Quantitative Determination of Lateral Concentration and Depth Profile of Histidine-Tagged Recombinant Proteins Probed by Grazing Incidence X-ray Fluorescence (original) (raw)
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Langmuir, 2002
Protein two-dimensional (2D) crystallization on lipid monolayers is a powerful method for structure determination. This method has been extended using the specific and strong interaction between histidine residues (of an overexpressed protein) and Ni 2+ ions tethered at the headgroup of synthetic lipids. Understanding and then improving the process of adsorption and crystallization of proteins on a lipid monolayer are prerequisites for the production of large and well-ordered crystals of any soluble or membrane His-tagged proteins. These large high-quality arrays are necessary for structural studies at high resolution. We have investigated the steps of adsorption and 2D crystallization of His-HupR using three different lipids: (i) 2-(bis-carboxymethyl-amino)-6-[2-(1,3-di-O-oleyl-glyceroxy)-acetyl-amino] hexanoic acid nickel-(II) (Ni-NTA-DOGA), which has been previously used, and two specifically designed Ni 2+ -chelating lipids, (ii) Ni-NTA-BB, which has two branched (B) alkyl chains and (iii) Ni-NTA-BF, a nonsymmetrical lipid with one branched (B) and one fluorinated (F) chain. These three lipids, when spread at the air-water interface, exhibit various fluidity properties. The adsorption and crystallization process have been monitored in situ and in real time using a variety of complementary techniques such as ellipsometry, shear rigidity measurements of the monolayer, and Brewster angle microscopy, and we have also developed X-ray reflectivity analysis to investigate the evolution of the electron density profile of the lipid-protein monolayer. Electron microscopy observations of the protein-lipid layers were also performed. We have found that the fluidity of the lipid monolayer has a marked influence on the rates of protein adsorption and crystallization of His-HupR. When Ni-NTA-BB is used to form the monolayer, it accelerates the process of protein adsorption and the protein crystallization is three times faster than when Ni-NTA-DOGA is used. (1) Uzgiris, E. E.; Kornberg, R. D. Nature 1983, 301, 125. (2) Jap, B. K.; Zulauf, M.; Scheybani, T.; Hefti, A.; Baumeister, W.; Aebi, U.; Engel, A. Ultramicroscopy 1992, 46, 45. (3) Lebeau, L.; Schultz, P.; Célia, H.; Mésini, P.; Nuss, S.; Klinger, C.; Olland, S.; Oudet, P.; Mioskowski, C. In Handbook of nonmedical applications of liposomes;
Soft Matter, 2008
Interactions of the antimicrobial peptide protegrin-1 (PG-1) with phospholipid monolayers have been investigated by using grazing incidence X-ray diffraction (GIXD) and specular X-ray reflectivity (XR). The structure of a PG-1 film at the air-aqueous interface was also investigated by XR for the first time. Lipid A, dipalmitoyl-phosphatidylglycerol (DPPG) and dipalmitoyl-phosphatidylcholine (DPPC) monolayers were formed at the air-aqueous interface to mimic the surface of the bacterial cell wall and the outer leaflet of the erythrocyte cell membrane, respectively. Experiments were carried out under constant area conditions where the pressure changes upon insertion of peptide into the monolayer. GIXD data suggest that the greatest monolayer disruption produced by PG-1 is seen with the DPPG system at 20 mN/m since the Bragg peaks completely disappear after introduction of PG-1 to the system. PG-1 shows greater insertion into the lipid A system compared to the DPPC system when both films are held at the same initial surface pressure of 20 mN/m. The degree of insertion lessens at 30 mN/m with both DPPC and DPPG monolayer systems. XR data further reveal that PG-1 inserts primarily in the head group region of lipid monolayers. However, only the XR data of the anionic lipids suggest the existence of an additional adsorbed peptide layer below the head group of the monolayer. Overall the data show that the extent of peptide/lipid interaction and lipid monolayer disruption depends not only on the lipid composition of the monolayer, but the packing density of the lipids in the monolayer prior to the introduction of peptide to the subphase.
The structure of supported lipid bilayers formed on a monolayer of nanoparticles was determined using a combination of grazing incidence X-ray and neutron scattering techniques. Ordered nanoparticle arrays assembled on a silicon crystal using a Langmuir-Schaefer deposition were shown to be suitable and stable substrates for the formation of curved and fluid lipid bilayers that retained lateral mobility, as shown by fluorescence recovery after photobleaching. A comparison between the structure of the curved bilayer assembled around the nanoparticles with the planar lipid membrane formed on the flat underlying silicon oxide surface revealed a ~5 Angstrom thinner bilayer on the curved interface, resolving the effects of curvature on the lipid packing and overall bilayer structure. The combination of neutron scattering techniques, which grant access to sub-nanometre scale structural information at buried interfaces, and nanoparticle-supported lipid bilayers, offers a novel approach to i...
