Author Archives: Bronwen Dekker

Membrane protein protocols

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While browsing recently published papers in other journals, I came across Supramolecular fishing for plasma membrane proteins using an ultrastable synthetic host-guest binding pair (published in Nature Chemistry). The idea here is that proteins on the surface of the cell are chemically modified by reaction with 1-trimethylammoniomethylferrocene (AFc). After cell lysis, the modified proteins bind to beads coated with cucurbit-7-uril. Cucurbit-7-uril and AFc interact to form an ultrastable host-guest binding pair. The captured proteins are then recovered either by treatment with a strong competitor or by heating to 95 C in buffer conditions.

I thought that this was rather pleasing! It reminded me of a protocol we had published on the metabolic labeling of glycans with azido sugars, and got me to thinking: “What sorts of protocols do we have for looking at membrane proteins?”.

Using our Browse functionality plus a PubMed search I came up with 17 that I thought would be relevant (please forgive me (and let me know!!) if I have missed something vitally important). These protocols can be roughly divided into those working with a specific protein that you already know (the majority), and those where you are working with membranes from whole cells. When looking at the structure and function of purified membrane proteins, one of the main challenges seems to be the design of a matrix such that you can get meaningful data from proteins that are in conditions as similar as possible to the cell membrane itself.

These are some notes that I made while going through the protocols:

Expression and purification

Preparative scale expression of membrane proteins in Escherichia coli-based continuous exchange cell-free systems

– This protocol includes methods for preparing E.coli s30 extracts and T7RNAP.

GFP-based optimization scheme for the overexpression and purification of eukaryotic membrane proteins in Saccharomyces cerevisiae

– This protocol includes a method for transforming a gene-vector construct into S. cerevisiae.

– Purification is via a His-tag

Purification of recombinant membrane proteins tagged with calmodulin-binding domains by affinity chromatography on calmodulin-agarose: example of nicotinamide nucleotide transhydrogenase

Looking mostly at structure

A general protocol for the crystallization of membrane proteins for X-ray structural investigation

– His-tagged proteins are expressed in E.coli. The membranes are isolated and solubilised. Purification on a nickel column is followed by removal of the his-tag and size exclusion chromatography. Crystallisation is by hanging-drop vapour diffusion.

Crystallizing membrane proteins using lipidic mesophases

Bicelle samples for solid-state NMR of membrane proteins

– Isotope labelled proteins are analysed in bicelles. The lipid bilayers of these are aligned perpendicular to the magnetic field forming a liquid crystalline phase.

Synthesis of TOAC spin-labeled proteins and reconstitution in lipid membranes

– The protein is synthesised via FMOC chemistry. When analysed by electron paramagnetic resonance, the TOAC residue accurately reports the position, orientation and dynamics of the peptide backbone near the labelled site.

Atomic force microscopy and spectroscopy of native membrane proteins

– The authors use purple membrane from Halobacterium salinarum as an example. The stylus of the AFM is used to obtain the topography of the membrane as well as to mechanically manipulate the protein. Measurements taken in the process of unfolding the protein provide information regarding the molecular interactions within it.

The fluorescence protease protection (FPP) assay to determine protein localization and membrane topology

– This protocol uses GFP-fusion proteins. The link will take you to a cartoon showing a single cell before and after exposure to digitonin and trypsin.

Site-directed alkylation of cysteine to test solvent accessibility of membrane proteins

– Each residue in the protein is systematically mutated to cysteine. The proteins are reacted with [N-ethyl-1-^14^C]ethyl-maleimide, separated by SDS–PAGE and analysed using a PhosphoImager.

Looking mostly at function using specific proteins

Gel chromatography and analytical ultracentrifugation to determine the extent of detergent binding and aggregation, and Stokes radius of membrane proteins using sarcoplasmic reticulum Ca2+-ATPase as an example

– This method uses the radioactive detergent ^14^C-n-dodecyl-β-D-maltoside. The Stokes radius is determined either by size-exclusion chromatography or by ultracentrifugation sedimentation velocity analysis.

Using patterned supported lipid membranes to investigate the role of receptor organization in intercellular signaling

Expression cloning and radiotracer uptakes in Xenopus laevis oocytes

– This protocol is at the border of a few sections as it uses a functional assay (uptake of a radiotracer appropriate to the type of membrane protein that you are interested in) to either find new proteins or work what mutations will result in changes in transport activity.

– cRNA is injected into the oocytes.

Two protocols that I wasn’t certain how to classify

The preparation and development of cellular membrane affinity chromatography columns

– The membrane proteins are isolated by centrifugation (usually from a transfected cell line containing a target protein) and attached to a stationary phase.

