14 research outputs found
Improving success rates for in meso crystallization using integral membrane proteins and membrane protein mimetics in the bicontinuous cubic phase
© 2016 Dr. Leonie van 't HagThe novel in meso crystallization method has facilitated the structural determination of several biologically relevant integral membrane proteins using macromolecular X-ray crystallography. To date, successful crystal growth from the lipid cubic phase is the bottleneck step in this process. An improved understanding of this technique can lead to increased success rates and more 3D structures of membrane proteins being solved, which are important for rational drug design for a wide range of diseases. Chapter 1 of this thesis outlines the relevant background about in meso crystallization, the cubic phase nanostructure, membrane proteins and peptides, methods for characterization and an outline of the thesis structure.
While the mechanism of in meso crystallization is still not well understood, the bulk bicontinuous cubic phase has been well studied and is thought to be essential for crystallization in meso. In order to improve success rates for in meso crystallization we therefore need to understand the effect of multiple components on the cubic phase nanostructure within the context of a crystallization trial. An extensive review of the literature on the effect of additives, such as cholesterol and phospholipids that modify the fluidity of the lipid bilayer and soluble components like PEG and ions that are present in crystallization screens on the cubic phase nanostructures is therefore included in this thesis in Chapter 2. Lyotropic liquid crystal engineering design rules were established that can be used to improve the experimental design for in meso crystallization, since they will facilitate selection of the best lipid bilayer composition, cubic phase nanostructure and crystallization screen components. Crystallization screen conditions that control the water activity and rate of water evaporation are also favorable to maximize the likelihood of obtaining large and well-ordered membrane protein crystals.
The effect of integral membrane proteins and peptides on the cubic phase nanostructure is equally of importance, as protein/peptide-lipid combinations that do not retain the cubic phase may not be suitable for in meso experiments. An improved understanding of the factors controlling peptide and protein encapsulation can also help to maximize protein loading, which is important to achieve supersaturation in the system to induce crystallization. These factors were studied using a wide range of different proteins and peptides, including synthetic WALP peptides with varying hydrophilic domain length, which are membrane protein mimics (Chapter 3). The Ag43 (Chapter 4) and BamA-E (Chapter 5) proteins from Gram-negative bacteria were also studied. It was found that the hydrophilic domain size and charge were the most important factors governing the changes in the lipid mesophase nanostructure. For this reason, cubic phase water channels that are larger than the hydrophilic domain of the protein may be preferred for in meso crystallization. In addition, screening of protein charges such that the Debye length is < 1 nm may be of use in preventing charge-induced swelling and disruption of the cubic phase. Hydrophobic mismatch and the diameter of the hydrophobic domain, the specific amino acid sequence of the proteins and local charges were also found to have an effect. This suggests that a strategy of matching of the hydrophobic segment of the lipid to the hydrophobic protein domain should also be used.
It was observed in Chapters 3 and 4 that there were significant differences in protein and peptide α-helical and β-sheet secondary structures when present in detergent micelles compared to the lipid membrane of the cubic phase. Even though these conformational changes may be small, they could affect membrane protein function. The activity of the Neisserial amphiphilic protein Lipid A PEA Transferase (NmEptA) upon encapsulation in the cubic phase was therefore studied directly in Chapter 6. Transfer of the phosphoethanolamine (PEA) headgroup of different phospholipids that were doped in the cubic phase membrane indicated the enzyme was active, even though the large hydrophilic domain must be confined within the nanoscale water channels. This reaffirms that cubic phase water channels larger than the hydrophilic domain of the protein might be favorable for encapsulation. The enzymatic reaction could also be followed using high-throughput techniques, reinforcing the prospect of using high-throughput sample preparation and analysis methods as a drug screening tool in meso.
