Name: contrast-matching detergent in small-angle neutron scattering experiments for membrane protein structural analysis and ab initio modeling
Uploaded: 2018-10-21T12:30:00
Description: Watch this Scientific Journal Video about Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling at JoVE.com

The Office of Biological and Environmental Research supported research at ORNL&#39;s Center for Structural Molecular Biology (CSMB) and Bio-SANS using facilities supported by the Scientific User Facilities Division, Office of Basic Energy Sciences, US Department of Energy. Structural work on membrane proteins in the Lieberman lab has been supported by NIH (DK091357, GM095638) and NSF (0845445).

1. Prepare and Submit a Neutron Facility Beam Time and Instrument Proposal
<ol>
	<li>Consult online resources for identifying neutron scattering facilities that provide general user neutron beam time access, such as Oak Ridge National Laboratory (ORNL). A map of neutron facilities and information about neutron research worldwide is available online.46&#160;Be aware that these facilities typically have regular calls for proposals; this determines when the next beam time will be available. Neutron...

Structural biology researchers take advantage of complementary structural techniques like solution scattering to obtain biochemical and structural details (such as overall size and shape) from biomolecules in solution. SANS is a particularly attractive technique for determining low resolution structures of membrane proteins, a core focus of modern structural biology and biochemistry. SANS requires quantities of purified proteins comparable to those of crystallographic trials (1 mg/sample). The recently expanding commerci...

