Wednesday, 28 June 2017

Luciferase versus GFP: A lighthearted molecule for July

Related imageThe summer brings out the best in most of us (purely based on the evidence of the greater number of smiling people on the platform when I catch the train!). So, I thought about choosing a molecule that reflected this mood. I could have looked amongst the proteins that are the targets of psychotic drugs, or I could have gone for sunlight capturing molecules involved in photosynthesis. In January of 2016, I discussed a number of photo-activated proteins after a thrilling seminar from the Biochemist Tomas Carrel. See here. This month I have chosen the enzyme luciferase, a key element in the generation of light in insects such as the glow worm and the  fire fly (Photinus pylaris). I hope you agree that these creatures (notwithstanding the general unpopularity of most insects), make most people smile!

Image result for lucifer paintingThe name luciferase, has its origins in the Latin for "bringer of light" (think lucid or elucidate). You might be familiar with the Biblical archangel Lucifer, who defied God, and went on to establish an alternative post-mortem retreat for those of a slightly unorthodox disposition. As Mark Twain (or J.M. Barrie?) famously commented: I'd choose Heaven for the climate and Hell for the company! I assume Lucifer lit up the general conversation in Hades? Alternatively, if you have read any Charles Dickens or Arthur Conan Doyle, you will know that the "nickname" for a match was a "lucifer". Let's now have a look how luciferase generates light and how the properties of the enzyme have been incorporated into a biological detection technology that is used both in a discovery and diagnostics mode. The reaction, catalysed by all luciferases is as follows.

luciferin + ATP → luciferyl adenylate + PPi

luciferyl adenylate + O2 → oxyluciferin + AMP + light

Light is produced because the reaction forms oxyluciferin in an electronically excited state. The reaction releases a photon of light as oxyluciferin returns to the ground state (in this case, the "quantum mechanical" state of a system having the lowest possible potential energy. The expression is also used in Biochemistry to define the lowest free energy state of substrate(s) in an enzyme catalysed reaction, usually with respect to the transition state and the products of the reaction). Firefly luciferase generates light from luciferin in a multistep process. First, D-luciferin is adenylated by ATP to form luciferyl adenylate and pyrophosphate. Following this "activation" by ATP, luciferyl adenylate is oxidized by molecular oxygen to form a dioxetanone ring. A decarboxylation reaction yields the excited state of oxyluciferin, which tautomerizes between the keto-enol form (at a given pH and temperature, all carbonyls have a tendency to shift between these two forms: you can read more here). The reaction finally emits light as oxyluciferin returns to the ground state. [I shall return to the important topic of "excitation" of molecules and its importance in Biological systems in a separate post.]

This is quite a complicated phenomenon without a background in undergraduate Biochemistry, Chemistry or Biophysics, so don't worry if it leaves you a little baffled. Think of the dyes that colour your clothes, or a bright blue copper sulphate solution. It is sometimes possible to re-organise electrons in a molecule in response to visible and uv light. This can result in a portion of the visible spectrum being removed by the molecule, which results in a very specific colour of a solution of the molecule. The process involves light energy in the form of photons, re-organising specific electrons in the molecule, followed by their return to the "unexcited" state which can be accompanied by the emission of a colour change, a fluorescence  emission or phosphorescence. There are specific "pathways" that are described in quantum mechanics that account for these phenomena and why different molecules choose one over the other, or none at all! I shall attempt to write a post on these important phenomena in the near future, since they are particularly important in the mechanism of photosynthesis.

The protein molecule (shown left from a dinoflagellate) comprises two major structural units. The blue (mainly) beta barrel sits beneath the alpha-helical arrangement, with the adenylate and the chromophore positioned at the junction of the two domains. On binding the reactants the domains come together to exclude water, which increases the half life of the "excited" state of the oxyluciferin. The details vary a little from species to species and this leads to a variation in the wavelength of the emitted light. One mechanism proposes that the colour of the emitted light depends on whether the product is in the keto or enol form. The mechanism suggests that red light is emitted from the keto form of oxyluciferin, while green light is emitted from the enol form of oxyluciferin. This is not proven, but the logic relates to the well established connection between resonance structures and the energetics of absorption of light in the visible and uv spectrum. There are some other ideas, but even though a consensus hasn't yet been reached, all mechanisms will probably connect the local (molecular) environment with the stabilisation of the excited state (see below RHS).

