INSANE IN THE MEMBRANE: HOW TO OVERCOME PROBLEMS WHEN STUDYING MEMBRANE PROTEINS.

Approximately 30% of the genes in the human genome code for membrane proteins, and yet we know relatively little about these molecules. Less than 1% of structures currently in the protein data bank correspond to membrane proteins, but this is not because a lack of interest but because working with these molecules is challenging and difficult. I would even dare to say that scientists that work with membrane proteins are unique, they are the bravest and more patient of us all.

Since many membrane proteins sit at the surface of cells, they are readily available to small molecule drugs circulating in the blood, thus they are important pharmaceutical targets. It is therefore not surprising that over 60% of small molecule drugs bind to membrane proteins.

Membrane proteins are unfortunately notoriously difficult to handle and study since they are designed to sit within the hydrophobic environment of the lipid bilayer. Common issues include:

• -        Expression
• -        Extraction
• -        Solubilization
• -        Purification
• -        Biophysical studies

RELIEVING THE COMPLEXITY OF STUDYING A MEMBRANE COMPLEX 

Working with membrane proteins is still based on trials and errors. From cloning and expression (choosing the right microorganism, what tags to use, setting conditions for overexpression), solubilization (testing different detergents) and reconstitution in artificial membranes, but it also depends on choosing the right tools, which can greatly ease your job, save a lot of time and money and more importantly that can retrieve valuable and precise information as in the case of biophysical techniques used to study their structure and function (molecule binding affinity and specificity, interaction with other molecules, etc).

In a beautiful study published on 2014 on JBC, Sabine Ebbensperger et al.¹ from Goethe University showed how these problems were overcome in the study of the membrane complex TAP.

THE TAP COMPLEX

When I was a kid I had chickenpox and it was a mess. I was so unlucky that the virus infected my brain and caused me encephalitis leaving me with some vision problems. Anyway, at the end it was me who won the battle and nowadays I still think how fascinating it is to overcome a viral infection. But how does that happen? Well, the immune system can destroy cells that are infected with viruses or otherwise damaged, when cytotoxic T-lymphocytes recognize antigenic peptides presented by MHC class I molecules (MHC I) on the cell surface. But how on earth does an antigenic peptide, coming from viruses or damaged cells, end up in the cell surface?

Well long story told short, antigenic peptides are degraded by the proteasome in the cytosol while MHC I molecules fold and assemble within the endoplasmic reticulum (ER). As a consequence it is necessary to translocate peptides from the cytosol into the ER lumen and this function is performed by the transporter associated with antigen processing (TAP) which uses the energy from ATP. TAP also acts as a scaffold for the final stage of MHCI assembly, i.e. peptide binding.

TURNING OFF THE TAP… HOW CLEVER CAN A VIRUS BE

TAP is a heterodimer formed by the subunits TAP1 and TAP2. Its membrane domains form the peptide-binding site, whereas ATP hydrolysis occurs at the nucleotide-binding domain (NBD), which energizes peptide translocation the ER lumen by membrane domains.

But of course, virus are smart and have evolved proteins that interfere with this pathway!!!!...... and…….. TAP is one viral target that is exploited for example by the herpes simple virus (HSV) ICP47 small protein. ICP47 inhibits peptide binding to TAP, but does not affect ATP binding. Yet ICP47 does not behave like a normal peptide as it is not translocated across the membrane and it remains associated with TAP.

THE CHALLENGE: TO STUDY THE TAP COMPLEX

PURIFICATION

In this work, TAP was expressed in yeast, with each subunit affinity tagged (His₁₀ and StrepII) to facilitate purification, and fusioned to fluorescent protein tags which allow direct visualization of the target during expression, solubilization and purification and can speed up the optimization of these processes. Way to go.

During the solubilization stage, membrane proteins are extracted from the lipid membrane to an aqueous environment by the use of detergents, which disintegrate the lipid bilayer while incorporating lipids and proteins in detergent micelles. Then, proteins re ready to be purified.

