Name: from constructs to crystals &#8211; towards structure determination of &#946;-barrel outer membrane proteins
Uploaded: 2016-07-04T13:00:00
Description: Watch this Scientific Journal Video about From Constructs to Crystals â€“ Towards Structure Determination of Î²-barrel Outer Membrane Proteins at JoVE.com

We would like to thank Herve Celia of the CNRS for providing the UV images and Chris Dettmar and Garth Simpson in the Department of Chemistry at Purdue University for providing the SONICC images. We would like to acknowledge funding from the National Institute of Diabetes and Digestive and Kidney Diseases and the Intramural Research Program at the National Institutes of Health. Additionally, we would like to acknowledge additional funding from the National Institute of General Medical Sciences (A.M.S. and C.J.), National Institute of Allergy and Infectious Diseases (N.N. 1K22AI113078-01), and the Department of Biological Sciences at Purdue University (N.N.).

1. Cloning and Expression
Note: To enable structural studies, sufficient quantities of highly purified protein must be prepared, and this generally starts with the cloning and overexpression of the target &#946;-barrel outer membrane protein (OMP) in E. coli (Figure 4). To date, all &#946;-barrel OMP structures, including those structures for mitochondrial VDAC, have been derived from bacterially expressed protein11. Here, general protocols are presented for ...

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&#946;-barrel OMPs serve essential roles in Gram-negative bacteria, mitochondria and chloroplasts and are important targets for structural analysis that offer a wealth of information about essential molecular mechanisms at the outer membranes of these respective organelles. However, producing enough sample for structural analysis is not always straightforward and therefore, a general pipeline is presented for the production of sufficient quantities of target &#946;-barrel OMPs for structure determination, explaining in d...

&#946;-barrel OMPs can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria1-3. While they serve similar roles as &#945;-helical proteins, they have a very different fold consisting of a central membrane-embedded &#946;-barrel domain ranging from 8-26 anti-parallel &#946;-strands with each strand being intimately connected to the two neighboring strands (Figures 1 and 2). The first and last strands of the &#946;-barrel domain then interact with one another, almost exclusively in an anti-parallel fashion (except for mitochondrial VDAC), to close and seal the &#946;-barrel domain from the surrounding membrane. All &#946;-barrel OMPs have extracellular loops of varying sequence and length which play an important role in ligand interactions and/or protein-protein contacts, with these loops sometimes being as large as 75 residues, such as found in Neisserial transferrin binding protein A (TbpA)4. &#946;-barrel OMPs can also have N-terminal or C-terminal periplasmic extensions which serve as additional domains for the protein&#39;s functional purpose (e.g., BamA5-7, FimD8,9, FadL10). While many types of &#946;-barrel OMPs exist11, two of the more common types are described below as examples for those less familiar with the field, (1) TonB-dependent transporters and (2) autotransporters.
TonB-dependent transporters (e.g., FepA, TbpA, BtuB, Cir, etc.) are essential for nutrient import and contain an N-terminal plug domain consisting of ~150 residues that is found tucked inside a C-terminal 22-stranded &#946;-barrel domain embedded into the outer membrane12 (Figure 3). While this plug domain prevents substrate from freely passing through the barrel domain, substrate binding induces a conformational change within the plug domain that leads to pore formation (either by plug rearrangement or by partial/full ejection of the plug) which can then facilitate substrate transport across the outer membrane into the periplasm. TonB-dependent transporters are especially important for the survival of some pathogenic strains of Gram-negative bacteria such as Neisseria meningitidis that have evolved specialized transporters that hijack nutrients such as iron directly from human host proteins4,13,14.
Autotransporters belong to the type V secretion system of Gram-negative bacteria and are &#946;-barrel OMPs that consist of a &#946;-barrel domain (typically 12-strands as with EstA and EspP) and a passenger domain that is either secreted or presented at the surface of the cell15,16 (Figure 3). These &#946;-barrel OMPs often serve important roles in cell survival and virulence with the passenger domain serving either as a protease, adhesin, and/or other effector that mediates pathogenesis.
Structural methods such as X-ray crystallography, NMR spectroscopy, and electron microscopy (EM) allow us to determine models for the OMPs at atomic resolution which can in turn be used to decipher exactly how they function within the outer membrane. This invaluable information may then be used for drug and vaccine development if applicable. For example, transferrin binding protein A (TbpA) is found on the surface of Neisseria and is required for pathogenesis because it directly binds human transferrin and then extracts and imports the iron for its own survival. Without TbpA, Neisseria cannot scavenge iron from the human host and are rendered non-pathogenic. After the crystal structure of human transferrin bound to TbpA4 was solved, it became much clearer how the two proteins associated, what regions of TbpA mediated the interaction, what residues were important for iron extraction by TbpA, and how one might develop therapeutics against Neisseria targeting TbpA. Therefore, given the importance of &#946;-barrel OMPs in Gram-negative bacteria for survival and pathogenesis, as well as in mitochondria and chloroplast function, and the need for additional structural information about this unique class of membrane proteins and the systems in which they function, general protocols are presented with the overall goal of expressing and purifying target OMPs at high levels for characterization by structural methods.

