MBP Squared – Biophysics Outreach

Currently in my post doctorate at the University of Kent, our group participates in outreach at a local school. The project is called MBP2 (Myelin Basic Protein Squared). We engage with students once a semester (up to 3 times a year) to an audience of 17-18 year olds. The aim is to provide the students with the fundamentals of research science as well as carrying out novel, ground breaking work.

Annotated Neuron Cell
Neuron cell highlighting several structures. The myelin sheath insulates the axon stopping any cross-signalling. Image from Wikipedia Commons.

What is Myelin?

Myelin is a lipid rich substance that surrounds neuron axons. This is fundamental to insulating neurones to send and receive signals. The signals provide control for our movement, vision and sensations such as touch. An example of neurones are wires which make connections between different pieces of hardware. An important aspect of wires are the plastic insultation (myelin sheath). Cross-signalling and short circuits happen without proper insulation of the wires. The equivalent in the human body is loss of movement, vision, urinary, bowel and sensations leading to serious disorders such as multiple sclerosis. Multiple sclerosis affects around 100,000 people in the UK1 (10,000 more people than a packed Wembley stadium).

cake-roll-pdv-MBP
Comparison between a chocolate Swiss roll and a neuron. A chocolate Swiss roll, left, is constructed from a chocolate cake wrapped with chocolate ice filling. The neurone, right, consists of an axon centre surrounded by a myelin sheath. Myelin basic protein holds the myelin sheath together. Image adapted from Public Domain Vectors.

Myelin basic protein is one of the fundamental proteins in forming the insulting layer, myelin sheath. This layer forms like a Swiss roll around the neurone. The chocolate icing is the myelin basic protein holding the myelin sheath together.

An auto-immune response can develop if the myelin basic protein becomes exposed in high concentrations. Exposure can occur due to myelin sheath damage. Myelin basic protein is unrecognised by and stimulates the immune system which leads to an auto-immune response. This starts a negative cycle of demyelination of neurones. One possible cause is the addition of a phosphate group to an amino acid on the myelin basic protein causing unfolding of the protein2. An example is where the chocolate icing becomes watery leading to the chocolate cake unfolding.

What we are doing

Using biophysics techniques, changes in protein structures can be measured and quantified. The biophysics technique the students carry out in the classroom is quartz crystal microbalance (QCM).

QCMMassChange
Diagram of a quartz crystal microbalance. Left – The thickness, h, is equal to the mass, m. The thickness produces a reference resonance, λ, by apply alternating voltage. Right – The thickness, h + Δh, changes the resonance, λ. The mass is now m + Δm. Illustration adapted from Biolin Scientific.

The QCM can measure nanogram changes on the surface of the crystal. This is measured by the change in the frequency of the crystal, λ. The frequency change can be convert to mass change using the Sauerbrey equation3.

SausburyEq
Sauerbrey equation3 – Calculation of mass using a quartz crystal microbalance. The mass change, Δm, is calculated based on constant, C, multiplied by the negative of the change in frequency, -Δf.

Water, fats and proteins deposit on the crystal surface where the change in frequency produces nanogram weight changes. Using this principle, material can be measured as bound or unbound on the surface after a washing step. Changes in protein structure affecting binding will change the protein structure. The change in protein structure will change how the protein embeds and interfaces within a cell membrane. This is important to consider when measuring myelin basic protein embedding the lipid bi-layer.

LipidBiLayer
Lipid bi-layer formed of two lipid membranes. The hydrophobic tails interface where the hydrophilic heads remain on the outside of the membrane. Image from Wikipedia Commons.

We can model a cell membrane by creating a lipid bi-layer (also known as phospholipid bilayer). A lipid bi-layer form membranes where hydrophobic (water hating) tails interface while the hydrophilic (water loving) heads create an inner and outer membrane.

Purified myelin basic proteins with and without addition of phosphate groups can be tested systematically on the lipid bi-layer. Binding of myelin basic protein can be tested as the proteins are deposited under flow. If the frequency remains the same after a washing step, the protein is likely bound and integrated within the membrane. If the frequency increased, it is likely the protein was not bound to the lipid surface. These modified derivatives of myelin basic protein are produced by other MBP2 groups.

Furthermore, we can use the mass change to calculate the molecule per area (molecule/cm2) or area per molecule (cm2/molecule). This is important we have a lipid bi-layer formed as well as calculating how many myelin basic protein derivatives have affinity for the layer. We know the weight of a molecule based on the kilodalton (kDa). Kilodalton is a unit of weight generally used for a single protein or polymer. Additionally we know the area as it is limited to the gold electrode on the crystal which we measure the frequency change of.

LayerPacking
Ping pong ball layer as either tightly packed or loosely packed layer. The tightly packed layer, left, has no pockets where the layer may have overlap. The loosely packed layer has pockets and gaps between the ping pong balls. More ping pong balls can be added to fill and complete the layer.

We can think of this as a swimming pool with ping pong balls on the surface. The number of ping pong balls determines whether we have a loosely packed surface where we can see pockets or if ping pong balls cover the entire surface. In addition to measuring the lipid deposition, we can measure the affinity for certain mutations of myelin basic proteins to the lipid bi-layer.

Neutron Reflection Diagram
Diagram of neutron scattering. The setup consists of a source producing neutrons which are directed in a beam indicated by a red arrow. The slit controls the shape of the neutron beam. The sample is the measured material which reflects neutrons which are measured by the detector. The angle of reflected beam is the same as the incident beam as this is specular reflection. The shape of the reflected neutrons provides information on the thickness, density and roughness of all substrate films on the surface.

As QCM is an indirect analysis of the surface, we teach the students about a direct measurement called neutron reflectometry4. Neutron reflectometry can measure smaller than a nanometre where myelin basic protein is between 5-25 nm depending on protein folding. The setup consists of a source producing neutrons, a slit to control the beam shape, the sample we measure and a detector to measure the neutrons reflected. This is specular reflection which means the incident beam has an equal angle to the reflected beam. The neutrons have different momentum vectors depends on which substrate layer was reflected, denoted as qz. The shape of the reflectivity profile provides information on the surface structure such as thickness, density and roughness of any substrate film layers. A way to think of this is a rainbow where light is scattered forming different colours. In neutrons, there are different layers which reflect neutrons at different momentums.

The students have the opportunity to fit the reflected neutron data. The raw data requires fits to each film layer building upon the prior layer. This requires a lot of minor adjustments to thickness, roughness, scattering length density and hydration. The students then export data on the layer thickness of a lipid bi-layer and myelin basic proteins.

The setup is similar to QCM where a bi-layer is formed and proteins are deposited. Neutron reflectometry can measure whether the protein is embedded in the lipid bi-layer, located on the lipid surface or is not present after a washing step. QCM can only provide a mass change but no information how the protein has embedded in the lipid bi-layer. A paper here from our group principle investigator measuring myelin basic protein within a lipid bi-layer5.

References

  1. Multiple Sclerosis Trust (2020). Prevalence and incidence of multiple sclerosis. Online. Accessed 14/01/2020.
    https://www.mstrust.org.uk/a-z/prevalence-and-incidence-multiple-sclerosis
  2. Boggs, J. M., Rangaraj, G., Gao, W., & Heng, Y. M. (2006). Effect of phosphorylation of myelin basic protein by MAPK on its interactions with actin and actin binding to a lipid membrane in vitro. Biochemistry, 45(2), 391–401. https://doi.org/10.1021/bi0519194
  3. Sauerbrey, G. (1959). Verwendung von Schwingquarzen zur Wägung dünner Schichten und zur Mikrowägung. Zeitschrift Für Physik, 155(2), 206–222. https://doi.org/10.1007/BF01337937
  4. ISIS Neutron and Muon Source (2019) Neutron Reflectometry. https://www.isis.stfc.ac.uk/Pages/Reflectometry.aspx
  5. Raasakka, A., Ruskamo, S., Kowal, J., Barker, R., Baumann, A., Martel, A., … Kursula, P. (2017). Membrane Association Landscape of Myelin Basic Protein Portrays Formation of the Myelin Major Dense Line. Scientific Reports, 7(1), 4974. https://doi.org/10.1038/s41598-017-05364-3
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