Tuesday, February 18, 2014

Hyperpolarization without persistent radicals

The paper for this week is

"Hyperpolarization without persistent radical for in vivo real-time metabolic imaging"


Eichhorn, Takado, Salameh, Capozzi, Cheng, Hyacinthe, Mishkovsky, Roussel and Comment

PNAS 2013 110 18064



There is a lot going on in this short and well-written paper.  I highly recommend that you read it yourself, because I don't quite know where to start.  I guess I have to start somewhere, so let's jump into metabolic imaging.

For most organic chemists busy dissolving their precious compounds into 500 uL of CDCl3 and transferring to 5 mm NMR tubes, it might be a shock to learn that using high end MRI scanners, it is possible to take spatially- and temporally-resolved spectra on mice (or humans).  Yes.  You can lay in a (high end) MRI scanner and someone can take an NMR spectrum of a specific anatomical region, say the brain or the heart.  Some organic chemist should ask the question - why doesn't my department buy an MRI scanner and let me (and all my lab mates) set up a reaction flask inside and take NMR spectra of little regions of my flask as the reaction proceeds?  Frankly, the spectra are crummy relative to conventional NMR.  One trick used to improve spectral quality is to use 13C-labeled material and record 13C NMR spectra.  Going back to biological samples, it is possible to measure the real-time, spatially-resolved conversion of substrates into metabolites in this technique.  This technique is called metabolic imaging. 

Sensitivity is one big problem with metabolic imaging, leading various groups (and companies, etc.) to borrow any available tools to increase signal-to-noise.  One powerful tool that is all the rage in solid state NMR of biological macromolecules these days is dynamic nuclear polarization or DNP. 

One of the best explanations of DNP is on the website of Bridge12, a company that sells hardware to do this type of experiment.


By the way, I'll mention that Bridge12 maintains a nice literature blog at http://blog.bridge12.com/

To summarize briefly, a sample is mixed with persistent radicals.  Because the unpaired electron has a much larger gyromagnetic ratio, the population difference between spin-down and spin-up states is larger, which explains why EPR is so much more sensitive than NMR.  The trick behind DNP is to use microwave irradiation to saturate the EPR signal of the persistent radicals and transfer the polarization to the nuclei in the sample, much like the classic steady state NOE experiment.  This transfer leads to an enhancement of signal.

For any clinical application, though, DNP is hard to pull off.  Because you cannot inject radicals into a patient, you have to filter them out after transfer.  Meanwhile everything is relaxing and some of the signal enhancement that you have fought so hard (and paid so much) to get is lost.  You get about a minute.  But in this minute, impressive results have been collected, allowing real-time detection of metabolic intermediates in vivo.  The authors assert that "most preclinical developments have focused on samples of neat pyruvic acid (PA) to which suitable persistent radicals are added."  PA is important because "this molecule has a central position in the glycolytic pathyway" and "is a powerful marker for cancer metabolism."   

As the title of the publication suggest the authors of this paper describe a way to take advantage of the sensitivity gains of DNP without persistent radicals.  They make tiny beads of neat PA and extract an electron by UV irradiation to make a radical.  They do the DNP experiment to enhance the 13C signal of PA.  When these beads are dissolved in water, all you get is hyperpolarized pyruvate, pyruvate hydrate and acetate (and CO2 gas).  All this extra signal make it possible to record spatially and temporally resolved 13C spectra inside a mouse and watch the formation of lactate and breakdown of pyruvate!

What do the authors actually do?

They devise a clever experimental setup so that droplets of 2 +/- 0.5 uL of pyruvic acid are dripped one-by-one into a 3 mm EPR tube (http://www.wilmad-labglass.com/Products/705-PQ-6-25/) sitting inside a EPR dewar (http://www.wilmad-labglass.com/Products/WG-850-B-Q/) filled with liquid nitrogen.  Each drop is flash frozen into a little bead ~1.5 mm in diameter.  Approximately a dozen beads are collect in each EPR tube.  I picture bubble tea in my head.  Then each EPR tube filled with frozen beads and liquid nitrogen is irradiated for 1 hour using a high-power 365-nm LED array.

What happens in this setup?  First, radical are created.  Figure 1B and C show the X-band EPR spectrum of natural abundance PA beads and [1-13C] PA beads at 77 K after 1 hour of UV irradiation.