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			<td height="40" style="height:40px;width:175px;">Crystallization Robot</td>
			<td style="width:159px;">TTP Labtech, Art Robbins</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;">Any should work here, except for LCP crystallization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">PCR thermocycler</td>
			<td style="width:159px;">Eppendorf, BioRad</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:175px;">Media Shaker</td>
			<td style="width:159px;">New Brunswick, Infors HT</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">UV-vis spectrometer</td>
			<td style="width:159px;">Eppendorf</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:175px;">SDS-PAGE apparatus</td>
			<td style="width:159px;">BioRad</td>
			<td style="width:227px;">1645050, 1658005</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">SDS-PAGE and native gels</td>
			<td style="width:159px;">BioRad, Life Technologies</td>
			<td style="width:227px;">4561084, EC6035BOX (BN1002BOX)</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">AkTA Prime</td>
			<td style="width:159px;">GE Healthcare</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">AkTA Purifier</td>
			<td style="width:159px;">GE Healthcare</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Microcentrifuge</td>
			<td style="width:159px;">Eppendorf</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Centrifuge (low-medium speed)</td>
			<td style="width:159px;">Beckman-Coulter</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Ultracentrifuge (high speed)</td>
			<td style="width:159px;">Beckman-Coulter</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">SS34 rotor</td>
			<td style="width:159px;">Sorvall</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Type 45 Ti rotor</td>
			<td style="width:159px;">Beckman-Coulter</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Type 70 Ti rotor</td>
			<td style="width:159px;">Beckman-Coulter</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Dounce homogenizer</td>
			<td style="width:159px;">Fisher Scientific</td>
			<td style="width:227px;">06 435C</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Emulsiflex</td>
			<td style="width:159px;">Avestin</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Dialysis tubing</td>
			<td style="width:159px;">Sigma</td>
			<td style="width:227px;">D9652</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">LCP tools</td>
			<td style="width:159px;">Hamilton, TTP Labtech</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">VDX 24 well plates</td>
			<td style="width:159px;">Hampton Research</td>
			<td style="width:227px;">HR3-172</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:175px;">Sandwich plates</td>
			<td style="width:159px;">Hampton Research, Molecular Dimensions</td>
			<td style="width:227px;">HR3-151, MD11-50 (MD11-53)</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:175px;">Grace Crystallization sheets</td>
			<td style="width:159px;">Grace Bio-Labs</td>
			<td style="width:227px;">875238</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">HiPrep S300 HR column</td>
			<td style="width:159px;">GE Healthcare</td>
			<td style="width:227px;">17-1167-01</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Q-Sepharose column</td>
			<td style="width:159px;">GE Healthcare</td>
			<td style="width:227px;">17-0510-01</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:175px;">Crystallization screens</td>
			<td style="width:159px;">Hampton Research, Qiagen, Molecular Dimensions</td>
			<td style="width:227px;">-</td>
			<td style="width:244px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:175px;">Gas-tight syringe (100 mL)</td>
			<td style="width:159px;">Hamilton&#160;</td>
			<td style="width:227px;">????</td>
			<td></td>
		</tr>
	</tbody>
</table>

from constructs to crystals &#8211; towards structure determination of &#946;-barrel outer membrane proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

YiuR is a TonB dependent iron transporter that is a putative vaccine target against Yersinia pestis. It was originally identified using a microarray assay. Here, the steps that were taken to determine the structure of YiuR using X-ray crystallography are outlined (Figure 9). For cloning, the DNA sequence of YiuR (minus the N-terminal signal sequence) was PCR amplified from genomic DNA and subcloned into a vector containing an N-terminal pelB signal sequence and 1...

