The alpha-helical bundle is the most common type of fold for membrane proteins. Their diverse functions include transport, signalling, and catalysis. While structure determination is much more difficult for membrane proteins than it is for soluble proteins, it is accelerating and there are now 586 unique proteins in the database of Membrane Proteins of Known 3D Structure. However, we still have quite a poor understanding of how membrane proteins fold. There is increasing evidence that it is more complicated than the two-stage model proposed in 1990 by Popot and Engelman.

The machinery that inserts most alpha-helical membrane proteins is the Sec apparatus. In prokaryotes, it is located in the plasma membrane, while eukaryotic Sec is found in the ER. Sec itself is an alpha-helical bundle in the shape of a pore, and its structure is able both to allow peptides to pass fully across the membrane, and also to open laterally to insert transmembrane helices into the membrane. In both cases, this occurs co-translationally, with translation halted by the signal recognition particle until the ribosome is associated with the Sec complex.

Voorhees, R. M. et al. (2014) Cell, 157(7), 1632–43

If helices are inserted during the process of translation, does folding only begin after translation is finished? On what timescale are these folding processes occuring? There is evidence that a hairpin of two transmembrane helices forms on a timescale of miliseconds in vitro. Are helices already interacting during translation to form components of the native structure? It has also been suggested that helices may insert into the membrane in pairs, via the Sec apparatus.

There are still many aspects of the insertion process which are not fully understood, and even the topology of an alpha-helical membrane protein can be affected by the last part of the protein to be translated. I am starting to investigate some of these questions by using computational tools to learn more about the membrane proteins whose structures have already been solved.

At the last meeting before Christmas I covered the article by DeKosky et al. describing a new methodology for sequencing of paired VH-VL repertoire developed by the authors.

In the recent years there have been an exponential growth of available antibody sequences, caused mainly by the development of cheap and high-throughput Next Generation Sequencing (NGS) technologies. This trend led to the creation of several publicly available antibody sequence databases such as the DIGIT database and the abYsis database, containing hundreds of thousands of unpaired light chain and heavy chain sequences from over 100 species. Nevertheless, the sequencing of paired VH-VL repertoire remained a challenge, with the available techniques suffering from low throughput (<700 cells) and high cost. In contrast, the method developed by DeKosky et al. allows for relatively cheap paired sequencing of most of the 10^6 B cells contained within a typical 10-ml blood draw.

The work flow is as follows: first the isolated cells, dissolved in water, and magnetic poly(dT) beads mixed with cell lysis buffer are pushed through a narrow opening into a rapidly moving annular oil phase, resulting in a thin jet that coalescences into droplets, in such a way that each droplet has a very low chance of having a cell inside it. This ensures that the vast majority of droplets that do contain cells, contain only one cell each. Next, the cell lysis occurs within the droplets and the mRNA fragments coding for the antibody chains attach to the poly(dT) beads. Following that, the mRNA fragments are recovered and linkage PCR is used to generate 850 bp cDNA fragments for NGS.

To analyse the accuracy of their methodology the authors sequenced paired CDR-H3 – CDR-L3 sequences from blood samples obtained from three different human donors, filtering the results by 96% clustering, read-quality and removing sequences with less than two reads. Overall, this resulted in ~200,000 paired CDR-H3 – CDR-L3 sequences. The authors found that pairing accuracy of their methodology was ~98%.

The article also contained some bioinformatics analysis of the data. The authors first analysed CDR-L3 sequences that tend to pair up with many diverse CDR-H3 sequences and whether such “promiscuous” CDR-L3s are also “public” i.e. they are promiscuous and common in all three donors. Their results show that out of 50 most common promiscuous CDR-L3s 49 are also public. The results also show that the promiscuous CDR-L3s show little to no modification, being very close to the germline sequence.

Illustration of the sequencing pipeline

The sequencing data also contained examples of allelic inclusion, where one B-cell expresses two B cell receptors (almost always one VH gene and two distinct VL genes). It was found that about ~0.5% of all analysed B-cells showed allelic inclusion.