Membrane proteins are encoded by an estimated 30% of all genes1 and represent a strong majority of targets for modern medicinal drugs.2 These proteins perform a wide array of vital cellular functions,3&#160;but despite their abundance and importance &#8212;&#160;only represent about 1% of total structures deposited in the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank.4 Due to their partially hydrophobic nature, structural determination of membrane-bound proteins has been exceedingly challenging.5,6,7
As many biophysical techniques require monodisperse particles in solution for measurement, isolating membrane proteins from native membranes and stabilizing these proteins in a soluble mimic of the native membranes has been an active area of research in recent decades.8,9,10 These investigations have led to the development of many novel amphiphilic assemblies to solubilize membrane proteins, such as nanodiscs,11,12,13&#160;bicelles,14,15&#160;and amphipols.16,17&#160;However, the use of detergent micelles remains one of the most common and straightforward approaches for satisfying the solubility requirements of a given protein.18,19,20,21,22,23,24,25&#160;Unfortunately, no single detergent or magic mixture of detergents currently exists that satisfies all membrane proteins; thus, these conditions must be empirically screened for the unique requirements of each protein.26,27
Detergents self-assemble in solution above their critical micelle concentration to form aggregate structures called micelles. Micelles are composed of many detergent monomers (typically ranging from 20-200) with hydrophobic alkyl chains forming a micelle core and hydrophilic head groups arranged in a micelle shell layer facing the aqueous solvent. The behavior of detergents and micelle formation has been classically described by Charles Tanford in The Hydrophobic Effect,28&#160;and sizes and shapes of micelles from commonly used detergents in membrane protein studies have been characterized using small-angle scattering.29,30&#160;Detergent organization about membrane proteins has also been studied, and the formation of protein-detergent complexes (PDCs) is expected with detergent molecules surrounding the protein in an arrangement that resembles the neat detergent micelles.31
One added advantage in using detergents is that the resulting micelle properties can be manipulated by incorporating other detergents. Many detergents exhibit ideal mixing, and select properties of mixed micelles may even be predicted from the components and ratio of mixing.22&#160;However, the presence of detergent can still present challenges for biophysical characterizations by contributing to the overall signal. For example, with X-ray and light scattering techniques, signal from detergent in the PDC is practically indistinguishable from protein.32&#160;Investigations with single-particle cryo-electron microscopy (cryo-EM) typically rely on trapped (frozen) particles; structural details of the protein are still obscured by certain detergents or a high concentration of detergent which adds to the background.33&#160;Alternate approaches toward interpreting the full PDC structure (including the detergent) have been made through computational methods which seek to reconstruct the detergent around a given membrane protein.34
For the case of neutron scattering, the core-shell arrangement of detergent in the micelle produces a form factor which contributes to the observed scattering. Fortunately, solution components can be altered such that they do not contribute to the net observed scattering. This &#34;contrast matching&#34; process is achieved by substituting deuterium for hydrogen to achieve a scattering length density that matches that of the background (buffer). A judicious choice of detergent (with available deuterated counterparts) and their ratio of mixing must be considered. For detergent micelles, this substitution can be performed using a detergent with the same head group but having a deuterated alkyl chain (d-tail instead of h-tail). Since the detergents are well-mixed,35&#160;their aggregates will have a scattering length density that is the mole-fraction weighted average of the two components (h-tails and d-tails). When this average contrast is consistent with that of the head group, the uniform aggregate structures can be fully matched to remove all contributions to observed scattering.
We present here a protocol to manipulate the neutron contrast of detergent micelles by incorporating chemically identical detergent molecules with deuterium-labeled alkyl chains.19,36,37&#160;This permits complete simultaneous contrast matching of micelle core and shell, which is a unique capability of neutron scattering.35,38&#160;With this significantly refined level of detail, contrast matching can enable otherwise unfeasible studies of membrane protein structures. Additionally, this contrast-matching approach could be extended to other systems involving detergent, such as polymer exchange reactions39 and oil-water dispersants,40&#160;or even other solubilizing agents, such as bicelles,41&#160;nanodiscs,42&#160;or block copolymers.43&#160;A similar approach as outlined in this manuscript, but employing a single detergent species with partial deuterium substitutions on the alkyl chain and/or head group, was recently published.37&#160;While this can be expected to improve the random distribution of hydrogen and deuterium throughout the detergent compared to the approach presented here, the limited number of available positions on the detergent for substitution and two-step detergent synthesis required poses additional challenges for consideration.
Steps 1 and 2 of the protocol detailed below often overlap since initial experiment planning must be done to submit a quality proposal. However, proposal submission is considered here as the first step to emphasize that this process should be started well in advance of a neutron experiment. It should also be noted that a prerequisite step, which should be demonstrated by the proposal, is to have biochemical and physical characterization (including purity and stability) of the sample supporting the need for neutron studies. A general discussion of small-angle neutron scattering (SANS) is beyond the scope of this article. A brief but thorough introduction is available in the reference work Characterization of Materials by Kaufmann,44 and a comprehensive textbook focused on biological small-angle solution scattering has recently been published.45 Further recommended reading is given in the Discussion section. Small angle scattering uses the so called scattering vector Q as the central quantity that describes the scattering process. This article uses the widely accepted definition Q = 4&#960; sin(&#952;)/&#955;, where &#952; is half the angle between incoming and scattered beam and &#955; is the wavelength of the neutron radiation in Angstroms. Other definitions exist that use different symbols such as &#39;s&#39; for the scattering vector, and that may differ by a factor 2&#960; or by using nanometers in place of Angstrom (see also discussion of Figure 10).

