BIO 3 - Proteins

Question 1

What are examples of the functions that proteins can have in the cell?

Answer 1

• Selective transport of species (Proteins move specific molecules across cell membranes, regulating the cell’s internal environment)
  -conversion of chemical into mechanical energy

structural support, biochemical catalysts, hormones, enzymes, building blocks, and initiators of cellular death.

do not provide energy or storing genetic information

Question 2

How does proteins manage within very short timescales to fold into complex 3D shapes?

Answer 2

Proteins fold into complex 3D shapes quickly due to a funnel-like energy landscape that guides them to the correct structure with speed and precision.

Question 3

Why do proteins fold?

Answer 3

• Proteins fold due to numerous weak interactions.
• Favorable interactions are somewhat offset by entropy
  loss during folding.
• The hydrophobic effect, a significant factor in folding, primarily involves changes in entropy rather than energy.

Question 4

What are Intrinsically Disordered Proteins (IDPs) and how do they differ from folded proteins?

Answer 4

IDPs: associated with signaling and regulatory processes, have many different binding partners, and often fold upon binding.
Folded proteins: catalysis and transportOther proteins are folded as isolated molecules in solution.

Question 5

*What is the Levinthal paradox of protein folding and how is it resolved?

Answer 5

The Levinthal paradox questions how proteins fold quickly despite numerous possible shapes. It’s resolved by the concept of a funnel-shaped energy landscape guiding proteins to their native structure efficiently.

Question 6

*What are the favourable and unfavourable interactions and energetic contributions that determine protein folding?

Answer 6

Favorable interactions in protein folding include
- hydrogen bonds
- hydrophobic interactions
- van der Waals forces
-electrostatic interactions.
These stabilize the folded structure.

Unfavorable interactions involve:
- the loss of entropy when the protein folds, particularly due to the confinement of water molecules in the hydrophobic core.

Overall, favorable interactions dominate, driving the protein towards its native conformation.

Question 7

*What are intrinsically disordered proteins?

Answer 7

Intrinsically Disordered Proteins (IDPs) are proteins that lack a well-defined three-dimensional structure under native conditions. Unlike folded proteins, IDPs do not adopt a single stable conformation but instead exist as dynamic ensembles of interconverting conformations. These proteins are often associated with regulatory and signaling functions in biological systems and can interact with multiple binding partners.

I molekylærbiologi er et iboende forstyrret protein et protein, der mangler en fast eller ordnet tredimensionel struktur, typisk i fravær af dets makromolekylære interaktionspartnere, såsom andre proteiner eller RNA

Question 8

*In what contexts is the limited solubility of proteins a problem?

Answer 8

• can lead to disease due to loss of function and/or gain of toxic functions.
• in biotechnology and pharmacology. An important aim of protein design is to make proteins more stable and more soluble.

Question 9

*What are examples of protein misfolding/aggregation diseases?

Answer 9

Examples of protein misfolding/aggregation diseases include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis (ALS), and prion diseases like Creutzfeldt-Jakob disease.

Question 10

*What is liquid-liquid phase separation of proteins and what is its role in disease and biological function?

Answer 10

The spontaneous formation of two protein phases from a single solution is called liquid-liquid phase separation and this process is thought to lead to the formation of membrane-less organelles and perhaps also facilitate the formation of toxic protein aggregates in some cases.

Liquid-liquid phase separation is driven by weak interactions between proteins and other biomolecules, such as RNA. The parameter Χ (greek Chi) expresses the relative interaction strength between solvent and protein. A positive Χ means that protein-protein interactions are more favourable than protein solvent interactions, which is a pre-requisite for phase separation. Entropy of mixing always favours a solution and therefore the overall behavior is determined by the interplay between Χ and the entropy of mixing.
Many organelles in eukaryotes are not bounded by membranes (“membrane-less organelles”). They form and dissolve in a dynamic manner by phase separation and play important roles in cellular stress response and gene regulation.

