Protein X is a key suspect in causing disease Y. So how do you study protein X?
The Biologist’s take
If you are a biologist you might overexpress your protein of interest in cells, maybe tagged with GFP. Can you see where it is located? Maybe you have another protein tagged in red, and in some locations within the cell you see yellow, which must mean the proteins co-localize and maybe even interact right?
You would probably also perform a western blot, to see if there are any other proteins that are up or down regulated? All these results confirm your hypothesis, so time to jump to mouse models. You overexpress the protein in mice and sure enough, the animals display symptoms similar to the human disease. Great, you are now just one step away from curing the disease; all you need to figure out is why is this protein causing the disease…
But for that, you need a lot more information about the protein; what is its structure? What is its function? How does it function? What other partners help it to function? What else contributes to its structure?
Never before have we had so much information at our fingertips. In the post-genomic world the information we have on protein sequences has increased dramatically, yet the information relating to function and structure is lagging far behind – see figure 1. This is why we need to embrace disciplines and techniques that allow us to close this gap.
Figure 1: Data available from biological databases; green - protein sequences, blue - protein function, red - protein structure.
The need for cross-disciplinary research is increasingly necessary to answer the many questions that continue to evade us as we hunt for the cure to any number of insidious diseases such as Cancer, Alzheimer’s and Diabetes.
As more and more students undertake their PhD however, it is very easy to focus in on one area of expertise. Ask an in vivo biologist about structural biology and chances are they will look at you with uncertainty. Ask an old school biochemist to perform some cell culture experiments and you may well hear a line about how complicated that system is, you would never get a clear answer to your question.
Compounding these issues is the language and terminology surrounding the different techniques. Common abbreviations in one field may mean something different in another. Even words with seemingly only one meaning, can mean different things to different people (e.g. what is an oligomer? What is a small amount of material? What do you mean when you say something is “small”?).
This makes it daunting when we need to consider results or analysis which usually sits within another field than our own. But to solve the riddle of our protein we need the complete picture – so let’s take a small step into biophysical chemistry together.
What is biophysical chemistry?
Biophysical chemistry aims to use physics (and chemistry) to explain what is happening in more complex biological systems. It combines molecular biology, bioinformatics, medical biology, chemistry, physics and mathematics to answer biological questions. In many ways you can imagine using it to answer fundamental scientific questions, yet putting those answers into a much broader picture of understanding biology and disease.
Using biophysical techniques, researchers usually address questions relating to how a molecule folds and what the 3D structure looks like. They investigate molecular interactions, how molecules bind and the kinetics of these events.
Ultimately the goal is to get down to the finest detail possible, whether this is single molecule, or down to the atomic level and explain the forces that govern life.
Why should a biologist care about biophysical chemistry?
Just as someone working within biophysical chemistry or biochemistry thinks working at the cellular or organism level involves way too many variables to truly understand what is going on, so too do many biologists question how much information can really be gathered by looking at single molecules - after all, that is not what happens in real life.
A biologist will look at a blot and can see a protein is up or down regulated, they can record in real time the trafficking of a fluorescent cargo within a cell or between cells, they can observe disabilities in their animal models. Those things are real and tangible.
However, show them reduced data from a small angle neutron scattering experiment and explain that that small dip they see is because their model lipid vesicle is no longer spherical but rather elliptical, that is harder to comprehend. Try and explain it further by going into all the mathematical modelling that went into coming up with that explanation and things just get worse, “You mean to say that you are basing this on equations and not “real” experiments?”
But these questions do increase our knowledge. We do need to know how our protein of interest folds and what its 3D structure looks like. We need to know what it’s preferred binding partners are and what conditions promote or suppress binding. Once we know this information, we can start to modify the behavior of the protein and manipulate it to function in a way we desire.
The Biophysics toolkit
With this toolkit you should be able to get meaningful insights on the proteins and peptides, not to mention lipids and nucleic acids you’re working on.
Perhaps the most widely used technique in science, and certainly the earliest used, the interaction of electromagnetic radiation with matter allows us to see the world around us.
These vary in complexity of running tests and results, but include;
- UV/Visible spectroscopy
- Circular dichroism
- Vibrational spectroscopy
Unlike the spectroscopic techniques mentioned above, mass spectrometry does not rely on electromagnetic radiation, but rather splits and analyses molecules based on size and charge. Molecules travel through a vacuum chamber, their path controlled by electric and magnetic fields, ultimately giving detailed information on molecular structure and interactions.
How do molecules move through solution? Do molecules change the way the solution flows? Using hydrodynamic techniques you can gain information on both the structure of molecules and different properties they may have.
- Analytical centrifugation
- Sedimentation equilibrium
- Microfluidic Diffusional Sizing (MDS)
- Dynamic Light Scattering (DLS)
Thermodynamics and interactions
Thermodynamics describes the interplay between enthalpy and entropy. That is the tendency for things to exist at low energy (think rolling down a hill) vs the effect of thermal motion at the molecular level that tends to have the opposite effect (heating pushes things uphill). In understanding the thermodynamic contributions to a system we can better understand how these forces regulate protein structure and interactions.
- Differential scanning calorimetry
- Isothermal titration calorimetry
Studying thermodynamics tells us what should happen at equilibrium, but rarely does the biological world exist at equilibrium, it is constantly moving. Kinetics tells us how fast things are moving.
- Enzyme kinetics
- Surface plasmon resonance
These techniques are used to characterize and purify molecules based on their size, charge and other properties.
- Chromatography (usually linked to a second system for hydrodynamic or spectroscopic analysis of the fractions)
The scattering of waves (be they electromagnetic or particle) by molecules allow us to obtain an image of that molecule. But first we need to reconstruct that image from the scattered waves by understanding the amplitude of the waves and the phase contrast.
- X-ray diffraction
- Small-angle scattering
- Electron microscopy
While all the techniques mentioned are capable of describing molecules in detail, they rely on the averaging a large number of molecules. Single molecule techniques allow us to monitor just a single molecule, more akin to what might happen in the cell.
- Atomic force microscopy
- Optical tweezers
- Single molecule fluorescence
As biologists we should appreciate the information which biophysical chemistry techniques can provide us, as it can sometimes be a vital missing piece of the puzzle. By embracing these techniques, we can see new angles of a problem which we couldn’t before. Ask the right questions, and you’ll get the right answers.