<table border="0" cellpadding="0" cellspacing="0" width="848">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">Amicon Ultra MWCO 50KDa concentrator&#160;</td>
			<td style="width:209px;">EMD Millipore</td>
			<td style="width:108px;">UFC905096</td>
			<td style="width:135px;">labware</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium citrate dibasic</td>
			<td>Fisher Scientific</td>
			<td>A663</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ammonium sulfate</td>
			<td>EMD Millipore</td>
			<td>2150</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bioflo 310 Bioreactor System</td>
			<td>Eppendorf</td>
			<td>M1287-2110</td>
			<td>equipment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium chloride dihydrate</td>
			<td>Acros</td>
			<td>423525000</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carbenicillin</td>
			<td>IBI Scientific</td>
			<td>IB02025</td>
			<td>antibiotic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chloramphenicol</td>
			<td>EMD Millipore</td>
			<td>3130</td>
			<td>antibiotic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cobalt (II) chloride</td>
			<td>Acros</td>
			<td>AC21413-0050</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Copper (II) sulfate</td>
			<td>Acros</td>
			<td>AC19771-1000&#160;</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Deuterium oxide</td>
			<td>Sigma-Aldrich</td>
			<td>756822</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Drierite Gas Purifier</td>
			<td>W.A. Hammond Drierite Co. Ltd.</td>
			<td>27068</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EDTA, disodium, dihydrate</td>
			<td>EMD Millipore</td>
			<td>4010</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Emulsiflex-C3</td>
			<td>Avestin</td>
			<td>EF-C3</td>
			<td>equipment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#196;kta Purifier UPC100</td>
			<td>GE Healthcare&#160;</td>
			<td></td>
			<td>equipment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td>Sigma-Aldrich</td>
			<td>G5516</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HiPrep 16/60 Sephacryl S-300 HR column</td>
			<td>GE Healthcare&#160;</td>
			<td>17116701</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Imidazole</td>
			<td>VWR</td>
			<td>97064-622</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">IPTG</td>
			<td>Teknova</td>
			<td>I3325</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iron(III) chloride hexahydrate</td>
			<td>MP Biochemicals</td>
			<td>ICN19404590</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LB Agar Miller</td>
			<td>Fisher Scientific</td>
			<td>BP1425-2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Magnesium sulfate heptahydrate</td>
			<td>VWR</td>
			<td>97062-134</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">Manganese(II) sulfate monohydrate</td>
			<td>Acros</td>
			<td>AC20590-5000</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MaxQ 6000 Incubated/Refrigerated Shaker</td>
			<td>Thermo Scientific</td>
			<td>SHKE6000-7&#160;</td>
			<td>equipment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">n-Dodecyl-d25-&#946;-D-maltopyranoside</td>
			<td>Anatrace</td>
			<td>D310T</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">n-Dodecyl-&#946;-D-maltopyranoside</td>
			<td>Anatrace</td>
			<td>D310A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium phosphate monobasic</td>
			<td>VWR</td>
			<td>97062-346</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:396px;">RC 6 Plus Centrifuge</td>
			<td>Thermo Scientific Sorvall</td>
			<td>46910</td>
			<td>equipment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SIGMAFAST protease inhibitor cocktail tablets, EDTA-free</td>
			<td>Sigma-Aldrich</td>
			<td>S8830</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S3014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>795429</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium phosphate dibasic</td>
			<td>Sigma-Aldrich</td>
			<td>S7907</td>
			<td>medium component</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile 25mm syringe filter with 0.2&#181;m PES membrane</td>
			<td>VWR</td>
			<td>28145-501</td>
			<td>labware</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile disposable bottle top filter with 0.2&#181;m PES membrane</td>
			<td>Thermo Scientific</td>
			<td>596-4520&#160;</td>
			<td>labware</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Superdex 200 10/300 GL&#160;</td>
			<td>GE Healthcare&#160;</td>
			<td>17517501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Superose-12 10/300 GL column&#160;</td>
			<td>GE Healthcare&#160;</td>
			<td>17517301</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrospec 10 Cell Density Meter</td>
			<td>GE Healthcare&#160;</td>
			<td>80211630</td>
			<td>equipment</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zinc sulfate monohydrate</td>
			<td>Acros</td>
			<td>AC38980-2500&#160;</td>
			<td>medium component</td>
		</tr>
	</tbody>
</table>

contrast-matching detergent in small-angle neutron scattering experiments for membrane protein structural analysis and ab initio modeling

