by performing a complete and detailed analysis of the neutron scattering data, we prove the validity of the theoretical assumptions required by the methyl group activation model used to analyse the observed spectral response.

Neutron reflectivity measurement of protein A–antibody complex at the solid-liquid interface

Highlights • The orientation of IgG4 adsorbed at the solid-liquid interface was probed.• A chromatography resin was mimicked by attaching protein A to a silica surface.• Neutron reflectivity was used to measure protein A and adsorbed IgG structures.• Protein A-modified silica was blocked with either BSA or PEG before IgG adsorption.• Adsorbed IgG extended up to 230 Å from the surface, depending on blocking strategy.