A great approach of this work and that makes a huge difference is that the TAP complex was then purified by tandem affinity of its subunits and detected by Multi Color Fluorescence Size Exclusion Chromatography (MC-FSEC), that as FSEC is used to enable tracking of individual subunits through expression, solubilization and purification steps. But what makes it better than FSEC though is its ability to detect multiple subunits of membrane protein complexes, such as TAP1 and TAP2, simultaneously and to analyse their behaviour in numerous conditions. It allows rapid assessment of the correct assembly and stoichiometry under the conditions tested and as the authors illustrated, it is suitable for the study of an hetero-oligomeric membrane protein complex.

RECONSTITUTION OF TAP IN NANODISCS

Reincorporation of purified membrane proteins into an artificial membrane continue to be crucial in studying the function and structure of these molecules. The necessity for reconstitution arises because many membrane proteins express their full activity only when correctly oriented and inserted in a lipid bilayer. 

Nanodiscs (ND) are self-assembled discoidal fragments of lipid bilayers 8-16 nm in diameter, stabilized in solution by two amphipathic helical scaffold proteins.

Now, nanodiscs provide several key advantages:

1.     Small size compared to liposomes

2.     Stoichiometry and composition of membrane protein and lipids can be controlled precisely.

3.     Substrate, ligand and protein interactions can be studied in close-to-native environment with access to both sides of the membrane protein complex.

TAP was successfully reconstituted in nanodiscs, with TAP1 and TAP2 subunits in nanodiscs detected by MC-FSEC. Moreover, the reconstitution procedure maintained TAP function (peptide binding and ATP hydrolysis) and gave important information like the annular lipid belt surrounding the TAP complex in nanodiscs is essential for high affinity IPCP47-TAP interaction.

PEPTIDE BINDING ANALYSIS BY MICROSCALE THERMOPHORESIS (MST)

Although biophysical methods have been successfully applied to an array of soluble protein targets they have failed in one way or another when applied to membrane proteins.

MST reports on a direct ligand-protein interaction and it is based on the differential movement of free fluorescent peptides versus and bound fluorescent peptides in a temperature gradient, induced by an infrared laser.

Accordingly, the change in fluorescence was used to reflect the concentration of peptide-TAP complexes. This technique was used to analyze peptide binding of TAP reconstituted in nanodiscs. More specifically it retrieved information about:

• -      Peptide binding affinity of TAP (Equilibrium dissociation constants K_D for nanodiscs-reconstituted TAP as well as detergent-solubilized TAP were obtained).
• 
  
• -        Specificity of peptide-binding.
• 
  
• -        Mode of action of the viral inhibitor ICP47 (using competition microscale thermophoresis it was demonstrated that ICP47 competes with peptides for the TAP binding pocket).

All in all, the combination of nanodiscs, MC-FSEC and MST worked very well in the study of a membrane complex. It certainly eased the job and showed that good information can be obtained from a difficult study model, if attention to the available technologies is paid.

MST holds promises to the study of membrane proteins and complexes and for the analysis of pharmaceutical-relevant molecules directed towards them.

1.     Eggensperger S, Fisette O, Parcej D, Schäfer LV, Tampé R. J Biol Chem. 2014 Nov    28;289(48):33098-108.

Parallels between men in love and electrophoretic mobility

“Men adjust their walking speed to match their romantic (female) partner's pace — a phenomenon not seen when guys walk with female friends”¹.

What does it possibly have to do with electrophoretic mobility??? Well, think about it. That same effect is pretty much what you can see in a classic EMSA (Electrophoretic Mobility Shift Assay), when a rather physical non-loving kind of interaction between nucleic acids and proteins is observed.

Well known is the relationship between electrophoretic mobility and size: bigger molecules have slower mobility than small ones. In the case of men, much of what determines walking speed is height: the longer your legs are, the faster you're likely to walk — a fact that means men, on average, have a higher optimal speed than women do.