For readers unfamiliar with EPR, it helps to think of EPR in terms of NMR, except EPR focuses on unpaired electrons.  The x-axis is magnetic field instead of frequency (or ppm) because in EPR the magnetic field is swept during the experiment.  The y-axis is the first derivative of absorbance instead of absorbance.  At the point on the x-axis where the signal switches from positive to negative is the maximum of the absorbance.  If you study Fig 1B, you will see four such crossing.  If you study the intensity of the bands you can convince yourself that the absorbance spectrum is a quartet.  The reason is that a delocalized unpaired electron is coupled to three equivalent protons (the methyl group of PA).  As a control, the authors also make beads with 13C enrichment at the 1 position.  In this case, the unpaired electron is coupled to one 13C nuclei and 3 equivalent protons.  The absorbance spectrum is a doublet of quartets.  If you study the EPR spectrum in Fig 1C you can convince yourself that you see a doublet of quartets. 

What is actually going on in the beads upon UV irradiation in not consequential for this publication.  To quote the authors "in aqueous solution, PA undergoes efficient photodecarboxylation. ... The radicals produced by the low-temperature UV irradiation of the pure acid  ... are most likely related to intermediary products postulated for this photodecarboxylation mechanism."  In summary it does not matter why, but the authors can produce PA radical at concentrations of 15 mM.  They get the concentration from quantitative EPR.

Approximately seven frozen beads of [U-13C] PA are dissolved in ~500 uL of D2O to make a 900 mM solution, which is transferred to 5 mm NMR tube.  The authors record a 1D 13C NMR spectrum with no 1H decoupling on a 400 MHz NMR.  The interscan delay equals 180 s and the number of scans equal 512 for a total acquisiton time ~17 hours.  The result is shown in Fig. 2.

The authors assign several 13C resonances to pyruvate, pyruvate hydrate, acetate and CO2.  The authors interpretation of this result is that upon dissolution, PA radical decomposes to CO2 and acetate!  There is no EPR signal upon dissolution and the 13C T1 are the same as non-UV irradiated PA.  Together these data indicate that there is no radicals and these samples can be injected or perfused into live animals.

To get hyperpolarization, though, you still have to do the microwave saturation experiment.  The authors state that using their system, 13C polarization of 10% can be achieved in 2.5 h at 5 T and 1.2 K using 50 mW microwave power.  I have no idea if that is impressive or not.  It sounds very expensive, though.  By way of comparison, using traditional persistent radicals, 13C polarization of 60% can be achieved in the same amount of time.

To demonstrate the potential of their method, the authors collect in vivo metabolic images on a mouse.  80 mM hyperpolarized pyruvate solutions are prepared from UV-irradiated PA beads.  A 300 uL bolus was injected into mouse femoral vein.  A 13C spectrum localized in the mouse head was measured every 3 s for 75 s.  Holy crap!  A 13C spectrum every three seconds!  Figure 4A shows the
spectra.  The inset spectrum is a sum projection (I assume).    

What is more is that the authors can collect metabolic images 5 mm^3 spatial resolution, 3 s time resolution.  Fig. 4B and C show 13C images using interleaved selective excitations of pyruvate (B) and lactate (C) superimposed on 1H anatomical images of the mouse head.  

Figure 4 is very impressive and it is clear why the authors think they are onto something.  Endogenous DNP is clearly a huge step towards routine clinical applications.  I look forward to following this field. 


researchblogging.org citation code

Eichhorn TR, Takado Y, Salameh N, Capozzi A, Cheng T, Hyacinthe JN, Mishkovsky M, Roussel C, & Comment A (2013). Hyperpolarization without persistent radicals for in vivo real-time metabolic imaging. Proceedings of the National Academy of Sciences of the United States of America, 110 (45), 18064-9 PMID: 24145405


Monday, February 10, 2014

Papers I am reading ...

It has been a big week at Sit and Spin.  Last Tuesday morning just after I posted my review of an Angew. Chem. paper on NMR of very large protein complexes (http://sitspinnmr.blogspot.com/2014/02/1-mega-dalton-and-beyond.html), I jumped over to twitter to publicize the blog.  I don't remember the exact chain of events, but eventually my blog was mentioned by @SeeArrOh, an organic chemist and blogger (http://www.justlikecooking.blogspot.com) with a wide readership.  As a consequence, the blog got more hits in one day than I had in the previous three months combined!  

In parallel, I saw an interesting news article on Nature (http://www.nature.com/naturejobs/science/articles/10.1038/nj7486-123a?WT.ec_id=NATUREjobs-20140206) highlighting the correlation between citations in research blogs and citations in the scientific literature.  This article reminded me why I got into the blogging game.  I think there is a lot of great research being done by hard-working and thoughtful scientists in the field of NMR.  I am concerned that the broader community of chemists is not aware of this work, or at least as aware as they are about work in other branches of chemistry.  Perhaps, in some small way, this blog can help to remedy this problem.  