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the investigation of biological materials, biofuel processing, and bio-inspired materials covering nanometer to micrometer length scales. The methods presented here for investigating physical properties (i.e., size and shape) of membrane proteins (here, MmIAP, an intramembrane aspartyl protease from Methanoculleus marisnigri) in solutions of micelle-forming detergents are well-suited for this small-angle neutron scattering instrument, among others. Other biophysical characterization techniques are hindered by their inability to address the detergent contributions in a protein-detergent complex structure. Additionally, access to the Bio-Deuteration Lab provides unique capabilities for preparing large-scale cultivations and expressing deuterium-labeled proteins for enhanced scattering signal from the protein. While this technique does not provide structural details at high-resolution, the structural knowledge gap for membrane proteins contains many addressable areas of research without requiring near-atomic resolution. For example, these areas include determination of oligomeric states, complex formation, conformational changes during perturbation, and folding/unfolding events. These investigations can be readily accomplished through applications of this method.

A beam time and instrument proposal should clearly convey all information needed to the review committee so that a valid assessment of the proposed experiment can be made. Communication with an NSS is highly suggested for inexperienced users. The NSS can assess initial feasibility and guide proposal submission to emphasize feasibility, safety, and the potential for high-impact science. The information provided in the proposal should include background information and context for the signi...

Watch this Scientific Journal Video about Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling at JoVE.com

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

This protocol demonstrates how to obtain a low-resolution ab initio model and structural details of a detergent-solubilized membrane protein in solution using small-angle neutron scattering with contrast-matching of the detergent.

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling

The biological small-angle neutron scattering instrument at the High-Flux Isotope Reactor of Oak Ridge National Laboratory is dedicated to the ...