In Chapters 7 and 8, the mechanism for in meso crystallization was studied. It is shown in Chapter 7 that small-angle neutron scattering can be used to study the location of peptides within the contrast-matched cubic phase. A preferential location of membrane proteins at the flat points of the cubic phase was suggested in several modelling studies. This arrangement was suggested to be a driving force for nucleation during in meso crystallization. No enrichment was observed, however, for the model WALP21 and WALPS53 peptides at the flat points or most negative Gaussian curvature saddle points of the diamond cubic phase of phytanoyl monoethanolamine indicating the importance of characterizing proteins with different lengths or physicochemical characteristics. The work presented in this thesis is the first time that contrast-matching techniques for neutron scattering were used for experiments with the bicontinuous cubic phase. This study is therefore expected to provide new insights into the assembly of these complex hybrid protein-lipid materials and to assist further studies.
In Chapter 8, the proposed mechanism for in meso crystallization was explored during crystallization of the single transmembrane α-helical peptide DAP12-TM. Small-angle X-ray scattering results with a micro-sized beam were found to be consistent with the proposed mechanism for in meso crystallization; crystallization occurred from the gyroid cubic mesophase via a highly oriented local lipid lamellar phase. No other direct experimental evidence for this mechanism has been published since a single study on bacteriorhodopsin in 2007. A new experimental protocol using equipment available in standard crystallography laboratories should enable further testing of the proposed mechanism using different proteins. Diffraction peaks at wide angles related to the peptide crystals were also observed, which are of potential use in locating crystals in meso, as they are often difficult to observe within the lipid cubic phase.
In Chapter 9 the conclusions and future perspectives of this thesis are presented. The research includes important new insights with respect to lipid–protein interactions, the location of amphiphilic peptides when encapsulated in the bicontinuous cubic phase and the mechanism of in meso crystallization. Advances in our understanding of the relationship between the lipidic material and the encapsulated protein as a result of this work should lead to improvements in experimental design for in meso crystallization and subsequently a higher success rate for this promising technique
Drying of African leafy vegetables for their effective preservation: the difference in moisture sorption isotherms explained by their microstructure
The problem of malnutrition and nutrition deficiency, as well as droughts that lead to reduction in food supply and starvation, is well documented for Sub-Saharan Africa. Reducing post-harvest losses of five species of African leafy vegetables (ALVs) by preservation through drying is studied herein. Energy efficient gentle drying conditions using superabsorbent polymers and a temperature of 40 °C were shown to preserve most leaf structures and vitamins. The microbial safe moisture content of the ALVs was found to be ≤14% dry basis. Dried Slender Leaf and Nightshade leaves could be rehydrated to the equilibrium moisture content of fresh leaves upon dry storage, while it was not possible for Jute Mallow, Cowpea and Amaranthus. This was attributed to different palisade parenchyma cell lengths. An increased amount of starch granules as observed in the microstructure of Cowpea and Nightshade leaves is suggested to explain their fibrous texture upon cooking. These results show that the ALVs can be effectively preserved using the same drying method and that this can be used to fight micro-nutrient deficiencies during droughts.ISSN:2042-6496ISSN:2042-650
Membrane Protein Structures in Lipid Bilayers; Small-Angle Neutron Scattering With Contrast-Matched Bicontinuous Cubic Phases
. Deuterated vesicles can be used to obtain the radius of gyration of membrane proteins, but protein-protein interference effects within the vesicles severely limits this method such that the protein structure cannot be modeled. We show herein that different membrane protein conformations can be distinguished within the lipid bilayer of the bicontinuous cubic phase using contrast-matching. Time-resolved studies performed using SANS illustrate the complex phase behavior in lyotropic liquid crystalline systems and emphasize the importance of this development. We believe that studying membrane protein structures and phase behavior in contrast-matched lipid bilayers will advance both biological and pharmaceutical applications of membrane-associated proteins, biosensors and food science
