I would like to explain a bit about the experiments I am doing and their relevance to HD research. Not everyone is a protein biochemist (its a pretty niche skill set) so I hope this primer allows you to get a bit more of an idea about the kind of work I am attempting with this project; structural studies of the huntingtin protein.
Structure-function relationships of proteins – why is this important for HD research?
Proteins are the molecules in our bodies which “do” things, whether that’s digesting food molecules, receiving neuronal signals in the brain, or acting as structural scaffolds in our cells. In a similar way to how we tailor-make tools of different shapes and sizes to perform specific tasks, proteins are tailor-made for their functions in our bodies by evolution. This means that the shape, size and other structural features of a protein molecule are likely important in understanding how the protein molecule works in our bodies. Protein biochemists (like me!) refer to this as the structure-function relationship of proteins.
Each gene of our DNA encodes the instructions for how to make a specific protein, a string of different amino acids in a precise order. In the case of huntingtin, the protein made from the corresponding huntingtin gene, we know exactly what the gene sequence is and what the long string of amino acids this encodes is too. We also know how the HD gene mutation found in HD patients would change the amino acid sequence of the huntingtin protein. However, we currently know very little about the 3D properties of huntingtin protein, how this long string of amino acids is shaped nor how this is changed when we have the disease-related expanded huntingtin gene. Understanding more about the structure-function relationship of huntingtin will help us learn more about why the HD mutation causes disease which will hopefully lead to more therapeutic opportunities.
One of the ways protein biochemists can begin to understand the 3D structure of protein molecules is called limited proteolysis which I explain in the next section. This is the experiment that I am currently working on with the huntingtin protein; I hope to have some exciting data to share with you soon!
Protein molecules consist of tens, hundreds or sometimes thousands of amino acids which join together forming a polypeptide chain. The polypeptide chain is folded into coils, loops and strands which can then assemble into more complicated folded structural units.
Protein Structure – from amino acids to 3-dimensional structures
Many proteins consist of modular assemblies of these structural units or domains where domains are linked by less well ordered regions of the polypeptide chain. This can be thought of as different sized and shaped beads on a string, where the beads in their various forms represent the different modular architecture of the protein domains, and the string in between the beads is the unfolded linker regions of the polypeptide chain.
To work out what the domain assembly of a protein looks like, we can use enzymes which cut at specific motifs in the protein amino acid sequence. These enzymes work much better at cutting the protein at unfolded or less structured regions and so will preferentially cut the protein at the linker regions between domains. This results in a series of protein fragments corresponding to the individual domain units of the protein structure. This experiment does not aim to completely chop up a protein molecule, so is called limited digestion or limited proteolysis.
Protein domain structure analysis by limited proteolysis
A – 3D folded protein domains are like beads on a string, linked by unfolded linker regions. B – the limiting enzyme can attack and break the polypeptide chain in the linker regions which are less well folded than the domains. C – following the limited proteolysis, individual domains are now found in the reaction mix, no longer joined together by the linker regions.
After cutting a protein sample with an enzyme, we can use a technique which separates out the fragments according to size. One such method is called gel electrophoresis. By passing the fragment samples through a gel matrix, the gel acts like a sieve, holding back the bigger fragments, whilst the smaller ones squeeze through the matrix and can travel further through the gel.
Separating the domains by size
Gel electrophoresis separates out the domains according to size. The larger domains are stuck at the top of the gel as they are less able to move through the gel matrix compared to the smaller domains found at the bottom of the gel.
Next, we can cut out the separated fragments and analyse them using mass spectrometry. This allows us to work out which part of the original complete protein sequence, the long string of amino acids, they correspond to. By assembling all of this information together we can build a model of where the domains are found in the protein molecule, which regions of the protein are structured, which are unfolded and compare this to what we know about other protein structures to try and infer other features of the protein.