Watch this Scientific Journal Video about From Constructs to Crystals â€“ Towards Structure Determination of Î²-barrel Outer Membrane Proteins at JoVE.com

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

The overall goal of this method is to obtain milligram quantities of outer membrane beta-barrel proteins that will crystallize and defract. This method can help answer key questions in the membrane protein folding and transport field, such as how beta-barrel proteins are inserted in cell membranes. The main advantage of this technique is that it is robust for beta-barrel proteins.Generally, individuals new to this method will struggle, because choice of detergents for purification, crystallization, and defraction is challenging. Jeremy Guerin, a postdoc from Susan Buchanan's laboratory, will be demonstrating the procedure, in addition to myself and colleagues in the lab. Begin this procedure with construction of a T7 expression vector containing the codon optimized target outer membrane protein, or OMP gene, for in vivo expression to the membrane as described in the text protocol.Transform the construct into an expression strain of bacteria for expression by pipetting one microliter of plasmid into 50 microliters of BL21(DE3)chemically competent cells, and mix gently by pipetting up and down. Incubate on ice for 30 minutes. Following incubation, heat pulse at 42 degrees Celsius for 30 seconds using a water bath, and then place back on ice for one minute.Add one milliliter of pre-warmed SOC media and shake at 37 degrees Celsius for one hour at 1000 RPM using a bench-top shaker incubator. Following incubation, plate 100 microliters of cells onto LB Agar plates containing an appropriate antibiotic and incubate overnight at 37 degrees Celsius, inverted. Next, perform small-scale expression tests by inoculating five milliliters of LB containing antibiotic culture with a single colony.Repeat for five to 10 colonies. Grow at 37 degrees Celsius with shaking to an optical density at 600 nanometers of approximately zero point six. Induce expression of the target OMP by adding five microliters of one molar IPTG to each culture tube and allow to grow an additional one to two hours.To compare expression levels for all the colonies, centrifuge one milliliter of each culture for one minute at 15, 000 times G, using a microcentrifuge. Remove the supernatant and resuspend the cells in 200 microliters of 1X SDS-PAGE loading buffer. Heat at 100 degrees Celsius for five minutes, and then centrifuge again at 15, 000 times G for five minutes.Analyze the samples using SDS-PAGE by pipetting 20 microliters into each well of a ten per cent gel. Run the gel for 35 minutes at a constant 200 volts. Finally, harvest the cells by centrifugation at 6000 times G for ten minutes.Resuspend the cells in lysis buffer at a ratio of five milliliters per gram of cell paste. Lyse the cells using a French press or cell homogenizer. Then, spin the lysed cells at 15, 000 times G for 30 minutes at four degrees Celsius to remove unlysed cells and cell debris.Transfer the supernatant to a clean tube and centrifuge again at high speed for one hour at four degrees Celsius. The resulting pellet is the membrane fraction which contains the protein of interest. Using a Dounce homogenizer, resuspend the membrane fraction.First, transfer the membranes and then add solubilization buffer at 2X concentration without detergent. Pour the resuspended membranes into a graduated cylinder and add water until a final concentration of 1X solubilization buffer is reached. Transfer the sample to a beaker and add detergent slowly to a final concentration of approximately 10 times the critical micelle concentration.Then, stir for 0.5 to 16 hours at four degrees Celsius, depending on how easily the target protein is extracted from the membranes. Finally, centrifuge the solubilized sample at 300, 000 times G for one hour at four degrees Celsius. Prepare a one to five milliliter IMAC column or use a pre-packed column.Perform subsequent chromatography steps at four degrees Celsius. Flush with water to remove any traces of preservatives. If using an automated purification system, install the column according to manufacturer's instructions.Prepare 500 milliliters of IMAC buffer A without imidizole and 250 millilliters of IMAC buffer B with one molar imidizole. Equilibrate the IMAC column using 10 column volumes of IMAC buffer A.Add imidizole to the protein sample to a final concentration of 25 millimolar and mix. Then, load the sample onto the equilibrated IMAC column at two milliliters per minute.Collect the flow-through. Next, wash the IMAC column with five column volumes each of increasing concentrations of imidizole, using buffer B.Collect the washes in two milliliter fractions. Elute the sample with a final concentration of 250 millimolar for five column volumes and collect the eluted sample in two milliliter fractions.Analyze the flow-through, the wash fractions, and the elution fractions using SDS-PAGE analysis based on their absorbance at 280 nanometers. Pool the fractions containing the target beta-barrel OMP as verified by SDS-PAGE analysis. Then, remove the 6X histadine tag by adding TEV protease to the pooled sample and incubating overnight at four degrees Celsius with gentle rocking.Load the sample protease solution onto an IMAC column again to separate the target beta-barrel OMP from the cleaved tags and any uncleaved sample. The flow-through will contain the cleaved sample lacking the tag. To prepare for crystallization, load the sample onto a gel filtration column into a buffer containing the detergent to be used for crystallization.Collect one milliliter fractions and analyze using SDS-PAGE analysis based on their absorbance at 280 nanometers. Pool those fractions containing the target protein, and then concentrate to approximately 10 milligrams per milliliter. Prior to preparing samples, assemble the gel apparatus.Use pre-cooled gel running buffer or run the gel in the cold room. Filter the sample using a 0.22 micron centrifugal filter to remove particulates and precipitation. Then, pipette 0.25 microliters of the sample into two 1.5 milliliter micro centrifuge tubes.Label one as boiled"and the other as RT"Next, add 9.75 microliters of the sample buffer to each tube and mix by pipetting. To both samples, also add 10 microliters of 2X SDS loading buffer and mix gently by pipetting. Boil the boiled"sample at 95 degrees Celsius for five minutes, while leaving the RT"sample at room temperature.Then, spin the boiled"sample briefly. Next, load 20 microliters of both samples onto the pre-assembled native gel. After running the gel for 60 minutes at a constant 150 volts remove the gel and soak in staining solution to visualize the results.Proceed with crystallization trials in detergent, bi-cells, and lipidic cubic phase, using previously published Jove methods. Crystallization heads are visualized under a UV microscope to verify that the crystals are, indeed, protein and not solved, lipid or detergent. Lead crystals are screened using a home source or at the synchrotron to see if the crystals defract.After data collection of crystals that defract well, process the data using processing software, such as HKL2000. Transfer resulting files to a software suite for structure determination, such as Phoenix Suit, and perform molecular replacement for initial phasing and structure determination. The crystal structure of YIUR was determined at 2.6 Angstrom resolution using this method.Once mastered, this technique can be done in two weeks, if it is performed properly. After watching this video, you should have a good understanding of how to express and purify bacterial outer membrane proteins for structural studies.