This method can help answer key questions about integral membrane proteins such as the oligomeric state, their size, overall shape and low resolution structure in solution. Solution studies of membrane proteins requires stabilizing molecules like detergents. This technique has the advantage that it makes detergents invisible and highlights the signal from the membrane protein.The implications of this technique extend to many biological and medical research questions because over 1/3 of proteins reside in membranes where they perform many vital cellular functions. Visual demonstration of this method is critical as the deuterium labeling and neutron scattering steps are difficult to learn because very few institutions offer training in these techniques. To begin, determine neutron scattering length densities or SLDs using the web application MULCh, modules for the analysis of contrast variation data.Open the contrast module by clicking contrast located in the left navigation pane. Provide text for a project title and enter the details below for two components as subunit one and subunit two. Enter the molecular formula for each component in the formula text box.Then enter the volume in cubic angstroms for each component in the box below volume. Finally, enter the details for buffer components. Click submit to perform the neutron contrast calculations.A results page is generated that provides a table of scattering length density and contrast match points as well as formulae for determining these parameters at any given percentage of D2O in the buffer. Review the scattering parameters with particular attention to the component contrast match points. Grow E.coli cells harboring an inducible expression vector for the target protein sequence in LB medium to an optical density at 600 nanometers or OD 600 of approximately one.Adapt E.coli cells to deuterium labeled medium prior to scale up for over expression of a deuterated protein by diluting the LB culture one to 20 in three milliliters of minimal medium. After growing to an OD 600 of approximately one, repeat the one to 20 dilutions using minimal medium containing 50, 75, and 100%D2O. Once adapted, continue growing the culture in a bioreactor to increase the yield of deuterated cell mass.As the D2O content is increased, growth rates will decrease. Proceed to harvest, lyse and purify cells as described in the text protocol. Next equilibrate a size exclusion column with greater than two column volumes of final exchange buffer using a fast protein liquid chromatography system.After injecting the sample, run the column in isolate fractions corresponding to the protein of interest. Reserve an aliquot of matched buffer for small angle neutron scattering or SANS background subtraction. To collect SANS data, first load samples and buffers into quartz cells.After ensuring that the beam shutter is closed, approach the sample environment area and place the quartz cells in the sample changer. Record the sample changer position for each sample cell. Check the area and ensure the beam path is free from obstruction before leaving the sample environment area.Then open the beam shutter. Execute a table scan for automated data collection. Follow the instructions for instrument operation provided by the neutron scattering scientist.Use Mantid Plot software and Python script for the reduction of SANS data from a 2D image to a 1D plot. To do so, log in to the remote analysis cluster and execute Mantid plot from the command line. Then open the provided user reduction script and place the corresponding scan numbers or identification for the sample in buffer pairs in the appropriate list.Execute the script to generate reduced data as a four column text file in the specified location. Right click the appropriate work space and Mantid plot and select plot with errors for an initial examination of the scattering profiles. Download the ATSAS software suite and use PRIMUS for data plotting, buffer scaling and subtraction and Guinier analysis.Launch the ATSAS, SAS data analysis application and load the reduced data files corresponding to the sample and buffer pair. To scale the buffer properly, select the data range at high Q where both profiles are similar in flat and click the scale button located under the operations tab. Increase the data range to view all points, click subtract to perform this operation.Perform a Guinier analysis of the buffer subtracted sample data using the analysis tab of the SAS data analysis application. Be sure the proper file is selected in the list and click radius of gyration. An automated attempt to perform a Guinier fit will be provided by clicking on the auto RG button.Expand the range of data used to include all of the low Q data and begin narrowing the data range by taking away high Q points until the Q max times RG limit is below 1.3. Use the plot of residuals to verify that data are linear in the fit range. Make small adjustments to the fit region and monitor the sensitivity of these values to the range of data used in the fit.Obtain the probability distribution function P of R in gnome. Now start the distance distribution wizard within the analysis tab. Obtaining a good fit to the data is essential to obtaining a quality model.Determine Dmax, the maximum interatomic distance within the molecule. Estimate a value for Dmax by unchecking the box to force Rmax equals zero and entering a large value for Dmax. The first X intercept in the plot of P of R yields this estimate.Make incremental changes to this Dmax value as well as the range of data used for number of points used to optimize the gnome fit to the data and the resulting P of R curve. Start the PRIMUS shape wizard from the dammif button on the analysis tab of SAS data analysis and use a manual selection of parameters. Define the Guinier range from the fit and proceed to the next step using the navigation buttons.Define fit values from the P of R plot and proceed to the next step. Provide parameters for the ab initio modeling process. Then initiate the process using the commit button.Overlay and superimpose the final sans envelope with a related high resolution model using supcomb within ATSAS. Place a copy of the high resolution PDB model to be fit to the SANS envelope in the working directory. Then execute supcomb from the command line using the PDB file names as two arguments with the template structure listed first.Finally visualize the ab initio model results using PyMOL, a 3D molecular graphics viewing program. Once the SANS envelope and high resolution structure have been superimposed, these models can be visualized using any molecular graphics viewing program. An overview of the PyMOL visualization process is shown.Once the PDB structure files have been opened in PyMOL, the models should be visible in the PyMOL viewer window. The representations of each model can be changed using the S button next to each file name. Here a surface representation is used for the SANS envelope and a cartoon representation is used for the protein backbone of the high resolution model.A suitable color scheme can be selected from the options available under the C button. For protein chains, a chainbow coloring provides a color gradient from N to C terminus to aid interpretation. Transparency is applied to the surface representation to allow better visualization of the protein structure within the envelope.For publication images, a white background is recommended. Once these visualization cues have been applied the 3D structures can be examined by clicking and dragging the structure. Manipulations of the perspective and molecule rotation and translation can be performed.While attempting this procedure it's important to remember to accurately match the neutron contrast of the detergent head and tail groups with the D2O H2O solvent. After its development, this technique paved the way for exploring the structure of intramembrane asparto proteases. These proteins are involved in the immune response, hepatitis C and Alzheimers disease.Don't forget that working in biochemical laboratories and at neutron beam facilities can be hazardous. Precautions such as wearing personal protective equipment and following all facility safety rules and radiological postings are mandatory while performing this procedure.