But the interesting thing is that researchers discovered that when a lovely-dovey couple walked together, the man slowed his pace to match his female’s optimal speed. In the same way, when nucleic acids interact with proteins, they slow down in an electrophoretic run compared to unbound nucleic acids. So you can say that we ladies are to proteins as men are to nucleic acids!

In 1981, while the world’s eyes where on Lady Di marrying prince Charles and Olivia Newton John’s hit ‘Physical’ was all over, a great technique to measure DNA-protein interactions, named EMSA, was published by two independent groups.

The research on protein-DNA interactions began in the early 1960s, when analyzing the binding of Lac and phage λ repressors to DNA. Back then, these complexes could be analyzed by a technique that arose from the discovery that certain membrane filters will retain DNA-protein complexes, but not free DNA². So, by quantifying the retention of radiolabeled DNA fragments mixed with varying amounts of a protein of interest, it became possible to determine the stoichiometry and binding affinity of a protein for a given sequence. Anyway, filter binding remained impractical for the characterization of less stable complexes and non-DNA-protein complexes.

At the very beginning of the 1980s, Arnold Revzin and Mark Garner, at Michigan State University, knew of a study that showed that the ternary transcription elongation complex—DNA bound to RNA polymerase with a nascent RNA chain—was sufficiently stable for visualization by gel electrophoresis³. Combining purified protein with DNA restriction fragments containing appropriate binding sites and then running the mixture on a polyacrylamide gel, Revzin and Garner observed an amazing result: protein-DNA complexes forming distinctly ‘shifted’ higher molecular weight bands on the gels. Thus was born the electrophoretic mobility shift assay (EMSA)⁴. 

But they were not the only one working on it. Michael Fried and Donald Crothers at Yale also had developed their version of EMSA. Initially, Fried had speculated that only free DNA would be amenable to electrophoresis, and that DNA-protein binding could be quantified by determining how much DNA did not enter the gel, a very interesting thought by the way. But what they saw instead was a variety of shifted bands that appeared to correlate with the number of repressor molecules bound to each DNA fragment. (That’s when Crothers said to Michael “forget what you’re doing-follow this up!”). Their paper also offered some important extensions of Garner and Revzin’s assay, using radioactive labeling rather than ethidium bromide staining to detect shifted bands, and demonstrating he capabilities of EMSA as a means for measuring the relative binding constants and stoichiometry of protein-DNA interactions⁵.

Despite its popularity and application depth, EMSA is typically limited to semiquantitative interaction analysis. Nowadays, MicroScale Thermophoresis (MST) appears a solution-based method with high sensitivity that provides reliable quantitative information on molecular interactions such as protein-nucleic acids, based on a simple protocol, making measurements very fast and efficient with low sample consumption. This technique relies on binding-induced changes in thermophoretic mobility, which depends on several molecular properties, including not only size, but also charge and solvation entropy⁶.

Science and lab techniques evolve, it can go from electrophoresis to MicroScale thermophoresis, but parallels among human behaviour and molecules will continue to impress me.

1.     Wagnild J. and Wall-Scheffler CM (2013). PLoS One 8(10): e76576. 

2.     Jones, G.W. and Berg, P. J. (1966). Mol. Biol. 22, 199–209.

3.     Chelm, B.K.and Geiduschek, E.P. (1979). Nucleic Acids Res. 7, 1851–1867.

4.     Garner, M.M. and Revzin, A. (1981). Nucleic Acids Res. 9, 3047–3060.

5.     Fried, M. and Crothers, D.M. (1981). Nucleic Acids Res. 9, 6505–6525.

6.     Seidel SAI, Dijkman PM, Lea WA, et al. (2013). Methods. 59(3): 301-315.

A trip to the past: Where the hell are the micropipettes??!!!!!!!!!!

Imagine you are taken by Michael J. Fox into a trip to the past and you travel back 50 years. How well do you think you would manage in a lab from the 60’s?Oh wait!! Where the hell are the micropipettes?!!!!!!!