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Determination of the Gas-phase Acidities of Oligopeptides

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus 1-6. Although extensive experimental studies on the acid-base properties of peptides have been carried out in the condensed phase, in particular in aqueous solutions 6-8, the results are often complicated by solvent effects 7. In fact, most of the active sites in proteins are located near the interior region where solvent effects have been minimized 9,10. In order to understand intrinsic acid-base properties of peptides and proteins, it is important to perform the studies in a solvent-free environment.
We present a method to measure the acidities of oligopeptides in the gas-phase. We use a cysteine-containing oligopeptide, Ala3CysNH2 (A3CH), as the model compound. The measurements are based on the well-established extended Cooks kinetic method (Figure 1) 11-16. The experiments are carried out using a triple-quadrupole mass spectrometer interfaced with an electrospray ionization (ESI) ion source (Figure 2). For each peptide sample, several reference acids are selected. The reference acids are structurally similar organic compounds with known gas-phase acidities. A solution of the mixture of the peptide and a reference acid is introduced into the mass spectrometer, and a gas-phase proton-bound anionic cluster of peptide-reference acid is formed. The proton-bound cluster is mass isolated and subsequently fragmented via collision-induced dissociation (CID) experiments. The resulting fragment ion abundances are analyzed using a relationship between the acidities and the cluster ion dissociation kinetics. The gas-phase acidity of the peptide is then obtained by linear regression of the thermo-kinetic plots 17,18.
The method can be applied to a variety of molecular systems, including organic compounds, amino acids and their derivatives, oligonucleotides, and oligopeptides. By comparing the gas-phase acidities measured experimentally with those values calculated for different conformers, conformational effects on the acidities can be evaluated.

