Pressure Relief: Reflections After Research

Monday 29 July 2019

With my first five weeks of research bound, folded and consigned to history, a pause to reflect on the summer is welcome. The goal of this first stage of research was to produce a pressure cell suitable for flash-freezing EPR samples. EPR, or electron paramagnetic resonance, is a method for studying molecules with unpaired electrons (or those to which we attach helpful ‘spin labels’), using the excitation of electron spin to glean information about the unpaired electron’s surroundings or, in the case of DEER (Double Electron-Electron Resonance), a distance distribution for the separation of two spin labels.
Though flipping a rock on textbooks of quantum mechanics sends matrices scurrying, Classical analogies, though necessarily flawed, can help with grasping the concept.

The reason that we can use EPR to investigate structures with free radicals is due to the magnetic moment of the unpaired electron, which arises due to a conserved quantity known as ‘spin’. Classically, a magnetic moment can be assigned to a conducting loop carrying a current. A loop with a large magnetic moment will experience a large torque, or ‘turning force’, so to speak, when at some arbitrary angle to the direction of an externally applied magnetic field. Also, the larger the magnitude of the magnetic dipole moment, the greater the potential energy of the dipole. The dipole will have its minimum potential energy when aligned with the direction of the magnetic field, and its maximum potential energy when counter aligned.
If we think of the electron as a Classical particle, say the omnipresent tiny billiard ball with finite radius, we can intuitively account for the electron’s magnetic moment:
Imagine the billiard ball, with a net charge of -1, isolated in space. Assume that the ball has a finite and uniform charge density. Now zoom in on a tiny portion of the outer surface of the billiard ball, which has some differential charge dq. Now set the ball spinning on its axis. The charge dq now travels in a circle around the axis of rotation of the ball. That is, it now constitutes a charge moving around a conducting loop, thereby possessing a magnetic dipole moment.
Quantum mechanics states that the spin angular momentum is quantised by the reduced Planck’s constant, with magnitude √(2&S(S+1)) , where S is the electron spin quantum number, determined by the secondary spin quantum number ms, an eigenvalue of the spin operator. The number of possible spin states is given by (2S + 1).
For the electron, with S = ½, this yields two possible spin states: + ½ and – ½, referred to as ‘spin-up’ and ‘spin-down’. Without an external magnetic field, these two states have the same associated energy. But when the external field is applied, the degeneracy is broken, the spin counteraligned with the direction of the external magnetic field possessing the larger associated energy. This loss of degeneracy is known as the electron Zeeman interaction.
One can think of this a little like a kayak paddle. In still air, there is no apparent difference between having one or the other edge of the blade leading. But place the paddle into a fast-flowing stream, and one must work to rotate the blade into the oncoming water. Once the blade is parallel with the racing fluid, it becomes stable; but lift the edge slightly, and the whole blade will rotate 180 degrees until parallel with the stream once again. Thus, the former state is high-energy, unstable; the latter, low-energy and stable.
The difference in energy between the two spin states may be bridged by the absorption of a microwave frequency photon, yielding an absorption spectrum more often presented as the first derivative. Alone, this trace carries limited information. However, various interactions fill in the blanks, including spin-nuclear hyperfine coupling, spin-spin dipolar fine coupling, spin-orbit coupling, spin-lattice coupling and the Fermi contact interaction. Though my current understanding of these interactions falls far short of where I would like, next year’s quantum mechanics courses will lay the foundations to explore these effects in greater detail.
June turned into something of an extended DIY project, punctuated by forays into quantum mechanics textbooks, convoluted adventures through product specification data and rather messy experiments with sealants. Throughout the threading of minuscule silicone pistons, proud returns to room 135 with boxes of dry ice and apologetic requests at the workshop, I came to know that a level of ignorance really can be beneficial. Not ignorance of process, theory or safety: one can never have enough of that kind of knowledge. I refer to the newness of a field, of poking one’s nose over the edge of something new and exploring its potential; a newness in which there are no imagined obstacles to hinder the testing of ideas. and where a dearth of expectations gives great excitement when the least usefulness arises from one’s efforts.
The hammer did not follow the humble stone, and so I found in these five weeks: half the challenge of construction lies in liberal application of, if not Ockham’s, perhaps Leonardo’s razor: in whittling down the fluff and frills of a first solution to something altogether simpler and easier to fix, remembering, simultaneously, that when an undergraduate’s first plans are put to the test, ‘cheap and cheerful’ is a prudent edict. Thus, a promising selection of valves were slashed to a simple tee, safety features reduced in complexity (if blowing out the bottom is undesirable, why, just blow out the top!) and incorporated into the few indispensable components to reduce cost and bulk. The end result is a design so straightforward that I wonder why it took so long to finalise; confirmation, perhaps, that the dissection of initial ideas has yielded a functional skeleton. From this, I learn that the process of refinement is only complete when the removal of just one piece causes the entire construct to crumble.

