TB2-1: understanding your protein Flashcards
Many biochemical experiments require pure samples of proteins for analysis. List 4 uses of pure protein in in vitro experiments (i.e. what is being determined as opposed to the method used)
- Strucutre determination
- Binding property determination
- Analysis of protein function
- Reconstituion of functional systems from components
Name 3 methods used for strucutral determination of protens that require a pure sample
-NMR
- crystallography
- cryo EM
Why is having a pure protein sample for in vitro (and in vivo) experiments so important? What happens when there isn’t a pure sample (broad two outcomes)?
-The better the sample, the increase in unambiguous data
(makes the experiment less ambiguous)
… Not having a purified protein:
- miss important findings
- attribute the wrong function to proteins
Name an example demonstrating how poor protein purification can cause a protein to be attributed to an incorrect function
Acyl transferase activity resulting from an impurity in endophilin purifications
(the impurity contained the function)
Describe the ‘acyl transferase/endophilin’ example that demonstrates how an impurity in protein purification can lead to a protein being attributed with an incorrect function
Originally believed that endophilin 1 (lipid) was able to convert lipids from outward to inward pointy cones (adopting very different curvatures). Therefore, the protein/enzyme endophilin was attributed with the function of affecting membrane curvature and acting as an acyl transferase.
Later paper showed the activity was caused by an E.coli protein contaminant (because the human protein of interest had been expressed in E.coli)
On a very basic level, why would you consider it important to know what type of protein you are investigating before making it and carrying out an experiment?
Different types of protein are made using different approaches
List 4 characteristics to consider when deciding what type of protein you have.
- single/multi-domain?
- intracellular, extracellular, integral membrane?
- folded, natively unfolded, partially folded?
- Post translational modification?
On a basic level, why would you want to consider whether a protein is intracellular, extracellular or an integral membrane protein before making it (and using it in experiments for investigation of the protein)?
These different proteins all have a different protein context which causes a difference in how you go about making and expressing them. This is because they have all evolved to survive in different environments
What key question should be considered when investigating a protein that has post translational modifications?
Do you want the protein to have this post translational modification in your experiments?
i.e. should you make the protein with these modifications or not?
Why might you not want a protein you’re investigating to have their post-translational modification in experiments you carry out in their investigation?
Having the post translational modification can lead to heterogeneity which causes difficulty in experiments due to the increased complexity?
What does “heterogeneity” mean?
“the quality or state of being diverse in character or content”
If you were investigating a virus protein (that binds to human surface proteins) would you want to include post-translational modifications or not? Why/why not? What is the post-translational modification?
Modification = glycosylation
You would want to include post-translational modifications.
This is because glycans (glycosylation events) are required for virus proteins to bind to human surface proteins (therefore essential for function)
List 4 reasons for why you want to make a protein for investigation (i.e. what is the purpose of the experiment)?
- for crystallisation
- to reconstitute a biological system
- to assess pathogen binding
- to dissect the funtion of different domains
If you are making a protein for the purpose of crystallisation, what should you consider when making the protein?
(more in a later module)
want to minimise disorder (of the crystal structure?)
e.g. glycans block crystal contacts meaning you are unable to solve
If you are making a protein for the purpose of reconstituting a biological system, what should you consider in the making of the protein?
You might want it to be as close to the native as possible…
e.g. include any naturally occurring modifications (glycosylations) and produce the whole protein (as opposed to e.g. a single domain)
If you are making a protein for the purpose of asssessing pathogen binding, what should you consider in the making of the protein?
(similar to previous question)
you would want it properly glycosylated and processed (i.e. include any post translational modifications)
In addition to keeping post-translational modifications (glycosylation) when studying virus proteins that bind to the human cell surface, what else might you consider for the making of your protein?
Probably only need to make the extracellular part of the molecule as this is the region that is bound to the virus (not the intracellular).
Useful because allows for a soluble context whereas if the whole protein was used, would require a membrane context
Other parts of the protein are unlikely to be affecting virus binding.
What is a fusion protein?
A protein made from a fusion gene which is made by joining two different parts of genes
Why would you want to add a fusion protein/peptide to modify your protein of investigation?
Useful for purification
e.g. His tag
List three ways you might wish to modify your protein of interest (i.e. consider before the making stage)
- adding fusion proteins or peptides
- choosing which section of the protein to produce
- making mutations to remove post-translational modifications
Why might you choose to only make part of your protein of interest? (3 points)
- Remove parts of the protein predicted to be disordered, felxible, that might interfere with protein function
- only express e.g. a single domain that mediates all the binding interaction of a multidomain protein
(this would make the experiment easier because it is simplified) - consider if membrane anchors or transmembrane helices are required
Using a single domain from a mutlidomain protein might make the experiment easier because it’s simplified, but what assumption has been made and how would you prove this assumption is true?
Assumption that the singular domain contains the whole function of the protein and mediates all the binding interactions
Prove with control experiments
When investigating a protein, what might you want to know about the protein structure before making the protein for use in experiments?
List 6 questions.
Is there a structure already available (in the pdb)?
Is there a secretion signal?
Are there transmembrane regions or anchors?
Are there post-translational modifications?
Are there regions of disorder?
What is the domain architecture?
Presence of a secretion signal in a protein is useful to predict what?
Useful to predict the location in which the portein is found
What do different secretion signals correspond to?
Different target destinations
Where is the signal peptide in eukaryotes located?
At the N terminal