Targeting protein phase separation is currently actively being pursued by a range of startups

So the role of LLPS in disease is that when it happen (that the two phases arrises) some disease can occur. for instance ALS

Question 11

*What is allostery?

Answer 11

Allostery is the regulation of a protein’s function by the binding of a molecule at a site separate from the active site, leading to a change in the protein’s activity.

Allostery is a direct and efficient means for regulation of biological macromolecule function, produced by the binding of a ligand at an allosteric site topographically distinct from the orthosteric site

Question 12

*What are enzymes and in what ways are they superior to non-biological catalysts?

Answer 12

Enzymes are biological catalytic proteins that accelerate chemical reactions within living organisms by lowering the activation energy required for the reaction to occur. They are superior to non-biological catalysts due to their specificity, efficiency, and regulation.

Key point: Enzymes catalyze reactions by binding the substrate and facilitating a specific reaction through e.g. weakening a given chemical bond. Enzymes often have metals in their catalytically active centers. The addition of metal centers increases the range of chemical reactivities that are accessible to proteins.

Question 13

*Be able to provide 2-3 examples of very important enzymes and their
roles in biology

Answer 13

Carbonic anhydrase: makes an already reasonably fast reaction even faster (up to 600000 reactions per second!). The ability to solubilize carbon dioxide makes this enzyme an attractive target for engineering to use it in carbon capture.

Nitrogenase:
Only enzyme able to convert elementary nitrogen into a water-soluble compound. Until the invention of the Haber-Bosch process, nitrogenase was the only significant source of biologically available nitrogen (lightning converts some nitrogen into soluble products as well), a limiting resource for the proliferation of life.

N2 is reduced to ammonia.

Cellulases:
enzymes that can act directly on solid cellulose and release sugar (glucose) units. This can be useful for biomass utilization.

PETases:
enzymes able to degrade man made PET plastics

Rubisco:
Enxyme convertig CO2 into energy rich molecules in plants. (photosynthesis)

Question 14

*How does an enzyme work?

Answer 14

Enzymes catalyze reactions by binding the substrate and facilitating a specific reaction through e.g. weakening a given chemical bond. Enzymes often have metals in their catalytically active centers. The addition of metal centers increases the range of chemical reactivities that are accessible to proteins.

Question 15

*Be able to give examples for biotechnological applications of enzymes.

Answer 15

application example for enzymes, the self-healing of concrete.

Carbon capture could be part of a global strategy to mitigate human induced climate change. Enzymes could play an important role in this field. This has been recognized by various funding bodies, such as the Novo Nordisk Foundation and research activities in this area are increased substantially.

Question 16

*What is directed evolution and how is it used to improve enzymes?

Answer 16

Directed evolution creates enzymes with new or improved properties through mutation and selection. While most mutations may not be beneficial, occasionally, variants with enhanced function emerge. However, destabilization can occur, necessitating strategies like stabilizing mutations. Overall, directed evolution is a powerful tool for tailoring enzymes for various applications.

uses various methods to gener ate a collection of random protein variants, called a library, at the DNA level.

Question 17

*What are antibodies and what are their roles in biology and their applications?

Answer 17

Antibodies are very large proteins that bind highly selectively and with high affinity to targets. They are able to interact with molecular signatures on the surfaces of pathogens.
They can target pathogens (such as a virus, bacterium) for destruction or directly inhibit their mechanism of pathogenicity.

Antibodies are protective proteins produced by your immune system. They attach to antigens (foreign substances) — such as bacteria, fungi, viruses and toxins — and remove them from your body.