Plans in progress: early ideas for the pressure cell arrangement

Following this little adventure among the flexible world of drawings and ideas, I was to learn another valuable lesson: the transition from idea to physical object takes time. Lead time. In this case, three weeks. I sat back, having completed week two of five, and evaluated the situation. Could I have avoided the inconvenience of equipment due to arrive on the final day of my project? Not without completing half of the project before it formally began. Quandary resolved, I turned to the unexpected task of filling a three-week vacuum: another opportunity to exercise self-leadership.
Cue the difficulties of freezing. Imagine the situation: a small biological sample, difficult to procure, confined to a 3 mm, frail quartz tube. Submerged in ethanol, in proximity to water. Frozen to -196 degrees Celsius. Spot the difficulties?
If the sections containing water are above the ethanol, how can we prevent the formation of a water/ethanol ice block around our precious sample? How to stop the ethanol invading the sample itself? What of sample recovery?
Previous work by Lerch and collaborators1 provided a partial solution to the problem of sample protection: cure silicone elastomer inside a sample tube, break the tube and cut the silicone, producing a convenient plug to separate the sample from the ethanol. However, this still left the issue of recovering the sample, not to mention the unfortunate demise of at least one pricey tube.
The ideas? Cut the silicone to size by hand (messy, ghastly fumes, would not recommend); use a cheap glass tube for the curing (the required dimensions are unavailable); insert a minuscule plastic bead to block the tube (the resulting curved meniscus will reflect microwaves). The one that works: 2 mm O-ring silicone, cut short and threaded on cotton; seal the end of the cotton with elastomer to block ethanol, and hey presto, a tiny sink plug fit for purpose.
The incompatibility of the water/ethanol duo had a straightforward, though slightly daunting solution: fill the Barocycler system with ethanol. A flammable liquid at 60,000 psi… not the makings of a sound night’s sleep. But factor in the low temperature of the system and the risk is greatly reduced.
But why all of this fuss to achieve high pressures? Imagine standing at one end of a dark room. At the other end is a stool, on which rests a small object, isolated, unapproachable. Without recourse to the tactile sense, how can one discover its function?
Simply switch on the light. Now one can see that the object is a Swiss army knife. This is akin to using EPR techniques to investigate the structure of a protein. The likely uses of the object are now apparent; but what is its precise functionality, and how might this be revealed without physically unfolding each attachment?

The barocycler, for generating pressure. Also a teacher of many lessons in troubleshooting and repair.

This is where pressure becomes helpful. Flood the army knife’s cavities with high pressure water, and the various files, blades and hooks will be pushed into the open. For a protein, the filling of cavities with a high pressure fluid enables the transition to a higher-energy, active state: a functional mode. Flash freezing then preserves this mode sufficiently long to probe its conformation with DEER.
These weeks of problem solving were enlivened by journal club meetings with my supervisor, Dr Janet Lovett’s, Electron Paramagnetic Resonance team, which provided a valuable glimpse of the organisation and teamwork needed to support long-term, interdisciplinary research projects. Attending the Centre of Magnetic Resonance annual meeting, I was struck by the extent of collaboration and breadth of expertise required to successfully refine and apply magnetic resonance techniques. In many ways, the days of solitary scientific research are no more: each discipline’s branch on the tree of knowledge has grown in girth until it cannot be spanned by a single pair of arms. The more questions that we answer, the more connections are unearthed between previously isolated disciplines, and the more relevant leadership becomes.
I extend my sincere thanks to Dr Janet Lovett for her advice and guidance; to the postgraduates of the Lovett lab for their warm welcome and patience; and to Lord Laidlaw, for granting me this opportunity to develop ingenuity, familiarity with protocol, and confidence in a research environment. The ability to walk into a department uncowed by the proximity of projects of daunting scope, to navigate through these juxtaposed nodes of endeavour to produce something of use in my own small corner, has galvanised my enthusiasm for not just research, but problem solving in a broader context.
1. Lerch, M.T., Yang, Z., Altenbach, C., Hubbell, W.L. (2015). Chapter Two – High-Pressure EPR and Site-Directed Spin Labeling for Mapping Molecular Flexibility in Proteins. Methods in Enzymology, 564, pp. 29-57.

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