Additionally, the Nature news article alerted me to the site www.researchblogging.org.  I am ashamed to admit that I was unaware of ResearchBlogging.org and underlying the movement underway in science communications.  This site is such a great aggregate of information.  I have spent so much time digging through it that I went ahead and applied for an account.  I would be honored if the editors include this blog.  

Of course with added attention there will be added pressure to write more posts.  It takes me a while to write a complete critique.  It probably takes almost as much time as it took me to make powerpoint slides for journal club in group meetings back when I was a post-doc.  And I only had to do that a few times per year!  I am trying to get a blog post out every other week.  

In case I don't get the opportunity to discuss all of them, here is a list of papers in my "to blog" pile.  I picked these out of the most prominent journals that publish NMR papers.  The criteria I use is the following: "Does the title and abstract seem interesting to a wide audience?"  If anyone out there finds something that I am missing, please contact me.  

Here is my list.  I'll try to get to two or three of these in the next month or so.

Hyperpolarization without persistent radicals for in vivo real-time metabolic imaging.

Eichhorn et al.

PNAS 2013 110 18064



NMR paves the way for atomic level descriptions of sparsely populated, transiently formed biomolecular conformers.

Sekhar A, Kay LE.

PNAS 2013 110 12867



Diastereotopic splitting in the 13C NMR spectra of sulfur homofullerenes and methanofullerenes with chiral fragments.

Tulyabaev AR, Tuktarov AR, Khalilov LM.
MRC 2014 52, 3

Biophysical aspects of cyclodextrin interaction with paraoxon.

Soni SD, Bhonsle JB, Garcia GE.
MRC 2014
Solution Structure of a G-quadruplex Bound to the Bisquinolinium Compound Phen-DC3.

Chung WJ, Heddi B, Hamon F, Teulade-Fichou MP, Phan AT.


Structure elucidation and NMR assignments of two unusual xanthones from Lomatogonium carinthiacum (Wulf) Reichb.

Wang Q, Bao B, Chen Y.

Structural elucidation and NMR assignments of two new pyrrolosesquiterpenes from Streptomyces sp. Hd7-21.

Liu DZ, Liang BW.

Automatic assignment of 1H-NMR spectra of small molecules.

Cobas C, Seoane F, Vaz E, Bernstein MA, Dominguez S, Pérez M, Sýkora S.

Tuesday, February 4, 2014

1 Mega-dalton and beyond ...

This week I'll take a look at a fascinating paper published in Angew. Chem. towards the end of 2013.  The title is "NMR spectroscopy of soluble protein complexes at one mega-dalton and beyond" by Mainz, Religa, Sprangers, Linser, Kay and Reif.



DOI: 10.1002/anie.201301215

Why should we care about protein complexes with a molecular mass greater than 1 mega-dalton?  Many of the most important biological processes in human health are mediated by large protein complexes.  To make matters worse, many of the most powerful tools available to chemists and biochemists, such as NMR, are not suitable for such systems.  What are the challenges in applying NMR spectroscopy to such large complexes?  In traditional protein NMR (aka solution state NMR) there are two issues: #1) There are too many signals in large complexes.  The resulting overlap makes it difficult to assign resonances and interpret data; #2) As the molecular mass increases, the spin-spin relaxation rate constant, R2 (= 1/T2) increases.  The resulting broad lines diminish sensitivity and resolution.  I'll note that many of the most important developments in NMR methodologies over the past 15 years have focused on addressing these limitations.  Examples include TROSY (along with perdeuteration) which fosters backbone resonance assignment of proteins with molecular mass of ~ 80 kDa and methyl-based labeling and methyl-TROSY experiments for side chain dynamics of very large systems (> 500 kDa).

In contrast to solution state NMR, for immobilized rigid solids spun rapidly at the "magic angle", the line width of resonances do not depend on molecular mass.  No matter how big the molecule, it is possible to acquire spectra with narrow lines (line width ~ 50 Hz) as long as the magic angle spinning (MAS) rate is greater than the anisotropic interactions that broaden lines in solids (such as dipolar coupling).  Fortunately, there has been impressive technical developments to miniaturize rotors and maximize spin rates.  The problem is that MAS solid state NMR (ssNMR) requires rigid solids, usually meaning crystals of protein complexes.