After suffering from tuberculosis as a soldier in the World War II, which probably saved his life, a one of a kind man named Heinrich Schnitger decided to study medicine…but not to practice medicine itself, but as he once said to control his health while protecting himself from incompetent doctors (told you... one of a kind!)

 At the age of 32 he joined the group of Theodor Bücher, Director of the Institute of Physiological Chemistry at the University of Marburg (Germany) as a post-doc. It was a time when pipetting by mouth was still the only method used.  Schnitger was assigned to work with the new anion exchange chromatography, used to measure phosphate-containing metabolites1. A gradient of up to 80% formic acid, following gravity flow, separated nucleotides and other anionic metabolites, which were collected in up to hundreds of fractions, often less than a millilitre in volume, for further analysis. Within a few weeks and to everyone's surprise, Schnitger developed a piston-driven pumping system, which replaced the gravity-driven flow of acid by more exact pump-controlled pressure…. And without any Google search!

While doing his routine work of aliquoting chromatography fractions for further analysis, Schnitger viewed micropipetting by mouth with great contempt. He eventually disappeared from the lab for a couple of days and came back with a self-designed tool to pipette microlitre volumes. Initially, Schnitger 'rebuilt' a tuberculin syringe by adding a spring to the piston that met an upward stop to define the pipetting volume. The syringe needle was replaced by a polyethylene (PE) tip, pulled from PE tubing. An air buffer separated the fluid from the syringe piston and confined it to the plastic tip. The clever features of Schnitger's device dramatically sped up and eased many other experiments as it enabled more accurate pipetting of all aqueous solutions.

And what did his boss do? Well, Bücher soon realized the enormous potential of this invention and as every great boss should do, he encouraged Schnitger to develop the pipette further while relieving him of his research work. Oh yeah!

Schnitger added various mechanical measures required for the exact and repetitive pipetting of small volumes. A major breakthrough was the introduction of a second spring, which allowed the piston to be pushed beyond the delivery point to blow out any residual fluid from the plastic tip.

 Schnitger also profited from the institute's excellent mechanical workshop, which was established by Bücher. On the basis of his prototypes, the workshop technicians produced copies for use in the lab.

Six months after he had built his first prototype, and conscious of its importance,Schnitger applied for a patent in Germany. His application, dated 3 May 1957, entitled “Vorrichtung zum schnellen und exakten Pipettieren kleiner Flüssigkeitsmengen” (Device for the fast and exact pipetting of small liquid volumes), was finally granted on 24 April 1961.

Soon, the medical supply company Eppendorf (Hamburg, Germany) bought the exclusive license for manufacturing and marketing the micropipette. Wilhelm Bergmann was responsible for developing the micropipette. It was further improved and most importantly, the newly available polypropylene (PEP) was used to create the tip which was ideal for single use. As an important addition, Bergmann created the 1.5 ml and 0.75 ml PEP centrifuge cups (the eppendorf tubes or eppitubes) with their snap-on tight cover as convenient vessels for transferring fluid with the micropipette, which quickly impressed laboratories worldwide. A microcentrifuge complemented this new toolset, in which the micropipette was to become an integral part of enzymatic assays together with the Eppendorf photometer. What an amazing combo right?!

Eppendorf failed to conquer this large market by focusing on technical perfection rather than on marketing. Eventually, Gilson Inc. (Middleton, WI, USA) realized its enormous market potential and created its own brand with a variable volume setting.

Schnitger did’t stop inventing, from a novel fraction colector to UV-micro-spectrophotometer with quartz optics to allow meaurements of 10 ul samples.

This man impressed me. I think just the invention of the micropipette would do for an entire PhD thesis. But what amazes me the most is how tiny problems would lead to such great technology improvements. Does any of us take time nowadays to solve problems like that ourselves? I was lucky enough to have my first lab experience in a lab where shelves were filled with hooses, springs and anything you can imagine for a ‘do it yourself’ experience that was encouraged by my mentor Dr Jaime Eyzaguirre Philippi. Though I must confess I wasn’t very good at it.