Finally, the authors looked at the occurrence of traits commonly associated with broadly Neutralizing Antibodies (bNAbs), produced to fight rapidly mutating pathogens (such as the influenza virus). These traits were short (<6 aa) CDR-L3 and long (11 – 18 aa) CDR-H3s. In total, the authors found 31 sequences with these features, suggesting that bNAbs can be found in the repertoire of healthy donors.

Overall this article presents very interesting and promising method, that should allow for large-scale sequencing of paired VH-VL sequences.

A long long time ago, in a galaxy far away, I gave a presentation about the state of my research to the group (can you tell I’m excited for the new Star Wars!). Since then, little has changed due to my absenteeism from Oxford, which means ((un)luckily) the state of work is by and large the same. Now, my work focusses on the effect that the translation speed of a given mRNA sequence can have on the eventual protein product, specifically through the phenomena of cotranslational folding. I’ve discussed the evidence behind this in prior posts (see here and here), though I find the below video a good reminder of why we can’t always just go as fast as we like.

So given that translation speed is important, how do we in fact measure it? Traditional measures, such as tAI and CAI, infer them using the codon bias within the genome or by comparing the counts of tRNA genes in a genome. However, while these have been shown to somewhat relate to speed, they are still solely theoretical in their construction. An alternative is ribosome profiling, which I’ve discussed in depth before (see here), which provides an actual experimental measure of the time taken to translate each codon in an mRNA sequence. In my latest work, I have compiled ribosome profiling data from 7 different experiments, consisting of 6 diverse organisms and processed them all in the same fashion from their respective raw data. Combined, the dataset gives ribosome profiling “speed” values for approximately 25 thousand genes across the various organisms.

Our first task with this dataset is to see how well the traditional measures compare to the ribosome profiling data. For this, we calculated the correlation against CAI, MinMax, nTE and tAI, with the results presented in the figure above. We find that basically no measure adequately captures the entirety of the translation speed; some measures failing completely, others obviously capturing some part of the behaviour, and then some others even predicting the reverse! Given that no measure captured the behaviour adequately, we realised that existing results that related the translation speed to the protein structure, may, in fact, be wrong. Thus, we decided that we should recreate the analysis using our dataset to either validate or correct the original observations. To do this we combined our ribosome profiling dataset with matching PDB structures, such that we had the sequence, the structure, and the translation speed for approximately 4500 genes over the 6 species. While I won’t go in to details here (see upcoming paper – touch wood), we analysed the relationship between the speed and the solvent accessibility, the secondary structure, and linker regions. We found striking differences to the observations found in the literature that I’ll be excited to share in the near future.

For this week’s journal club, I presented the paper by Goldman et al, “Mechanical force releases nascent chain-mediated ribosome arrest in vitro and in vivo”. The reason for choosing this paper is that it discussed an influence on protein folding/creation/translation that is not considered in any of today’s modelling efforts and I think it is massively important that every so often we, as a community, step-back and appreciate the complexity of the system we attempt to understand. This work focuses on the the SecM protein, which is known to regulate SecA (which is part of the translocon) which in turn regulates SecM. The bio-mechanical manner in which this regulation takes place is not fully understood. However, SecM contains within its sequence a peptide motiff that binds so strongly to the ribosome tunnel wall that translation is stopped. It is hypothesised that SecA regulates SecM by applying a force to the nascent chain to pull it past this stalling point and, hence, allow translation to continue.

To begin their study, Goldman wanted to confirm that one could advance past the stall point merely by the application of force. By attaching the nascent chain and the ribosome to nano-tweezers and a micro-pipette respectively they could do this. However, to confirm that the system was stalled before applying a (larger) force, they created a sequence which included CaM, a protein which periodically hops between a folded and unfolded state when pulled at 7pN, followed by the section of SecM which causes the stalling. The nano-tweezers were able to sense the slight variations in length at 7pn from the unfolding and refolding of CaM, though no continuing extension, which would indicate translation, was found. This indicated the system had truly stalled due to the SecM sequence. Once at this point, Goldman increased the applied force, at which point distance between the pipette and the optical tweezers slowly increased until detachment when the stop codon was reached. As well as confirming that force on the nascent chain could make the SecM system proceed past the stalling point, they also noted a force dependence to the speed with which it would overcome this barrier.