You may wish to compare the properties of luciferases with naturally fluorescent proteins such as the Green Fluorescent Protein (GFP for short). Can you think of the biological advantages for an organism emitting light? Maybe a useful exercise is to compare and contrast the applications of these enzymes in contemporary experimental molecular cell biology? Can you find glow worms and fireflies in the UK? Take a look at the survey.

Key points from the Blog. There are naturally occurring proteins (and small molecules) that have fascinating optical properties. Such properties are sometimes related to their requirement for energy to drive unfavourable reactions (DNA repair, photosynthetic electron transfer). In some cases, the natural glow of a fire-fly or the bright fluorescence of marine organisms has evolved for reasons that are not entirely understood. However, such beautiful natural phenomena attract Biochemists and they can lead to technologies that unlock hidden secrets in the behaviour of cells. Luciferases are used in a wide range of diagnostic and research methods, and I hope you agree with me that they are incredible molecules, however I think GFP currently holds the prize as the most important optical probe in contemporary biology.

Tuesday, 6 June 2017

Fighting back! Vancomycin (plus) my molecule for June

This month I decided to wait for the release of a paper that the press announced as an example of magic! Since all magic can be explained by science, and therefore science is not magic; I thought it would be appropriate to set the record straight. Dale Boger's group at The Scripps Institute in California published a series of chemical modifications to the "last resort" antibiotic vancomycin that impact not only on its potency, but also look to have made significant inroads to reducing the emergence of "resistance". The excellent final figure in their recent publication in Proceedings of the National Academy of Scienceis shown below. It reveals the complexity of the molecule and it identifies the chemical groups associated with its antibiotic properties. But first let me provide some background to vancomycin. 

Vancomycin was discovered in the same year that Watson and Crick discovered the double helical nature of DNA (1953), around 24 years after Fleming published the discovery of Penicillin. By 1953, resistance to penicillin treatment had become a real clinical issue, particularly in Staphylococcus aureus (recall MRSA). With the discovery of vancomycin (the vanquisher!), it looked like an alternative treatment for resistant strains was now in sight. In fact the drug was fast-tracked into hospitals and was in use just five years after its discovery.

Vancomycin is a naturally occurring heptapeptide, originally isolated from the organism Amycolatopsis orientalis, which was originally identified by the Harvard trained organic chemist, Edmund Kornfeld at Eli Lilly, working with soil samples collected from the jungles of Borneo by missionary workers! We now know that this seven amino acid peptide is synthesised by non-ribosomal protein synthesis (NRPS), after which it is chemically modified in a a complex series of secondary metabolism steps. the aromatic rings are a combination of modified phenyl-glycine and tyrosine which are chlorinated and glycosylated. The sequence of amino acids is 

(1) Leucine (2) Tyrosine (modified by hydroxylation) (3) Asparagine (4) Glycine (modified by phenyl-hydroxylation) (5) Glycine (modified by phenyl-hydroxylation) (6) Tyrosine (modified by hydroxylation) (7) Glycine (modified by addition of dihyxdroxylated benzene)

starting from the bottom RH corner and proceeding clockwise in the structural diagram at the top.

As you might imagine, this is too short a sequence to be synthesised as heptameric units on the ribosome (what is the shortest polypeptide to be synthesised in a mature form via the ribosome? and what are the constraints on chain length?). The role of vancomycin, a complex secondary metabolite in the physiology of Amycolatopsis orientalis, as with other antibiotics is presumably to serve as a defence against bacterial threats to its survival, but it also reveals that complex carbohydrates etc can be introduced into microbial polypeptide chains in a way that we usually associate with proteins in much more elevated species. The 7 modules of vancomycin (centred on each amino acid) are generated and "finalised" for function by a set enzymes that utilise ATP to provide the necessary energy through an adenylate intermediate. You can read more here about these enzymes and their genes. 

In my mind there are always three main questions that need addressing when trying to rationalise the mode of action of antibiotics. The first is pretty well understood and relates to the target (or targets). The second is less clear, and that is an understanding of the mechanism of killing versus growth arrest. The third question relates to the likelihood and mechanism of resistance. In the case of vancomycin and its synthetic derivatives, it would appear that there are three targets. The biosynthesis of the bacterial cell wall is essential for the normal growth of most bacteria. Vancomycin is a bivalent inhibitor of this process, interfering with two distinct enzymatic steps (shown in blue at 6 0'clock and 12 o'clock in the diagram). In addition, the positively charged moiety (bottom LHS) is thought to disrupt the cell membrane. The combination of these three weapons not only reduces the minimal inhibitory concentration (MIC) of vancomycin, but it also massively reduces the emergence of resistance in the target organism. So the Boger group have made significant progress in taking a natural product and improving it, in the great traditions of natural product chemistry and contemporary organic synthesis. It is now up to the molecular biologists to provide the explanations relating to cell death and ultimately the mechanisms of resistance, which will be when and not if!