The determination of the gas-phase acidities of cysteine-containing oligopeptides is described. The experiments are performed using a triple quadrupole mass spectrometer. The relative acidities of the peptides are measured using collision-induced dissociation experiments, and the quantitative acidities are determined using the extended Cooks kinetic method.

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue ...

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose, Lignin, and a Synthetic Polycation

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.

The objective of this research was to form synthetic plant cell wall tissue using layer-by-layer assembly of nanocellulose fibrils and isolated lignin assembled from dilute aqueous suspensions.&#160; Surface measurement techniques of quartz crystal microbalance and atomic force microscopy were used to monitor the formation of the polymer-polymer nanocomposite material.

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and ...

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for characterizing lyophilized formulations of protein therapeutics. Labeling followed by suitable proteolytic digestion allows the protein structure and interactions to be mapped with peptide-level resolution. Since the protein structural elements are stabilized by a network of chemical bonds from the main-chains and side-chains of amino acids, specific labeling of atoms in the amino acid residues provides insight into the structure and conformation of the protein. In contrast to routine methods used to study proteins in lyophilized solids (e.g., FTIR), ssHDX-MS and ssPL-MS provide quantitative and site-specific information. The extent of deuterium incorporation and kinetic parameters can be related to rapidly and slowly exchanging amide pools (Nfast, Nslow) and directly reflects the degree of protein folding and structure in lyophilized formulations. Stable photolytic labeling does not undergo back-exchange, an advantage over ssHDX-MS. Here, we provide detailed protocols for both ssHDX-MS and ssPL-MS, using myoglobin (Mb) as a model protein in lyophilized formulations containing either trehalose or sorbitol.

Here, we present detailed protocols for solid-state amide hydrogen/deuterium exchange mass spectrometry (ssHDX-MS) and solid-state photolytic labeling mass spectrometry (ssPL-MS) for proteins in solid powders. The methods provide high-resolution information on protein conformation and interactions in the amorphous solid-state, which may be useful in formulation design.

Amide hydrogen/deuterium exchange (ssHDX-MS) and side-chain photolytic labeling (ssPL-MS) followed by mass spectrometric analysis can be valuable for ...

Synthesis and Exfoliation of Discotic Zirconium Phosphates to Obtain Colloidal Liquid Crystals

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and engineers. Growth of such anisotropic nanocrystals through a simple chemical method is a challenging task. In this study, we fabricate discotic nanodisks of zirconium phosphate [Zr(HPO4)2&#183;H2O] as a model material using hydrothermal, reflux and microwave-assisted methods. Growth of crystals is controlled by duration time, temperature, and concentration of reacting species. The novelty of the adopted methods is that discotic crystals of size ranging from hundred nanometers to few micrometers can be obtained while keeping the polydispersity well within control. The layered discotic crystals are converted to monolayers by exfoliation with tetra-(n)-butyl ammonium hydroxide [(C4H9)4NOH, TBAOH]. Exfoliated disks show isotropic and nematic liquid crystal phases. Size and polydispersity of disk suspensions is highly important in deciding their phase behavior.

A two dimensional model material of discotic zirconium phosphate was developed. The inorganic crystal with lamellar structure was synthesized by hydrothermal, reflux, and microwave-assisted methods. On exfoliation with organic molecules, layered crystals can be converted to monolayers, and nematic liquid crystal phase was formed at sufficient concentration of monolayers.

Due to their abundance in natural clay and potential applications in advanced materials, discotic nanoparticles are of interest to scientists and ...

X-ray Powder Diffraction in Conservation Science: Towards Routine Crystal Structure Determination of Corrosion Products on Heritage Art Objects

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder diffraction (XRPD) is presented in detail via two case studies.
The first material under investigation was sodium copper formate hydroxide oxide hydrate, Cu4Na4O(HCOO)8(OH)2&#8729;4H2O (sample 1) which forms on soda glass/copper alloy composite historic objects (e.g., enamels) in museum collections, exposed to formaldehyde and formic acid emitted from wooden storage cabinets, adhesives, etc. This degradation phenomenon has recently been characterized as &#34;glass induced metal corrosion&#34;.
For the second case study, thecotrichite, Ca3(CH3COO)3Cl(NO3)2&#8729;6H2O (sample 2), was chosen, which is an efflorescent salt forming needlelike crystallites on tiles and limestone objects which are stored in wooden cabinets and display cases. In this case, the wood acts as source for acetic acid which reacts with soluble chloride and nitrate salts from the artifact or its environment.
The knowledge of the geometrical structure helps conservation science to better understand production and decay reactions and to allow for full quantitative analysis in the frequent case of mixtures.

