Name: Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling (Video) | JoVE Journal
Uploaded: 2018-10-21T12:30:00.000+00:00
Duration: 10 min 27 s
Description: Learn small-angle neutron scattering for membrane protein structural analysis using deuterium labeling and contrast-matching detergent. This method enables determination of size, shape, and oligomeric state while minimizing detergent interference.

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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).

The authors Volker S. Urban, Sai Venkatesh Pingali, Kevin L. Weiss, and Ryan C. Oliver are contracted through UT-Battelle with US DOE to support the Neutron Science User Program and Bio-SANS instrument at Oak Ridge National Laboratory used in this Article.
This manuscript has been co-authored by UT-Battelle, LLC, under contract DE-AC05-00OR22725 with the US Department of Energy (DOE). The US government retains and the publisher, by accepting the article for publication, acknowledges that the US government retains a nonexclusive, paid-up, irrevocable, worldwide license to publish or reproduce the published form of this manuscript, or allow others to do so, for US government purposes. DOE will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan.82

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

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...

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.

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

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

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Learn small-angle neutron scattering for membrane protein structural analysis using deuterium labeling and contrast-matching detergent. This method enables determination of size, shape, and oligomeric state while minimizing detergent interference.

Contrast-Matching Detergent in Small-Angle Neutron Scattering Experiments for Membrane Protein Structural Analysis and Ab Initio Modeling (Video) | JoVE Journal

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

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