Monday, 8 May 2017

Molecule of the Month of May 2017: Adhirons

As usual, I had a molecule almost ready to go, until I started reading an interesting thesis from one of (Professor) Mike McPherson's research group at the University of Leeds. Without disclosing any secrets, the thesis centred around a class of molecules called adhirons (see the left hand image from the Leeds group RCSB PDB). I thought I would put my reading to good use and share my enthusiasm for these remarkable polypeptides. On the one hand they offer an excellent opportunity to challenge the primacy of antibodies in high affinity, selective protein binding, but they also offer considerable insight into the evolutionary relationships between primary structure and the "encoded" tertiary interactions between protein surfaces. And as you know, it is the network of protein:protein interactions that expands the functionality of many eukaryotic genomes. Since "Molecule of the Month" aims to introduce you to a mixture of "Old Chestnuts" and "Young Turks", I also try to select molecules for the lessons they can teach about the relationship between protein structure and function in an evolutionary context.

Adhirons and antibodies have some things in common and some significant differences. I have written about IgG before, and rather than give details, I shall cite their general properties for comparative purposes here, since antibodies form a major part of any Biology curriculum in High School and above. The first point to make is that Adhirons are not found in Nature. However, they are molecules that have been considerably "informed" by Nature. They are derivatives of a group of protease inhibitors referred to as statins (but not the cardiovascular drugs), a class of cysteine protease inhibitors which are related to the stefins, studied in Sheffield in (Professor) Jon Waltho's structural biology group in the 1990s. The development of NMR for the determination of protein structure has been helped significantly by the existence in Nature of polypeptides of less than 100 amino acids (low molecular weight) that exist to keep hydrolytic enzymes, including proteases, nucleases and amylases in check. Such proteins (Tendamistat, Bovine Pancreatic Trypsin Inhibitor and Statins etc.) proved to be workhorses for method development and helped interpretation of the complex spectra derived from sophisticated NMR experiments. You can read Nobel laureate, Kurt W├╝thrich's overview of the development of NMR in structural biology here.

The adhiron used in Mike's group is derived from a plant statin family called the phytostatins. It was selected for its simplicity (no awkward post-translational modifications), and its intrinsic physical properties. It is stable at elevated temperatures (around 100 degrees C), it is extremely soluble (hundred of mg/ml solutions are not uncommon: many proteins drop out of solution above concentrations of 1mg/ml!), it has no sensitive disulphide bonds (which can lead to redox problems in use) and it is relatively low in molecular weight and monomeric, which means every application becomes simplified and that much more cost-effective. You can read more about the background to the development of adhirons here.

Let me compare adhirons to antibodies in respect of their general structure and properties. Both molecules comprise two elements: a variable region (not really a domain) and a constant domain. In IgG, the constant domain (Fc) provides the scaffolding, or support structure, upon which the variable (Fv) domains (they are paired, in most antibodies, which gives them their bivalent binding property) sit, comfortably awaiting the "arrival" of a complementary antigen. This structure is usually represented as a Y shape: the V is the variable region and the tail is the constant region, which interacts with other proteins (such as Fc receptors). In adhirons, the scaffold function is provided by the regular secondary structure elements beta sheets and an alpha helix. And as with the Fv region in IgGs, the selective binding function is found on a series of "loops". Time for a little diversion to explain the paradox between Emil Fischer's elegant "lock and key" model and the use of "loops" in high affinity (sub-nanomolar) recognition between proteins (the figure above shows a benzene ring in the active site of an imaginary enzyme....or a nut in a spanner!).