Anyways, I also love the hi-tech generation, with such incredible technologies such as cell cytometry, Mass- spec., Microscale thermophoresis, among so many others, with such an easy handling and beautiful outcomes, that make our scientific experience so much better.

1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1369176/

Do we take biochemical tools for granted? Long live the western blot!!

I often think that we, young scientists resemble to kids with cell phones or tablets in the way that they we rely on technology and learn it fast  but don’t really understand what comes behind the creation of such devices.  It is amusing by how the passage of time erases memory of the hard work that goes into developing ‘common use’ or new biochemical tools. That's why I'll write a little about the so commonly used and usefull Western Blot.

The name of this technique was given by W. Neal Burnette to keep the compass theme for naming blotting methods (Northern and Southern) and because his lab was located on the West Coast

Now, Western blotting was developed by three groups almost simultaneously and of course with some differences and so the credits should go to ALL of them: George Starck’s, Harry Towbin’s and W. Neal Burnette’s.

The Stark’s group was already famous for developing the RNA blotting technique called “Northern blot”, a name that came as a joke based on the DNA blotting technique called “Southern blot,” which in turn was named after its inventor, Edwin Southern. Stark’s lab became experts in making diazobenzyloxymethyl-paper or DBM-paper, which they use for Northern blots. They soon realized that there was a problem in detecting specific proteins and used the same chemistry as for Northern blotting, because they realized DBM-paper reacted with both nucleic acids and proteins. When the proteins transferred out of the gel, they covalently bound to the DBM-paper. “The surprise bit was that the immobilized protein reacted so nicely and specifically with antibodies” notes Stark (1). The Stark group published a technique for passively transferring proteins to DBM-paper which were then detected by radio-labeled Protein A. 

Meanwhile, Towbin, a postdoc in Switzerland was focusing on making antibodies against ribosomes for structure-function studies for his research project, but didn’t have a biochemical tool for the analysis. So he began to work out a method that would allow him to establish which antibody bound to which component of the ribosomal complex. By that point, DNA and RNA blotting methods were popular, so the idea of transferring proteins out of a gel and onto a membrane seemed natural. “It was in the air!” says Towbin (1). He says they knew that proteins, but not RNA, bound to nitrocellulose, so they separated ribosomal proteins on a polyacrylamide gel with urea as a denaturing agent and then electrophoretically transferred them onto nitrocellulose. Although they primarily focused on gels with urea as the denaturing agent, Towbin says they also got their approach to work with SDS. The Towbin group published a technique that is very similar to the one used today.  Their technique used electric current to transfer proteins to nitrocellulose membranes within the standard “immunoblotting sandwich.”  Additionally, they used secondary antibodies to detect bound proteins.

Burnette was unaware of the work done by the two groups as he was developing his approach but saw the Stark and Gordon groups’ papers in print while he was preparing his manuscript. But he felt that his method was different enough to press ahead. He focused on electrophoretically transferring proteins out of SDS-polyacrylamide gels onto nitrocellulose in a more quantitative manner. The Burnette group published an electrophoretically method of protein transfer to nitrocellulose which were then detected by radio-labeled Protein A.

The Starck and Towbin groups published almost simultaneously in 1979. The Burnette group, although submitting their paper that same year, it was rejected and published later on in 1981. However, Burnette is often cited as the developer of the western blot probably because he used a good marketing technique by choosing a clever name. Funny thing is that Burnette himself cites this naming of the technique as one of the reason his paper was rejected, since a reviewer said it was given a ‘flippant name’.

Interesting how a ‘common use’ technique involved so much work. Towbin once said “The younger generation of biologists takes the method for granted!”.

My advice: do not spend all of your time studying for exams and take some time to learn about the history and the people involved in the bench tools you use on a daily basis, take time to really understand what you are doing and how things work and be thankful that someone did an amazing job so you can do yours today.