ANTIBODIES: LARGE GLYCOPROTEINS (PROTEINS WITH SUGARS) THAT ARE ABLE TO INTERACT WITH MOLECULAR SIGNATURES ON THE SURFACE OF PATHOGENS, tHE BINDING BLOCKS SO THE PATHOGEN OR TOXIC AGENT ARE NEUTRALIZED,

APPLICATION: INDENTIFICATION OF PROTEINS, IN VIVO STAINING OF TISSUES AND CELLULAR STRUCTURES, DISEASE DIAGNOSTICS AND THERAPEUTICS (AGAINST NEURODEGENERATIVE DISEASES).

Question 18

Be able to perform simple stoichiometric calculations of antibody binding to target.

Question 19

*What are nanobodies and why are they better suited for some applications than classical antibodies?

Answer 19

single domain antibodies found only in camelids (Kameler og lamaer) and discovered originally in the context of a student’s lab practical.
- small size facilitates tissue penetration and renal excretion of free nanobodies
- high stability against proteases (enzymer som nedbryder proteiner ved at spalte proteinernes peptidbindinger.) and low pH could allow oral application

Question 20

*What is the key idea of protein display technologies, such as phage display?

Answer 20

In phage display, genotype and phenotype are linked through a phage virus. A phage is a virus that infects bacteria. The bacteria can be used to amplify (forstærke) the viruses. The DNA of the phage contains the DNA sequence of the antibody of interest and the surface of the phage virus contains the antibody protein itself.

Genteknologi:
Gen indsættes I bakteriofag => som viser proteinet på dens overflade (fænotype)=>
Leder til en klar sammenhæng mellem genotypen (genet der indsættes) og fænotypen (Proteinet der vises på overfladen)

Drug discovery, vaccine development

Phage display is a technique used in the study of protein interactions using bacteriophages, or viruses that infect bacteria. It works by inserting the gene encoding our protein of interest into a bacteriophage, causing it to express the protein on its surface, while simultaneously maintaining the gene inside of it. This results in a connection between the genotype and the phenotype of the protein of interest.

Here’s a simplified explanation of how a phage display protocol may be carried out:

1. A library of bacteriophages is created, by adding the genes coding for different proteins to each of the phages, causing them to express said proteins on top of them as a sort of protein hat.
2. This library of bacteriophages are exposed to selected targets. Only some bacteriophages will interact with the specific ligands on these targets, subsequently attaching themselves to them.
3. All unbound bacteriophages are washed away. Only bacteriophages expressing proteins, displaying affinity to the selected targets will remain.
4. The remaining bacteriophages are recovered through an elution step, such as for example affinity chromatography.
5. These recovered bacteriophages can then be used to infect new host cells for amplification.Finally, this 5-step cycle is repeated 2-3 times to increase the affinity between the expressed proteins and the selected targets.

Phage display is used in a wide range of applications, including drug discovery, vaccine development, and protein engineering. It is a powerful tool for identifying and isolating specific proteins or peptides from complex mixtures.

Question 21

*What are these protein display technologies mostly used for?

Answer 21

Drug discovery, vaccine development, protein engineering

Question 22

*Be able to perform simple calculations of amino acid sequence diversity of peptides.

Question 23

*Why is it difficult to determine the sequence of proteins, especially at low quantities?

Answer 23

Proteins are complex molecules made up of long chains of amino acids, and their sequences can vary widely. Determining the precise sequence requires advanced techniques that can accurately identify each amino acid in the chain.
Sample Heterogeneity: Biological samples often contain a mixture of different proteins, each with its own sequence. This heterogeneity can make it challenging to isolate and analyze specific proteins of interest, particularly when they are present in low quantities.

Sequence space of even small proteins is virtually infinite: 20100 for a 100 aa protein
Small changes in sequence (single point mutations) can lead to dramatic changes in
stability and solubility, which are difficult to predict accurately, even if the structure does not change
Only a small fraction of all possible sequences is likely to fold into functional structures

Question 24

*What is proteomics and what experimental method is it mostly based on?

Answer 24

Proteomics:
- suite of methods used to identify proteins in complex mixtures, based on comparison with data bases.

It is also possible to determine the sequence of new proteins that are not yet included in data bases, by building up the sequence from very short fragments.

liquid chromatography-mass spectrometry, forkortet LC-MS

Question 25

*What are the advantages and disadvantages of chemical synthesis and recombinant production of proteins?

Answer 25

Ribosomal Synthesis:

Advantages: Flexibility in protein complexity, high purity (error rate 1:10000), wide availability of natural amino acids.
Disadvantages: Limited incorporation of non-natural amino acids.
Chemical Synthesis:

Advantages: Incorporation of non-natural amino acids.
Disadvantages: Limited to peptides and small proteins, purity challenges.

Question 26

*How are proteins degraded? What are PROTACS?

Answer 26

At the end of a protein’s lifetime, it is tagged with (multiple copies of) a small protein, ubiquitin.

This ubiquitination is a signal that the protein should be degraded by a large molecular machine, the proteasome. The amino acids of the protein are recycled.
Once a protein is no longer needed, or when it is damaged, it is targeted for degradation and broken down into amino acids by the proteasome.

PROTACS are a relatively novel drug modality that targets problematic proteins for degradation by linking them up to the proteasome.

PROTACS are bi-functional small molecule drugs that bring a target protein together with the ubiquitination enzyme, such that the protein is targeted for degradation.

Question 27

*What is self-assembly and what is its role in biology? Be able to give some examples for structures that form through self-assembly.

Answer 27

Molecular self-assembly is the process by which molecules adopt a defined arrangement without guidance or management from an outside source. Intramolecular self-assembly is also called “folding”. Self-assembly relies on the presence of a multitude of weak, non-covalent, reversible interactions that constantly form and break and allow the system to find the thermodynamically most stable state. Examples include virus capsid assembly, protein filament formation, lipid bilayer formation, ribosome formation etc.

Some examples of the marvelous structures that can form through self-assembly: virus capsids, the ribsome, cellular filamentous networks. Biological systems are organized by molecular self-assembly from the bottom up, i.e. the structures to be formed are encoded in the chemical properties of the molecules.

Question 28

*Be able to give some examples for sustainable sourcing of food proteins.

Answer 28

Meat Cell Cultures: Animal cells cultured in lab for meat production. Sustainable and ethical alternative to traditional meat.

Recombinant Animal Protein from Microorganisms: Animal proteins produced by genetically engineered microorganisms. Sustainable and scalable protein source.

Fungal Proteins (Mycoproteins): Proteins from fungi like Quorn. Sustainable meat substitute with lower environmental impact.

Insect Protein: Protein from edible insects. Eco-friendly, nutritious alternative to traditional livestock

Question 29

*Be able to give some examples for functional materials generated from proteins.

Answer 29

Silk: Proteins from silkworms or spiders can be used to produce silk fibers with exceptional strength, elasticity, and biocompatibility. Silk proteins are also used in medical sutures, tissue engineering, and drug delivery systems.

Question 30

*Why is a living cells so much more complex than what the DNA alone can encode?

Answer 30

Key point: Digital information of DNA increases significantly in complexity once at the functional (protein level). The information content and information processing ability of a living cell cannot be understood simply by looking at the DNA. The rest of the cellular structures and components need to also be considered. This is the hardware on which the DNA software is executed.

How is the digital information stored in DNA converted into the analog complexity of protein function and ultimately all cellular processes?

chemical diversity of amino acids much higher than of nucleotides; chemical complexity implicitly encoded in amino acid sequences
sequence information in combination with the interactions that amino acids can undergo leads to the emerging complexity of proteins
“information” stored in DNA does not depend on concentration or solution conditions, but (protein) “function” does
not DNA is the starting point of a new organism, but DNA + a fully functional cell (egg)
an organism cannot be re-constructed from DNA alone (see exercise in BIO 2)
complexity arises from the myriad of different ways how the relative rates of gene translations and protein activity can be regulated by the cell (see BIO 2)