The authors have developed a technique that they call FROSTY MAS to take advantage of the fact that for solids the line width is not a function of molecular mass, while avoiding the inconvenience of working with solid samples.  In FROSTY MAS ~40 uL of ~3 mM protein (40x10^-6 * 3x10^-3 * 1x10^6 = .12 g!) is dissolved in a buffer with 30-40% glycerol and loaded into a 4 mm rotor.  The protein is fully deuterated at non-exchangeable sites and only ~20% protonated at exchangeable sites.  Also, there is Cu(II)-EDTA in the solution.  This mixture is loaded into a solids probe and spun at 22 kHz.  Under these conditions rotational reorientation is impeded and the molecule behaves just as if it were a rigid solid lattice, allowing the authors to get narrow lines regardless of the protein molecular mass!  The objective of this paper is to demonstrate that the FROSTY MAS technique can be used to acquire 1H-detected backbone-based ssNMR experiments for resonance assignments of large protein complexes.

The protein that Mainz et al. apply their novel technique to is the 20S proteasome with 11S activation lids.  The 20S proteasome is a multi-subunit complex consisting of 4 heptameric rings.  This protein plays a vital role in maintaining cellular function via selective degradation of proteins.  Figure 1 explains the modular architecture of this protein (along with the molecular mass and rotational correlation time of each complex).

One trick the authors use to reduce the number of signals is that only the alpha subunit is isotopically enriched.  So overall, their spectrum will have as many signals as a 26 kDa protein (233 residues), even though it tumbles like a 1.1 MDa complex!

What are the authors results?  First, they record a proton-detected MAS spectrum for three complexes: the double heptameric ring (a7a7, 360 kDa), the full 20S proteasome (a7b7b7a7, 670 kDa) and the 20S-11S complex (1.1 MDa).  Figure 3 shows these results.  

The y-axis of these plots are normalized to the concentration of alpha subunit in the samples.  The observation is that the signal increases with molecular mass.  The authors interpretation is that the sensitivity increases due to "the reduced rotational mobility of the larger assemblies in the sedimented state."  In other words, the larger complex is more solid-like so the ssNMR tricks work better.  An alternative explanation that the authors address is that chemical exchange is responsible for the difference in signal intensity.  One could imagine that the a7a7 or a7b7b7a7 samples are in slow or intermediate exchange between two conformations and the addition of the 11S cap stabilized one state.  The traditional tools to address chemical exchange, at least for small molecules, are temperature and field.  The authors do not use these tools here.  To be honest, I do not really follow their argument (which takes ~1 paragraph and jumps from CP to TROSY), but at the end the authors dismiss the possibility that  dynamics on the us-ms or ns-us timescale could be responsible for the sensitivity increase.  

The other major result is the backbone resonance assignment of the alpha subunit of the 1.1 MDa complex.  Kay and co-workers have assigned the single ring a7 complex (180 kDa) in solution.  Supplemental figure S3 overlays the 1H-15N TROSY of this molecule (red) with the FROSTY-MAS 1H-15N correlation spectrum of the 1.1 MDa complex.  

This figure certainly highlights the impressive sensitivity and resolution of the FROSTY-MAS experiment.  The authors also record hCAhNH and hCOhNH (ssNMR equivalent of the HNCA and HNCO) of the 1.1 MDa complex.  These experiments, in combination with the assignments from the solution state, enable backbone resonance assignment of 108 or 227 non-proline amino acids in the alpha subunit.  The less optimistic description is that 119 residues are not assigned.  Of course, Kay and co-workers could not assign (some of) these residues in the solution state either, because of ms-us timescale dynamics.  At any rate, using the assignments they have, the authors can map the interface between the 11S activator and the alpha subunit using chemical shift perturbations.  Also they can use a program like TALOS+ to assess the secondary structure from the Ca chemical shift.  

Overall, this paper is a very exciting demonstration of NMR spectroscopy on protein complexes that are so large that it would have been unthinkable to try to study a few years ago.  My imagination could run wild dreaming up uses for this technique.  One example is the crowded cellular environment.  I am a bit concerned about the mass and solubility requirements for this technique.  It is not cheap to make > 100 g of triple labeled protein.  Also, as I understand it, any aggregation or amorphous solids in the sample will undermine FROSTY-MAS.  These concerns are minor, though.  This paper is outstanding and paradigm shifting!  For very large protein complexes we could see solid state NMR surpass solution in the very near future, particular when taken in combination with other emerging solid techniques, such as DNP.