1. http://www.asbmb.org/asbmbtoday/asbmbtoday_article.aspx?id=16084

Checkpoint mechanism preventing errors in chromosome segregation revealed

When you travel from one country to another you must go through a “border checkpoint”, a place where you and your goods are inspected before you can go any further. The same control mechanism can also be found inside the cells. One of the most crucial checkpoints in life occurs during cell division (termed ‘mitosis’ in somatic cells and ‘meiosis’ in germ lines). Faithful segregation of the genetic material is so important that errors in the distribution of individual chromosomes can cause some of the most terrible human diseases. A single mistake in the segregation of chromosome 18 during meiosis is responsible for Edwards syndrome.

Kinetochores are mega-molecular assemblies formed at the centromeres of chromosomes at the onset of cell division. Successful completion of segregation requires that sister kinetochores become attached to spindle microtubules, which are responsible for chromosome movement into opposite poles of the cell. This controlled kinetochore-microtubule attachment step constitutes the Spindle Assembly Checkpoint (SAC), which relies on the kinetochore-localized protein kinase Mps1.

Until recently, a key unresolved question was how SAC prevents cell division to proceed until all kinetochores are attached to microtubules. In June 12th issue of Science, two independent studies were published which revealed that the key checkpoint protein Mps1 compete with microtubules for binding to Ndc80c^1,2, a major microtubule receptor complex localized at the kinetochore, thus monitoring its attachment to microtubules. Even though both papers conclusions overlap, they followed different analytical approaches, what makes them interesting to analyze and compare.

First, using MicroScale Thermophoresis (MST), both groups demonstrated a direct interaction between Mps1 and Ndc80c with µM binding affinities in perfect agreement, and also corroborated that this interaction occurs in cells.

In a more detailed analysis, Ji et al. showed that Mps1 interacts directly with Ndc80c through two distinct motifs: NTE and MR motifs on Mps1 bound to Hec1 and Nuf2 subunits of Ndc80c, both of which contain binding sites for microtubules.

Moreover, both studies revealed that this interaction is phosphorylation-dependent: using MST, Hiruma et al. showed that phosphorylation at the NTE domain of Msp1 increases the affinity of this interaction at least 20 times, while Ji et al. showed that phosphorylation at the middle region MR domain of Msp1 increases affinity by a factor of 4, as measured by Isothermal Titration Calorimetry.

The main conclusion of both studies, regarding the competition of Mps1 and microtubules for binding to Ndc80c, was reached by two different approaches. On one hand Ji et al. analyzed the release of Ndc80c protein bound to beads containing Mps1 fragments by microtubules, which was further resolved with SDS-PAGE and quantified by immunoblot. On the other hand, Hiruma et al. used MST and analyzed the resulting binding curves for Ndc80c titrated against fluorescent labeled Mps1 fragments alone or with the addition of microtubules. These results show that both, semi-quantitative and quantitative analytical procedures respectively are complementary.

Both studies revealed a mechanism for sensing kinetochore-microtubule attachment and how this interaction inhibit production of the anaphase inhibitor SAC. The proposed model below shows that there are two types of Mps1-Ndc80c interactions at kinetochores: a major one involving the NTE-Hec1 interface and a minor one involving the MR-Nuf2 interface. Binding of microtubules to Ndc80c releases both and inhibits Mps1 signaling, thus allowing cell division to proceed. Moreover, increasing phosphorylation of Hec1 by Aurora B, progressively weakens MT binding³ and enhances Mps1 binding.

An interesting feature is that the weak, multisite Mps1-Ndc80c interactions explain the transient nature of Mps1 at kinetochores and the inability to detect these interactions in human cell lysates. Since MST quantifies interactions and provides binding affinities with high precision, this technology is perfectly suited to shed light on complex mechanisms such as chromosome segregation.

1.         Hiruma et al. Science. 2015 Jun 12; 348(6240): 1264-7

2.         Ji et al. Science. 2015 Jun 12; 348(6240): 1260-4

3.         Zhu et al. J Biol Chem. 2013 Dec 13; 288(50): 36149-59