When two perfectly complementary, rigid bodies come together, they first make a good "fit". This is the equivalent of placing the correct "spanner" on a "nut" (as shown above LHS), or a "Philips" screw driver into a suitable screw (such screws were invented by Henry F. Phillips over 80 years ago in Oregon and remain the most popular today). However, the nice fit must be stabilised in order for the interaction to be "productive". So the plumber or carpenter must push the screwdriver into the screw head or hold the spanner perpendicular to the nut (usually) before applying the force needed to turn both. This is the lock and key approach. As an amateur craftsman I have in my toolbox fixed width spanners and a selection of screwdrivers to match the screw sizes. I also have an adjustable spanner (above, RHS), to deal with the fact that sometimes, there are nuts that vary in diameter. The adjustable spanner has a mechanism for "fine tuning" the width of the spanner to fit a selection of nuts. This is an induced fit spanner! The latter "design" of spanner allows for an "evolutionary" change in the diameter of the nut, or it could be the availability of lactose versus glucose in the case of an enzyme. Sometimes we encode an enzyme for every substrate and sometimes we have enzymes that can accommodate substrates of varying dimensions. I am thinking here of short, medium and long chain acyl CoA dehydrogenases, for example.

It is less intuitive, to appreciate how two flexible surfaces can unite and organise rigidly (which is what enzyme active sites have to do most of the time). But think of a mixture of oil and water: although the two liquids are "mobile"; the interface is very sharp. Or think of pushing your hand into a soft glove. The other way I think of this challenging phenomenon is to consider protein folding during translation. As a polypeptide chain emerges from the ribosome it must find and adopt its tertiary structure pretty quickly (life and death is less than 30 minutes in E.coli!). So a disordered region, meeting a ligand or another protein surface will harness the forces of entropy and enthalpy in the same way as the folding polypeptdide. The role of the scaffold in antibodies and adhirons, serves to facilitate this: and experimental observations have shown that this strategy is really successful in other settings, including RNA binding and sequence-specific DNA recognition. It is this approach to molecular recognition that is adopted by adhirons. And this is translated into dissociation constants between adhirons and their targets that are often in the tens of picomolar range. [As a rule of thumb, you can estimate a dissociation constant (recall lower the molarity, the higher the affinity) from the average physiological concentration of the ligand: substrates for enzymes have Kds of a few mM, coenzymes are found at tens of micromolar and individual proteins and nucleic acids have Kds in the nM range)].

The variation in antibody specificity arises owing to the expression of a diverse set of variable regions that are linked to the constant domain (I wont discuss the mechanism here, but it is something you should read about). Adhirons can be generated in vitro that will specifically bind to many biological targets (although I expect not all with the same affinity), by randomising the loop regions that form the "spanner in the works" as statins block cysteine proteases. This can be achieved experimentally using an error prone DNA polymerase or some form of synthetic DNA, localised gene replacement method. The "library" of mutants can then be used to screen the target: a favourite technique for this is known as phage display, and you can read the details here. In this way, high affinity, highly selective adhirons can be generated to recognise target molecules with all of the advantages of a monoclonal antibody, but at a much lower cost (in view of the relatively simple methodology). The combination of a flexible interaction surface supported by a stable and relatively invariant scaffold domain in proteins, represents an interesting evolutionary concept that Nature has exploited on several occasions. (One such scaffold and loop structure is shown top left, which is part of the structure of thioredoxin, a widely distributed redox protein: the loop regions is shown in blue). Adhirons are not in the mainstream of molecular recognition reagents just yet, and the technologies for detection etc. that have grown up around antibodies are certainly not about to become obsolete, but these molecules certainly seem to me to offer some very significant opportunities for research and diagnostics in the future.

Summary of Key Points

Image result for life sciences utc logoFirst of all, I have picked a group of proteins (the adhirons) that have been derived by exposing a naturally occurring protease inhibitor to mutation in vitro. This results in a "library" of small proteins that are able to make very strong interactions with the target molecule. This feature is very similar to the highly specific recognition associated with antibodies. Since adhirons possess a set of small polypeptide loops (i.e. they show no rigid structure in solution), through which they make very strong interactions with other proteins, it is perhaps surprising that such flexible structures are so good at making these interactions. Adhirons, like antibodies comprise a stable scaffold upon which a set of flexible loops can specify strong intermolecular recognition. In fact we are beginning to appreciate that the strong interactions that stabilise substrates in the active sites of proteins seem to be less rigid (i.e. less lock and key and more induced fit), than we anticipated. Finally, this arrangement of molecules and cells with one part changing and one part constant seems to have been used on a number of occasions in Biology. The changeable region brings diversity of function and the constant region brings economy of function. Perhaps this could be discussed in class?