In Meso Crystallization: Compatibility of Different Lipid Bicontinuous Cubic Mesophases with the Cubic Crystallization Screen in Aqueous Solution
In
meso crystallization uses bicontinuous cubic lipidic mesophases
as matrices for the crystallization of membrane proteins. In this
work, we look at the impact of a screen specifically marketed as compatible
with the cubic mesophase, the Cubic crystallization screen (Emerald
BioSystems), on the cubic mesophases formed by three different lipids:
monoolein, monopalmitolein, and phytantriol. The Cubic screen was
found to be compatible with cubic mesophase retention under most crystallization
conditions for all three lipids studied. The effect of the individual
components comprising the multicomponent screen was deconvoluted in
two ways. Initially, the effect of specific polyÂ(ethylene glycol)
(PEG) and salt components on the cubic mesophase was determined using
small-angle X-ray scattering (SAXS). The effect of high-molecular-weight
(<i>M</i><sub>w</sub>) PEG was shown to dominate the phase
behavior within the screen. The effect of additional salts present
within the screen becomes important for low <i>M</i><sub>w</sub> PEG molecules. Finally, a recently developed multiple linear-regression
modeling method was shown to deconvolute the effect of individual
components within the screen effectively
Effect of Lipid-Based Nanostructure on Protein Encapsulation within the Membrane Bilayer Mimetic Lipidic Cubic Phase Using Transmembrane and Lipo-proteins from the Beta-Barrel Assembly Machinery
A fundamental
understanding of the effect of amphiphilic protein
encapsulation on the nanostructure of the bicontinuous cubic phase
is crucial to progressing biomedical and biological applications of
these hybrid protein–lipid materials, including as drug delivery
vehicles, as biosensors, biofuel cells and for <i>in meso</i> crystallization. The relationship between the lipid nanomaterial
and the encapsulated protein, however, remains poorly understood.
In this study, we investigated the effect of incorporating the five
transmembrane and lipo-proteins which make up the β-barrel assembly
machinery from Gram-negative bacteria within a series of bicontinuous
cubic phases. The transmembrane β-barrel BamA caused an increase
in lattice parameter of the cubic phase upon encapsulation. By contrast,
the mainly hydrophilic lipo-proteins BamB–E caused the cubic
phase lattice parameters to decrease, despite their large size relative
to the diameter of the cubic phase water channels. Analysis of the
primary amino acid sequence was used to rationalize this effect, based
on specific interactions between aromatic amino acids within the proteins
and the polar–apolar interface. Other factors that were found
to have an effect were lateral bilayer pressure and rigidity within
the lipid bilayer, water channel diameter, and size and structure
of the lipo-proteins. The data presented suggest that hydrophilic
bioactive molecules can be selectively encapsulated within the cubic
phase by using a lipid anchor or aromatic amino acids, for drug delivery
or biosensing applications
Transmembrane Complexes of DAP12 Crystallized in Lipid Membranes Provide Insights into Control of Oligomerization in Immunoreceptor Assembly
The membrane-spanning α helices of single-pass receptors play crucial roles in stabilizing oligomeric structures and transducing biochemical signals across the membrane. Probing intermolecular transmembrane interactions in single-pass receptors presents unique challenges, reflected in a gross underrepresentation of their membrane-embedded domains in structural databases. Here, we present two high-resolution structures of transmembrane assemblies from a eukaryotic single-pass protein crystallized in a lipidic membrane environment. Trimeric and tetrameric structures of the immunoreceptor signaling module DAP12, determined to 1.77-Å and 2.14-Å resolution, respectively, are organized by the same polar surfaces that govern intramembrane assembly with client receptors. We demonstrate that, in addition to the well-studied dimeric form, these trimeric and tetrameric structures are made in cells, and their formation is competitive with receptor association in the ER. The polar transmembrane sequences therefore act as primary determinants of oligomerization specificity through interplay between charge shielding and sequestration of polar surfaces within helix interfaces