contrast-matching-detergent-small-angle-neutron-scattering

Research

JoVE Journal

Biochemistry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the disordered and protease labile C-terminus of the protein. This manuscript describes the methods to purify recombinant Nsa1 from S. cerevisiae for structural analysis by both X-ray crystallography and SAXS. X-ray crystallography was utilized to solve the structure of the well-ordered N-terminal WD40 domain of Nsa1, and then SAXS was used to resolve the structure of the C-terminus of Nsa1 in solution. Solution scattering data was collected from full-length Nsa1 in solution. The theoretical scattering amplitudes were calculated from the high-resolution crystal structure of the WD40 domain, and then a combination of rigid body and ab initio modeling revealed the C-terminus of Nsa1. Through this hybrid approach the quaternary structure of the entire protein was reconstructed. The methods presented here should be generally applicable for the hybrid structural determination of other proteins composed of a mix of structured and unstructured domains.

Combining X-Ray Crystallography with Small Angle X-Ray Scattering to Model Unstructured Regions of Nsa1 from S. Cerevisiae

This method describes the cloning, expression, and purification of recombinant Nsa1 for structural determination by X-ray crystallography and small-angle X-ray scattering (SAXS), and is applicable for the hybrid structural analysis of other proteins containing both ordered and disordered domains.

Determination of the full-length structure of ribosome assembly factor Nsa1 from Saccharomyces cerevisiae (S. cerevisiae) is challenging because of the ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

Detection of Detergent-sensitive Interactions Between Membrane Proteins

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein interactions in many cases is associated with significant experimental challenges. In particular, sorting receptors interact with their protein cargo in the lumen of the membrane compartments often in a detergent-sensitive fashion, making co-immunoprecipitation of these proteins unusable. Binding of the sorting receptor sortilin to glucose transporter GLUT4 may serve as an example of weak luminal interactions between membrane proteins. Here, we describe a fast, simple, and inexpensive assay to validate the interaction between sortilin and GLUT4. For that, we have designed and chemically synthesized the myc-tagged peptide corresponding to the potential sortilin-binding epitope in the luminal part of GLUT4. Sortilin tagged with six histidines was expressed in mammalian cells, and isolated from cell lysates using Cobalt beads. Sortilin immobilized on the beads was incubated with the peptide solution at different pH values, and the eluted material was analyzed by Western blotting. This assay can be easily adapted to study other detergent-sensitive protein-protein interactions.

We describe a protocol for detection of detergent-sensitive interactions between membrane proteins using binding of the sorting receptor, sortilin, to the first luminal loop of the glucose transporter protein, GLUT4, as an example.

Our ability to explore protein-protein interactions is the key to understanding regulatory connections in the cell. However, detection of protein-protein ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Ion Exchange Chromatography (IEX) Coupled to Multi-angle Light Scattering (MALS) for Protein Separation and Characterization

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique.
Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.

Ion Exchange Chromatography IEX Coupled to Multi-angle Light Scattering MALS for Protein Separation and Characterization

This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.

Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination ...

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave pyrophosphate into two orthophosphate molecules, which is coupled with proton and/or sodium ion pumping across the membrane. Since no homologous proteins occur in animals and humans, mPPases are good candidates in the design of potential drug targets. Here we present a detailed protocol to screen for mPPase inhibitors utilizing the molybdenum blue reaction in a 96 well plate system. We use mPPase from the thermophilic bacterium Thermotoga maritima (TmPPase) as a model enzyme. This protocol is simple and inexpensive, producing a consistent and robust result. It takes only about one hour to complete the activity assay protocol from the start of the assay until the absorbance measurement. Since the blue color produced in this assay is stable for a long period of time, subsequent assay(s) can be performed immediately after the previous batch, and the absorbance can be measured later for all batches at once. The drawback of this protocol is that it is done manually and thus can be exhausting as well as require good skills of pipetting and time keeping. Furthermore, the arsenite-citrate solution used in this assay contains sodium arsenite, which is toxic and should be handled with necessary precautions.

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Here we present a screening method for membrane-bound pyrophosphatase (from Thermotoga maritima) inhibitors based on the molybdenum blue reaction in a 96 well plate format.

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave ...