Session 4.1g - Pre-Reading [Article] Flashcards
https://www.sciencemag.org/news/2016/07/protein-designer-aims-revolutionize-medicines-and-materials?utm_source=general_public&utm_medium=youtube&utm_campaign=vid-protein-fold-5886
What shapes can 3D printed proteins make?
- Rings
- Balls
- Tubes
- Cages
etc.
What has David Baker and his team been doing?
Figuring out how long strings of amino acids fold up into the 3D proteins that form the working machinery of life. Now, he and colleagues have taken this ability and turned it around to design and then synthesize unnatural proteins intended to act as everything from medicines to materials.
What applications has virtuoso proteinmaking yielded so far?
- An experimental HIV vaccine
- Novel proteins that aim to combat all strains of the influenza viruses simultaneously
- Carrier molecules that can ferry reprogrammed DNA into cells
- New enzymes that help microbes suck carbon dioxide out of the atmosphere and convert it into useful chemicals.
Cages of proteins can be made from as many as how many proteins?
120
How can proteinmaking be used to combat HIV?
To create an experimental HIV vaccine
How can proteinmaking be used to combat influenza?
To create novel proteins that aim to combat all strains of the influenza viruses simultaneously
How can proteinmaking be used to involve DNA?
To create carrier molecules that can ferry reprogrammed DNA into cells
How can proteinmaking be used for enzymes?
To create new enzymes that help microbes suck carbon dioxide out of the atmosphere and convert it into useful chemicals.
How can proteinmaking be used for carbon dioxide?
To create new enzymes that help microbes suck carbon dioxide out of the atmosphere and convert it into useful chemicals.
Cages can be assembled from as many 120 designer proteins.
What could this do?
This could open the door to a new generation of molecular machines.
What is the newfound ability of designing novel proteins akin to?
If the ability to read and write DNA spawned the revolution of molecular biology, the ability to design novel proteins could transform just about everything else
What implications could the design of novel proteins lead to?
Nobody knows the implications because it has the potential to impact dozens of different disciplines
How far back do efforts to predict how proteins fold and using that information to fashion novel versions date back?
Decades
When did biochemists at the U.S. National Institutes of Health (NIH) recognise that each protein folds itself into an intrinsic shape?
In the early 1960s
How can you lose a protein’s 3D structure?
Heat a protein in a solution and its 3D structure will generally unravel
What happens when you heat a protein in solution?
Its 3D structure will generally unravel
What happens after proteins are heated and structures unravelled?
The proteins refold themselves as soon as they cool.
Proteins can be heated and their 3D structure will generally unravel. However, as soon as they cool, the proteins refold themselves. What does this imply about their structure?
It implies that their structure stems from the interactions between different amino acids, rather than from some independent molecular folding machine inside cells
How do we know protein structure is dependent on interactions between amino acids rather than from independent molecular folding machinery inside the cell?
Heat a protein in a solution and its 3D structure will generally unravel. However, these proteins will refold themselves as soon as they cool
What is needed to be able to understand how an amino acid sequence would assume its final shape?
If researchers could determine the strength of all those interactions [between amino acids], they might be able to calculate how any amino acid sequence would assume its final shape
What is the protein folding problem?
Understanding how amino acid interactions lead to the final protein shape.
What is essential for life from DNA to proteins?
The machinery for building proteins is essential for all life on earth
What does building proteins begin with?
Building proteins begins with DNA’s genetic code.
What is the relationship between DNA, genes and amino acids?
In protein-coding regions of genes, each amino acid is encoded by three rungs of the DNA ladder.
How many amino acids make up the building blocks of proteins?
Twenty such amino acids make up the building blocks of proteins.
What is the first step in DNA becoming a protein?
Double stranded DNA is transcribed into single-stranded RNA, which is then sent to the ribosome where proteins are manufactured.
How does RNA become an amino acid?
A molecular machine called the ribosome translates each RNA coding sequence into an amino acid, building up a growing protein chain.
How are amino acids formed into a protein?
Forces between amino acids cause a linear chain to fold up on itself, creating a functional 3D protein.
How can proteins structures be configured experimentally?
- X-ray crystallography
- Nuclear magnetic resonance (NMR) spectroscopy
What is a disadvantage of using x-ray crystallography/NMR to determine protein structures?
It’s slow and expensive
How many proteins does the Protein Data Bank hold for known structures?
Only roughly 110,000 proteins - out of the hundreds of millions or more thought to exist
Why would it be beneficial to know a proteins’ shape?
Knowing the 3D structures of those other proteins would offer biochemists vital insights into each molecule’s function, such as whether it serves to ferry ions across a cell membrane or catalyze a chemical reaction
It would also give chemists valuable clues to designing new medicines
What is the next step after X-ray crystallography/NMR spec to studying protein shape?
So, instead of waiting for the experimentalists, computer modelers such as Baker have tackled the folding problem with computer models.
What are the two broad kinds of folding models?
- Homology models
- Ab initio modeling
How do homology models works?
These compare the amino acid sequence of a target protein with that of a template - a protein with a similar sequence and a known 3D structure
The models adjust their prediction for the target’s shape based on the differences between its amino acid sequence and that of the template
What is a disadvantage of the homology model?
But there’s a major drawback: There simply aren’t enough proteins with known structures to provide templates - despite costly efforts to perform industrial-scale x-ray crystallography and NMR spectroscopy.
How have they tried to combat
Despite costly efforts to perform industrial-scale x-ray crystallography and NMR spectroscopy there simply aren’t enough proteins with known structures to provide templates
Explain how x-ray crystallography can be used to provide protein templates but also explain the drawbacks of using this.
A scarcity of structure - X-ray crystallography and other techniques have determined the structures of only a fraction of proteins, though these can be used to model others.
Proteins in nature
30x10 grid
1 - Proteins in data bank
299 - Easily solved by comparative modeling
X-ray crystallography - 100,528
NMR - 10,054
Others - 1,036
What is ab initio modelling?
This calculates the push and pull between neighbouring amino acids to predict a structure.
What is Rosetta (ab initio modelling)?
Early on, Baker and Kim Simons, one of his first students, created an ab initio folding program called Rosetta, which broke new ground by scanning a target protein for short amino acid stretches that typically fold in known patterns and using that information to help pin down the molecule’s overall 3D configuration.
After Rosetta, what did Baker and the team produce?
Seeking more computing power, they created a crowdsourcing extension called Rosetta@home, which allows people to contribute idle computer time to crunching the calculations needed to survey all the likely protein folds. Later, they added a video game extension called Foldit, allowing remote users to apply their instinctive protein-folding insights to guide Rosetta’s search. The approach has spawned an international community of more than 1 million users and nearly two dozen related software packages that do everything from designing novel proteins to predicting the way proteins interact with DNA.
What is the drawback of Rosetta?
The software was often accurate at predicting structures for small proteins, fewer than 100 amino acids in length. Yet, like other ab initio programs, it struggled with larger proteins.
After computational modelling, what was proprosed?
- Technique first proposed in the 1990s - those were the early days of whole genome sequencing (beginning to decipher the entire DNA sequences of microbes and other organisms).
- Wondered whether gene sequences could help identify pairs of amino acids that, although distant from each other on the unfolded proteins, have to wind up next to each other after the protein folds into its 3D structure.
How can clues from genome sequences be used to predict protein structure?
Comparing the DNA of similar proteins from different organisms shows that certain pairs of amino acids evolve in tandem—when one changes, so does the other. This suggests they are neighbours in the folded protein, a clue for predicting structure.
Draw and label this diagram on amino acid sequences.
Amino acid sequences
- Conserved position
- Coevolved positions
- Variable position
Folded protein
- Amino acid pair is essential to function
- Amino acids change in sync, revealing tight link.
How can co-evolution of amino acids be used to work out protein function?
- Reasoned that the juxtaposition of those amino acids must be crucial to a protein’s function
- If a mutation occurs, changing one of the amino acids so that it no longer interacts with its partner, the protein might no longer work, and the organism could suffer or die.
- But if both neighboring amino acids are mutated at the same time, they might continue to interact, and the protein might work as well or even better.
How can co-evolution of protein sequences be investigated from different species?
- Certain pairs of amino acids necessary to a protein’s structure would likely evolve together.
- Researchers would be able to read out that history by comparing the DNA sequences of genes from closely related proteins in different organisms.
- If DNA revealed pairs of amino acids that appeared to evolve together, it would suggest that they were close neighbours in the folded protein.
How can co-evolution be used in ab initio modelling?
Amino acids that co-evolved together in different proteins suggests that they have an interaction in a protein together (thus, a mutation in one would mean that the other died, and the protein was no longer viable for life).
Put enough of those constraints on amino acid positions into an ab initio computer model, and the program might be able to work out a protein’s full 3D structure.
When was DNA sequencing and protein folding readily available?
1990s: not enough high-quality DNA sequence data
2000s: DNA sequences were flooding in due to gene-sequencing technology. Co-evolving amino acids from similar proteins could now be tracked.
How many protein families are there
Estimated 8000
What can protein folding be fast-tracked to solve?
“We were limited by what existed in nature. … We can now short-cut evolution and design proteins to solve modern-day problems.” - Baker
Why is it hard to create a vaccine for the influenza flu virus?
Flu viruses come in many strains that mutate rapidly, which makes it difficult to find molecules that can knock them all out
What do all strains of influenza virus contain?
Every strain contains a protein called hemagglutinin and a portion of the molecule known as the stem that remains similar across many strains
What does hemagglutinin do?
It is a protein on all strains of influenza flu viruses that helps it invade host cells
What is the stem on the influenza virus?
A portion of the molecule that remains similar across many strains.
How can developing a novel protein be useful in combating the influenza virus?
Flu viruses come in many strains that mutate rapidly, which makes it difficult to find molecules that can knock them all out. But every strain contains a protein called hemagglutinin that helps it invade host cells, and a portion of the molecule, known as the stem, remains similar across many strains.
Developing a novel protein that would bind to the hemagglutinin stem and thereby prevent the virus from invading cells could combat the virus.
How are novel proteins created?
- Design the protein
- Engineer microbes to make it
- Test in the lab
- Rework the structure if necessary, can take many rounds to complete e.g. 80 rounds
Can novel proteins be used to combat the influenza virus?
Yes - 4 February issue of PLOS ONE, the researchers reported that when they administered their final creation to mice and then injected them with a normally lethal dose of flu virus, the rodents were protected
How effective are novel proteins used to combat influenza in comparison to Tamiflu?
It’s more effective than 10 times the dose of Tamiflu
What is Tamiflu?
An antiviral drug currently on the market for the influenza virus
What is the benefit of using a novel designer protein as an antiviral drug?
It can be used universally as it can bind to a specific part of the virus that remains common in all strains.
Give 2 examples in medicine designer proteins can be used for?
- Creating a novel protein for the influenza virus
- Chopping up gluten for people with gluten sensitivity (or Coeliac disease)
Where is gluten found?
Wheat and other grains
What is found in wheat and other grains that commonly causes intolerance?
Gluten
What conditions cause people to have trouble digesting glucose?
- Coeliac disease
- Gluten sensitivity
What is Coeliac disease?
An inability to digest gluten
Other than breaking down the gluten via designer proteins, how else could they be used to treat Coeliac disease?
Self-assembling cages could be filled with drugs or therapeutic snippets of DNA or RNA that can be delivered to disease sites throughout the body.
How can designer proteins be used for research?
Baker et al. have also attached up to 120 copies of a molecule called green fluorescent protein to the new cages, creating nano-lanterns that could aid research by lighting up as they move through tissues.
Designer proteins can be used to edit genes. What applications could this hold?
- Bind to hormones
- Bind to drugs e.g. for heart disease
Designer proteins have been created to bind to which DNA-cutting enzyme?
Cas9
What is Cas9?
A DNA-cutting enzyme that is part of the popular CRISPR genome-editing system
What is CRISPR?
A family of DNA sequences that can be used to cut DNA.
Give an example of a CRISPR enzyme
Cas9
Novel proteins have been designed with the idea of binding to Cas9, the DNA-cutting enzyme. What could this allow?
The strategy could allow researchers or physicians to target the powerful gene-editing system to a specific set of cells—those that are responding to a hormone or drug
What could biosensors do as a clinical application for genetics?
Biosensors could also make it possible to switch on the expression of specific genes as needed to break down toxins or alert the immune cells to invaders or cancer
Biosensors could also make it possible to switch on the expression of specific genes for ___?
Give 2 examples
- break down toxins
- alert the immune cells to invaders/cancer
Why is it important to predict how an amino acid sequence will fold?
The ability to predict how an amino acid sequence will fold - and hence how the protein will function - opens the way to designing novel proteins that can catalyze specific chemical reactions or act as medicines or materials
How can the genes used to create new proteins be utilised?
Genes for these proteins can be synthesized and inserted into microbes, which build the proteins
What do the genes need to be inserted into to create new proteins?
Microbes
What can 2D arrays be used for?
2D arrays can be used as nanomaterials in various applications.
What can the information do to protein sequences?
Information can be coded into protein sequences, like DNA
How do antagonists work?
Antagonists bind to a target protein, blocking its activation.
What do channels in membranes do?
Channels through membranes act as gateways.
What is the use of creating a designer cage?
Cages can contain medicinal cargo or carry it on their surfaces.
What do biological sensors do?
Sensors travel throughout the body to detect various signals.
How have designer proteins been thought to utilise carbon dioxide?
Last year, his group and collaborators reported engineering into bacteria a completely new metabolic pathway, complete with a designer protein that enabled the microbes to convert atmospheric carbon dioxide into fuels and chemicals.
How can designer proteins be used for electronics?
Two years ago, they unveiled in Science proteins that spontaneously arrange themselves in a flat layer, like interlocking tiles on a bathroom floor. Such surfaces may lead to novel types of solar cells and electronic devices.
Although this is work in progress, what project is currently being tasked, involving designer proteins and DNA?
Baker’s team has designed proteins to carry information, imitating the way DNA’s four nucleic acid letters bind and entwine in the genetic molecule’s famed double helix. For now, these protein helixes can’t convey genetic information that cells can read. But they symbolize something profound: Protein designers have shed nature’s constraints and are now only limited by their imagination.