Modern high resolution X-ray powder diffraction (XRPD) in the laboratory is used as an efficient tool to determine crystal structures of long-known corrosion products on historic objects.

The crystal structure determination and refinement process of corrosion products on historic art objects using laboratory high-resolution X-ray powder ...

Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While metal-based nanoparticles are associated with synthetic and environmental hassles, cellulose introduces a green, sustainable alternative for nanoparticle synthesis. Here, we present the chemical synthesis and separation procedures to produce new classes of hairy nanoparticles (bearing both amorphous and crystalline regions) and biopolymers based on wood fibers. Through periodate oxidation of soft wood pulp, the glucose ring of cellulose is opened at the C2-C3 bond to form 2,3-dialdehyde groups. Further heating of the partially oxidized fibers (e.g., T = 80 &#176;C) results in three products, namely fibrous oxidized cellulose, sterically stabilized nanocrystalline cellulose (SNCC), and dissolved dialdehyde modified cellulose (DAMC), which are well separated by intermittent centrifugation and co-solvent addition. The partially oxidized fibers (without heating) were used as a highly reactive intermediate to react with chlorite for converting almost all aldehyde to carboxyl groups. Co-solvent precipitation and centrifugation resulted in electrosterically stabilized nanocrystalline cellulose (ENCC) and dicarboxylated cellulose (DCC). The aldehyde content of SNCC and consequently surface charge of ENCC (carboxyl content) were precisely controlled by controlling the periodate oxidation reaction time, resulting in highly stable nanoparticles bearing more than 7 mmol functional groups per gram of nanoparticles (e.g., as compared to conventional NCC bearing &#60;&#60; 1 mmol functional group/g). Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) attested to the rod-like morphology. Conductometric titration, Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), dynamic light scattering (DLS), electrokinetic-sonic-amplitude (ESA) and acoustic attenuation spectroscopy shed light on the superior properties of these nanomaterials.

Synthesis schemes to prepare highly stable wood fiber-based hairy nanoparticles and functional cellulose-based biopolymers have been detailed.

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While ...

From Molecules to Materials: Engineering New Ionic Liquid Crystals Through Halogen Bonding

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid crystals (ILCs). Taking advantages of the high specificity of XB for haloperfluorocarbons and the ability of anions to act as XB-acceptors, we obtained supramolecular complexes based on 1-alkyl-3-methylimidazolium iodides and iodoperfluorocarbons, overcoming the well-known immiscibility between hydrocarbons (HCs) and perfluorocarbons (PFCs). The high directionality of the XB combined with the fluorophobic effect, allowed us to obtain enantiotropic liquid crystals where a rigid, non-aromatic, XB supramolecular anion acts as mesogenic core.
X-ray structure analysis of the complex between 1-ethyl-3-methylimidazolium iodide and iodoperfluorooctane showed the presence of a layered structure, which is a manifestation of the well-known tendency to segregation of perfluoroalkyl chains. This is consistent with the observation of smectic mesophases. Moreover, all the reported complexes melt below 100 &#176;C, and most are mesomorphic even at room temperature, despite that the starting materials were non-mesomorphic in nature.
The supramolecular strategy reported here provides new design principles for mesogen design allowing a totally new class of functional materials.

A protocol for the synthesis of a new type of mesogens, based on the halogen-bonded supramolecular anion [CnF2n+1-I&#183;&#183;&#183;I&#183;&#183;&#183;I-CnF2n+1]&#8722;, is reported.

Herein, we demonstrate that a bottom-up approach, based on halogen bonding (XB), can be successfully applied for the design of a new type of ionic liquid ...

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping.

We present a technique for contactless micromanipulation of vesicles, using localized calcium ion gradients. Microinjection of a calcium ion solution, in the vicinity of a giant lipid vesicle, is utilized to remodel the lipid membrane, resulting in the production of membrane tubular protrusions.

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local ...