With this force dependence established, they pondered whether a domain folding upchain of the stall point could generate enough force that it could cause translation to continue. To investigate, Goldman created a protein that contained Top7 followed by a linker of variable length, followed by the SeqM stalling motif, which was in turn followed by GFP. Shown in the figure above, altering the length of the linker region defined the location of Top7 while it attempts to fold. A long linker allows Top7 to fold completely clear from the ribosome tunnel. A short linker means that a it can’t fold due to many of its residues being inside the ribosome tunnel. Between these extremes, however, the protein may only have a few residues within the tunnel and by stretching the nascent chain it may access them so as to be able to fold. In addition, Top7 was chosen specifically as it was known to fold even under light pressure. Hence, by newtons third, Top7 would fold even while its C terminus would be under strain into the ribosome, it in turn generates an equal and opposite force on the stalling peptide sequence within the heart of the ribosome tunnel, which should allow translation to proceed past the stall. Crucially, if Top7 folded too far away from the ribosome, this interaction would not occur and translation would not continue.

Goldman’s experiments showed that this is in fact the case; they found that only linkers of 15 to 22 amino acid would successfully complete translation. This confirms that a protein folding at the mouth of the ribosome tunnel can generate sizeable force (they calculate roughly 12pN in this instance). Now I find this whole system especially interesting as the I wonder how this may generalise to all translation, both in terms of interactions of the nascent chain with the side wall and the domain folding at the ribosome tunnel mouth. Should I consider these when I calculate translation speeds for example? Oh well, we need a reasonable model for translation while ignoring these special cases first before I really need to worry!

I recently had the pleasure of working for 11 weeks with the wonderful people in OPIG. I studied protein interaction networks and how we might discern the parts of the network that are important for disease (and otherwise). In the past, people have looked at differential gene expression or used community detection to this end, but both of these approaches have drawbacks. The former misses the fact that biological systems are rarely just binary systems or interactions. Community detection addresses this, but it in turn does not take into account the dynamic nature of proteins in the cell – how do their interactions change over time? What about interactions or proteins that are only present in some cells? Community detection tries to look at all proteins and ignores important context like this.

My aim was to develop approaches that combined these elements. We used Pearson’s correlation coefficient on gene expression data and community detection on an interaction network. We showed that the distribution of the correlation of pairs of genes is weighted towards 1.0 for those that interact compared to those that do not, and for those in the same community compared to those that are not – see the figure above. We went on to assign a “score” to communities based on their correlation in each set of expression data. For example, one community might have a high score in expression data from cells undergoing amino acid starvation. We ended up with a list of communities which seemed to be important in certain environmental conditions. We made use of functional enrichment – drawing on the lovely Malte’s work – to try and verify these scores.

I had a great time with some lovely people and produced something that I thought was very interesting. I really hope I see this work pop up again and get taken to interesting places! So long, and thanks for all the cookies!

Click here for some more pretty plots and a code repository (by request only).

Overall, the authors conclude that, where possible, datasets should be collected at RT, as the derived models offer a more realistic description of the biologically-relevant conformational ensemble of the protein.

Yesterday’s group meeting got transformed into a mock interview for the final evaluation step of my ERC starting grant application. To be successful with an ERC starting grant application one has to pass three evaluation steps.

The first step consists of a short research proposal (max 5 pages), CV, successful previous grant writing, early achievements track record, and a publication list. If a panel of reviewers (usually around 4-6) decides that this is “excellent” (this word is the main evaluation criterion) then the application is transferred to step two.

In step two a full scientific proposal is evaluated. The unfair procedure is that if step one is not successful then the full proposal is not even read (although it had to be submitted together with step one).

Fortunately, my proposal passed step one and step two. The final hurdle will be a 10 minutes interview + 15 minutes questions in Brussels where the final decision will take place.

I already had one mock interview with some of the 2020 research fellows (thanks to Konrad, Remi, and Laurel), one with David Gavaghan, and the third one took place yesterday with our whole research group.

After those three mock interviews I hope to be properly prepared for the real interview!