XANES Measurements for Studies of Adsorbed Protein Layers at Liquid Interfaces
Materials, 2020
X-ray absorption near edge structure (XANES) spectra for protein layers adsorbed at liquid interfaces in a Langmuir trough have been recorded for the first time. We studied the parkin protein (so-called E3 ubiquitin ligase), which plays an important role in pathogenesis of Parkinson disease. Parkin contains eight Zn binding sites, consisting of cysteine and histidine residues in a tetracoordinated geometry. Zn K-edge XANES spectra were collected in the following two series: under mild radiation condition of measurements (short exposition time) and with high X-ray radiation load. XANES fingerprint analysis was applied to obtain information on ligand environments around zinc ions. Two types of zinc coordination geometry were identified depending on X-ray radiation load. We found that, under mild conditions, local zinc environment in our parkin preparations was very similar to that identified in hemoglobin, treated with a solution of ZnCl2 salt. Under high X-ray radiation load, conside...
Phospholipid and Protein Monolayers
Japanese Journal of Applied Physics, 1995
We report on X-ray reflectivity and diffraction studies using monolayers of phospholipids and of the protein streptavidin specifically bound to monolayers. For phospholipids with the phosphocholine head group attached to the glycerol backbone via (flexible) ethylene oxide spacers, we demonstrate that the lateral interactions can be reduced by increasing of the spacer length. This is reflected in a reduction of the tilt angle of aliphatic tails. Diffraction of the protein layer can be observed in situ. The data reveal that positional order is merely short-ranged and that the structure can be changed reversibly via film compression and expansion.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1997
The energy-dispersive X-ray reflectometry and turbidity measurements are used to investigate the kinetics of concanavalin A binding onto the distearoylphosphatidylcholinerdistearoylphosphatidylethanolamine-maltobionamide Ž. DSPCrDSPE-mal or distearoylphosphatidylcholinerdistearoylphosphatidylethanolamine-maltotetrabionamide 1 Ž. DSPCrmal mixed monolayer at the air-water interface. The resulting adsorbed layer of this sugar-binding protein near 3 the membrane with one or three hexoses in the lipid head-group is 3.9 nm or 9.7 nm thick, respectively. The different thicknesses of the adsorbed layer can be correlated with the diverse orientations of the adsorbed proteins. These lay flat on the surface containing DSPE-mal and 'perpendicular' to the surface containing DSPE-mal. The monolayer structure is 1 3 little affected by concanavalin A binding, but the incorporation of sugar lipids decreases the chain tilt and the interfacial thickness marginally. The binding is quasi-exponential with the time constant between some minutes and several hours depending on the concanavalin A and vesicle concentrations in the bulk. The experimental resolution of the time-resolved measurements made with the laboratory-based instrument is 15 min and the spatial resolution is between 0.05 nm and 0.5 nm, depending on the electron contrast. It is estimated that the high-brilliance synchrotron X-ray source combined with the detection method outlined in this work, could permit the kinetic measurements on the timescale of-1 minute.
X-ray reflectivity study of cyclic peptide monolayers at the air-water interface
Israel Journal of Chemistry, 2005
The dynamic and living characteristics of monolayers at the air-water interface of a cyclohexapeptide (C6G) and a cyclooctapeptide (C8G), both composed of glutamic acid and 3-aminobenzoic acid subunits in an alternating sequence, were investigated using the Langmuir balance technique, Brewster angle microscopy (BAM), and X-ray reflectivity (XR). An alanine-containing cyclohexapeptide (C6A) was included in this study for comparison. All three cyclopeptides preferentially adopt an orientation parallel to the subphase at low surface pressure. Continuous compression then causes the molecules to flip to a perpendicular state, thus minimizing their molecular area. In contrast to C8G and C6A, a pronounced hysteresis observed during the compression-expansion cycle of C6G indicates that strong intermolecular interactions between the cyclopeptide rings occur in the monolayers of this peptide. This result is supported by BAM measurements that show the formation of crystallite structures for C6G at high surface pressures, whereas no structures were observed for C8G and C6A. These results indicate that C6G is able to self-assemble upon surface compression, an ability that is obviously critically dependent on the correct ring size and composition of the peptide. The presence of hydrogen bond acceptors in the side chains of C6G suggests that the structural stabilization of the monolayer is due to H-bonding, possibly between ring NH groups and side chain CO groups. Our in situ study thus provides a detailed understanding of the molecular dynamics and uninterrupted interfacial behavior of the three peptides in a real-time frame.