– Frontal affinity chromatography using labelled ligands is used to measure molecular interactions.

Detecting interactions with membrane proteins using a membrane two-hybrid assay in yeast

– This protocol is for the MYTH system shown in the figure below.

Proteomic-type protocols

Quantitative proteomic approach to study subcellular localization of membrane proteins

– The method used is called LOPIT (localisation of organelle proteins by isotope tagging).

– Cell membranes are separated into fractions by equilibrium density gradient centrifugation. Each fraction is labelled using a different iTRAQ reagent, and the samples are pooled.

Identification of membrane proteins from mammalian cell/tissue using methanol-facilitated solubilization and tryptic digestion coupled with 2D-LC-MS/MS

– The key features of this protocol are (1) the use of 60 % methanol to dissolve the membrane-protein sample, and (2) that the strong cation exchange chromatography is done off-line to allow for optimisation of the fractionation to improve the capacity of the LC-MS/MS analysis. This is significant, because it allows the identification of 1500-2500 unique proteins.

An introduction to Lab Groups

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A feature of the new, improved Protocol Exchange site is that each protocol is associated with a Lab Group. A full list of these Lab Groups can be access from the Lab Groups tab.

outstanding labgroup 3.PNG

Lab groups can be:

1. Closed (all of the Lab Groups in the above example). This means that you can only become a member of the Lab Group if you are invited by the Lab Group Owner.

2. Accepting Requests (identified by the link “Apply to group”). This means that any user of Protocol Exchange can request to join this Lab Group, this application will be accepted or declined by the Lab Group Owner.

3. Open (identified by the link “Join group”). This means that any user of Protocol Exchange can join this group.

If you are a member of a Lab Group you can share protocols, or make a list of publications associated with the group by entering the DOIs for each article.

kurgan lab group.PNG

The Kurgan Lab, for example, has uploaded 1 protocol and listed 30 publications.

Most of the Lab Groups on our site are from Academic Institutions, but it is also possible for Commercial organisations to create a Lab Group and upload their protocols.

Two example of these are:

Targefect Transfection Group, Targeting Systems

biocision

When their protocols are listed, for example, on the Browse section, it is clearly marked that they are contributed by suppliers.

supplier and community.PNG

Another defining feature of the Lab Group is that you can upload a logo! The maximum allowed dimenstions are 200 X 100 pixels (width x height). I have made a list of the labgroups that have logos below; some of them are rather beautiful!

Sherwood’s Lab, Duke University

Sorger Lab, Harvard Systems Biology

Suga’s Lab (Hiroaki Suga), The University of Tokyo

Wehkamp Lab, IKP Stuttgart

Zaffran group, INSERM unit 910, Université de la Méditerranée

Guaza´s Lab, Cajal Institute, Madrid

Haseeb’s Lab (King Saud University, Riyadh, Saudi Arabia)

Kurgan Lab (University of Alberta)

Ren Lab, Oregon Health & Science University

Sanquin

Targefect Transfection Group, Targeting Systems

biocision

………………………………………………………..

More information about Lab Groups can be found in the About page.*

If you find that you have a protocol on the Exchange that was migrated from the old Protocols Network, and you want to get connected with the group that was automatically created, please do get in touch at: protocol.exchange@nature.com.

How to insert images into an Exchange Protocol

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Today we published a rather beautiful protocol entitled: Crystallization of Cryptophane-A and Cryptophane-A Derivatives with and without Encapsulated Guests

It contains two figures both of which have been very elegantly incorporated into the main text, so I thought I would take this opportunity to show you how to achieve this.

1| Prepare your figure. In order for it to show as an image it has to be a jpeg, png or bitmap file. Unfortunately tif files will NOT show as images. If you want it to display nicely within the framed page, it should have a width of 560 pixels or less.

2| Assuming that you are in the process of sharing a protocol, upload your figure by going to the “Images and Attachments” page. Notice that both the Name (e.g. Figure 1) and the Title (Overall heading) are required fields.

screenshot 1.PNG

3| Click on the link “How do I enter formatted text, symbols, links and images?”, and expand the section: "URLS for files uploaded via the “Images and Attachments” page.

screenshot 2.PNG

4| Copy the link for the image that you are interested in inserting including the exclamation marks, and paste it into, for example, the Introduction.

e.g. This:

screenshot 3.PNG

will result in this

screenshot 4.PNG

If you wanted to insert the thumbnail with a link to the main figure (this would be a good idea if the main figure was wider than 560 pixels), then you could insert the following html into the text: <a href="https://url/for/the/fullsize/image"> <img src="https://url/for/the/thumbnail/image" > </a>