Deconvoluting the Effect of the Hydrophobic and Hydrophilic Domains of an Amphiphilic Integral Membrane Protein in Lipid Bicontinuous Cubic Mesophases
Lipidic bicontinuous cubic mesophases
with encapsulated amphiphilic
proteins are widely used in a range of biological and biomedical applications,
including in meso crystallization, as drug delivery vehicles for therapeutic
proteins, and as biosensors and biofuel cells. However, the effect
of amphiphilic protein encapsulation on the cubic phase nanostructure
is not well-understood. In this study, we illustrate the effect of
incorporating the bacterial amphiphilic membrane protein Ag43, and
its individual hydrophobic β<sup>43</sup> and hydrophilic α<sup>43</sup> domains, in bicontinuous cubic mesophases. For the monoolein,
monoalmitolein, and phytantriol cubic phases with and without 8% w/w
cholesterol, the effect of the full length amphiphilic protein Ag43
on the cubic phase nanostructure was more significant than the sum
of the individual hydrophobic β<sup>43</sup> and hydrophilic
α<sup>43</sup> domains. Several factors were found to potentially
influence the impact of the hydrophobic β<sup>43</sup> domain
on the cubic phase internal nanostructure. These include the size
of the hydrophobic β<sup>43</sup> domain relative to the thickness
of the lipid bilayer, as well as its charge and diameter. The size
of the hydrophilic α<sup>43</sup> domain relative to the water
channel radius of the cubic mesophase was also found to be important.
The secondary structure of the Ag43 proteins was affected by the hydrophobic
thickness and physicochemical properties of the lipid bilayer and
the water channel diameter of the cubic phase. Such structural changes
may be small but could potentially affect membrane protein function
Data from: Exploring the in meso crystallization mechanism by characterizing the lipid mesophase microenvironment during the growth of single transmembrane α-helical peptide crystals
The proposed mechanism for in meso crystallisation of transmembrane proteins suggests that a protein or peptide is initially uniformly dispersed in the lipid self-assembly cubic phase but that crystals grow from a local lamellar phase, which acts as a conduit between the crystal and the bulk cubic phase. However, there is very limited experimental evidence for this theory. We have developed protocols to investigate the lipid mesophase microenvironment during crystal growth using standard procedures readily available in crystallography laboratories. This technique was used to characterize the microenvironment during crystal growth of the DAP12-TM peptide using synchrotron Small Angle X-ray Scattering with a micro-sized X-ray beam. Crystal growth was found to occur from the Gyroid cubic mesophase. For one in four crystals a highly-oriented local lamellar phase was observed, providing supporting evidence for the proposed mechanism for in meso crystallisation. A new observation of this study was that we can differentiate diffraction peaks from crystals grown in meso, from peaks originating from the surrounding lipid matrix, potentially opening up the possibility of high-throughput SAXS analysis of in meso grown crystals
Data from: Exploring the in meso crystallization mechanism by characterizing the lipid mesophase microenvironment during the growth of single transmembrane α-helical peptide crystals
The proposed mechanism for in meso crystallisation of transmembrane proteins suggests that a protein or peptide is initially uniformly dispersed in the lipid self-assembly cubic phase but that crystals grow from a local lamellar phase, which acts as a conduit between the crystal and the bulk cubic phase. However, there is very limited experimental evidence for this theory. We have developed protocols to investigate the lipid mesophase microenvironment during crystal growth using standard procedures readily available in crystallography laboratories. This technique was used to characterize the microenvironment during crystal growth of the DAP12-TM peptide using synchrotron Small Angle X-ray Scattering with a micro-sized X-ray beam. Crystal growth was found to occur from the Gyroid cubic mesophase. For one in four crystals a highly-oriented local lamellar phase was observed, providing supporting evidence for the proposed mechanism for in meso crystallisation. A new observation of this study was that we can differentiate diffraction peaks from crystals grown in meso, from peaks originating from the surrounding lipid matrix, potentially opening up the possibility of high-throughput SAXS analysis of in meso grown crystals