European Biophysics Journal With Biophysics Letters, 2011
Studying membrane active peptides or protein fragments within the lipid bilayer environment is particularly challenging in the case of synthetically modified, labeled, artificial, or recently discovered native structures. For such samples the localization and orientation of the molecular species or probe within the lipid bilayer environment is the focus of research prior to an evaluation of their dynamic or mechanistic behavior. X-ray scattering is a powerful method to study peptide/lipid interactions in the fluid, fully hydrated state of a lipid bilayer. For one, the lipid response can be revealed by observing membrane thickening and thinning as well as packing in the membrane plane; at the same time, the distinct positions of peptide moieties within lipid membranes can be elucidated at resolutions of up to several angstroms by applying heavy-atom labeling techniques. In this study, we describe a generally applicable X-ray scattering approach that provides robust and quantitative information about peptide insertion and localization as well as peptide/lipid interaction within highly oriented, hydrated multilamellar membrane stacks. To this end, we have studied an artificial, designed β-helical peptide motif in its homodimeric and hairpin variants adopting different states of oligomerization. These peptide lipid complexes were analyzed by grazing incidence diffraction (GID) to monitor changes in the lateral lipid packing and ordering. In addition, we have applied anomalous reflectivity using synchrotron radiation as well as in-house X-ray reflectivity in combination with iodine-labeling in order to determine the electron density distribution ρ(z) along the membrane normal (z axis), and thereby reveal the hydrophobic mismatch situation as well as the position of certain amino acid side chains within the lipid bilayer. In the case of multiple labeling, the latter technique is not only applicable to demonstrate the peptide’s reconstitution but also to generate evidence about the relative peptide orientation with respect to the lipid bilayer.
Langmuir, 2001
We present isotherm and X-ray reflectivity (XR) measurements from Langmuir monolayers of a de novo synthetic di-R-helical peptide, consisting of two identical 31-residue, mostly R-helical peptide units joined by a disulfide bond at their amino-termini. Fitting the XR data to slab models shows that the dihelices lie in the plane of the interface at low pressures. The monolayers were insufficiently stable for study at high pressures, but Langmuir films based on a derivative of the peptide alkylated at its amino termini permitted investigations over a larger range of pressures. We observed an orientational transition, in which the R-helices begin by lying in the plane of the interface at low surface pressures and orient themselves approximately normal to the interface at high pressures. We draw the same conclusions from the XR data when we analyze it using box refinement, an iterative, model-independent method for recovering structure from XR data. Mixtures of these palmitoylated peptides with a fatty acid (palmitic acid) or a phospholipid (DLPE) behaved similarly. None of the systems produced peaks in the grazing incidence diffraction signal indicative of long-range ordering of the upright R-helices. Off-specular in-plane scattering measurements based on the difference signal between the peptide/DLPE mixture and pure DLPE suggest that the peptide achieves only liquidlike order within the plane. We discuss the implications and prospects for future work on designed peptide monolayers incorporating prosthetic groups that could be used to study electron transfer in proteins and provide a basis for biomolecular electronics applications.
The Journal of Physical Chemistry B, 2015
The influence of phospholipid oxidization of floating monolayers on the structure perpendicular to the global plane and on the density profiles of ions near the lipid monolayer has been investigated by a combination of grazing incidence X-ray fluorescence (GIXF) and specular X-ray reflectivity (XRR). Systematic variation of the composition of the floating monolayers unravels changes in the thickness, roughness and electron density of the lipid monolayers as a function of molar fraction of oxidized phospholipids. Simultaneous GIXF measurements enable one to qualitatively determine the element-specific density profiles of monovalent (K + or Cs + ) and divalent ions (Ca 2+ ) in the vicinity of the interface in the presence and absence of two types of oxidized phospholipids (PazePC and PoxnoPC) with high spatial accuracy (±5 Å). We found the condensation of Ca 2+ near carboxylated PazePC was more pronounced compared to PoxnoPC with an aldehyde group. In contrast, the condensation of monovalent ions could hardly be detected even for pure oxidized phospholipid monolayers. Moreover, pure phospholipid monolayers exhibited almost no ion specific condensation near the interface. The quantitative studies with well-defined floating monolayers revealed how the elevation of lipid oxidization level alters the structures and functions of cell membranes.