An example of a protocol where this has been done is: Aptamer-facilitated Protein Isolation from Cells

This:

screenshot 5.PNG

Resulted in this

screenshot 6a.PNG

News from Nature Protocols

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One of our featured protocols this month is for the partial reduction of electron-deficient pyrroles under both Birch (Li/NH3) and ammonia-free (Li/DBB) conditions using the formation of pyrrolines 2, 3 and 5 as examples (for reaction scheme click here). Pyrroles 2 and 5 have recently been published by Donohoe et al. as key intermediates in the of the biologically active natural products 1-epiaustraline, hyacinthacine A119and omuralide (also known as lactacystin lactone).

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Besides being an excellent protocol with beautiful photographs of reactions in progress, it is the first of our Nature Protocols to have associated with it two Network Protocols. These are for the preparation of the starting materials 1 and 4, namely the N-Boc pyrrole 2,5-methyl diester and the N-Boc pyrrole 2-ethyl ester.

Our Protocols Network has two components: protocols, such as these, that are uploaded by users (or suppliers); and comments that can be made both on the open access and the peer-reviewed content. If you would also like to participate, by uploading your favourite method or suggesting modifications to those that have already been published, you could follow the links from our homepage. A Guide to Authors for the Protocols Network also contains more information about this part of our content.

Bronwen (Nature Protocols)

References:

1. Donohoe, T.J. et al. Utility of the ammonia-free Birch reduction of electron-deficient pyrroles: total synthesis of the 20S proteasome inhibitor, clasto-lactacystin -lactone. Chem. Eur. J. 11, 4227–4238 (2005).

2. Donohoe, T.J. & Sintim, H.O. A concise total synthesis of ( )-1-epiaustraline. Org. Lett. 6, 2003–2006 (2004).

3. Donohoe, T.J., Sintim, H.O. & Hollinshead, J. A noncarbohydrate based approach to polyhydroxylated pyrrolidizines: total syntheses of the natural products hyacinthacine A1 and 1-epiaustraline. J. Org. Chem. 70, 7297–7304 (2005).

4. Donohoe, T.J., Sintim, H.O., Sisangia, L. & Harling, J.D. An efficient synthesis of lactacystin -lactone. Angew. Chem. Int. Ed. 43, 2293–2296 (2004).

News from Nature Protocols

Posted on by
Reply

One of our featured protocols this month is for the partial reduction of electron-deficient pyrroles under both Birch (Li/NH3) and ammonia-free (Li/DBB) conditions using the formation of pyrrolines 2, 3 and 5 as examples (for reaction scheme click here). Pyrroles 2 and 5 have recently been published by Donohoe et al. as key intermediates in the of the biologically active natural products 1-epiaustraline, hyacinthacine A119and omuralide (also known as lactacystin lactone).

<!–

—>

Besides being an excellent protocol with beautiful photographs of reactions in progress, it is the first of our Nature Protocols to have associated with it two Network Protocols. These are for the preparation of the starting materials 1 and 4, namely the N-Boc pyrrole 2,5-methyl diester and the N-Boc pyrrole 2-ethyl ester.

Our Protocols Network has two components: protocols, such as these, that are uploaded by users (or suppliers); and comments that can be made both on the open access and the peer-reviewed content. If you would also like to participate, by uploading your favourite method or suggesting modifications to those that have already been published, you could follow the links from our homepage. A Guide to Authors for the Protocols Network also contains more information about this part of our content.

Bronwen (Nature Protocols)

References:

1. Donohoe, T.J. et al. Utility of the ammonia-free Birch reduction of electron-deficient pyrroles: total synthesis of the 20S proteasome inhibitor, clasto-lactacystin -lactone. Chem. Eur. J. 11, 4227–4238 (2005).

2. Donohoe, T.J. & Sintim, H.O. A concise total synthesis of ( )-1-epiaustraline. Org. Lett. 6, 2003–2006 (2004).

3. Donohoe, T.J., Sintim, H.O. & Hollinshead, J. A noncarbohydrate based approach to polyhydroxylated pyrrolidizines: total syntheses of the natural products hyacinthacine A1 and 1-epiaustraline. J. Org. Chem. 70, 7297–7304 (2005).

4. Donohoe, T.J., Sintim, H.O., Sisangia, L. & Harling, J.D. An efficient synthesis of lactacystin -lactone. Angew. Chem. Int. Ed. 43, 2293–2296 (2004).