Lecture 5: Protein Engineering Flashcards
Protein engineering applications
- Pharmaceuticals
- Agriculture
- Research
- Bioenergy
- White biotech
Rational design of proteins
- Based on protein knowledge
- Structure
- Mechanisms
- Dynamics
- Natural variation
- Analogous to mechanical engineering
Random mutagenesis or directed evolutionary processes
- Library of potential mutations n screen them
- Important to test n isolate for targeted properties
- Once mutant of interest is isolated, characterize what it can do
PEGylation advantages in protein engineering
- Flexible hydrophilic coat → solubility
- Reduced accessibility → protease resistance n non-antigenicity
- Increased size → serum half life
Fluorophores
Fluorescent labelling → tracking location or dynamics
Uricase
- Important in the treatment of gout
- Poor half life in the body
- Attach PEG to it to combat this issue
- Effectively reduce the antigenicity (recognition by our immune system) -> lives longer in the blood stream
- Poor half life in the body
- Take uricase n attach by surface AA to produce PEG-uricase which will degrade uric acid thus getting rid of gout
Rational design of protein by site directed mutagenesis
- Couple of DNA oligonucleotides which will bind to DNA target of interest
- Specify mutant residues required for mutation
- PCR experiment where production of target is increased
- Enzyme to degrade the original template → end up w only required mutant DNA to transform into cells
Structure of DapF
- Changes an L form to a D form
- Heart is a cystine residue
- Cystines evolved in a disulfide bond
- Active site cystine 73 is disulfide bonded to cystine 217 as a result of oxidation upon purification, preventing any enzymological study (dead enzyme)
Rational design of protein by creating fusion proteins
- Creation
- Remove stop codon of first genes
- Ligate genes together in frame
- Include linker codons
Aims of creating fusion proteins
- Combine the properties of the components
- Addition of antibody Fc fragment to proteins increases serum half life
- Co-localize the components
- Set of enzymes that work in a reaction pathway
Considerations for creating fusion proteins
- Linker length n flexibility
- Distance b/w protein components
- Protease resilience
- Ability for domains to fold
What is the functional relationship between PBP2 and RodA in bacterial cells?
- PBP2 and RodA are two membrane proteins in bacteria that work together as part of the biosynthesis of the bacterial cell wall.
- They form a complex, and it was predicted that the C terminus of PBP2 is located in the cytoplasm, likely adjacent to the N terminus of RodA.
How was the spatial relationship between the C terminus of PBP2 and the N terminus of RodA investigated?
- A fusion protein with a small peptide linkage between the C terminus of PBP2 and the N terminus of RodA was created.
- The expressed protein was then used to analyze the spatial arrangement, providing insights into their proximity and interaction.
What technique was employed to solve the cyro EM structure of the PBP2-RodA chimera, and what key features were revealed?
- The membrane scaffold nanodisc was used to solve the cyro EM structure of the PBP2-RodA chimera.
- The structure revealed single transmembrane helices of RodA and identified a red helix that activates RodA, representing the active enzyme in the complex.
Directed evolution in the context of proteins
Using techniques that mimic natural evolution to accelerate the generation of protein variants with potentially new properties of interest
How can we generate variation?
- Introduce a mutation once every 100bp -> creates a library
- End up w lots of point mutations in the library which you then screen
- Shuffle bits of different genes together
- PCR n genetic based strategy
- Selectively take out lumps of DNA to give you variation
Aim of random point mutations
Introduce a random number of mutations into a target gene w the intention of selecting for a new property of that protein (e.g. solubility of heat stability)
Random point mutation methodology
- Thermostable DNA polymerase during PCR, which introduces incorrect nucleotides under specific conditions.
- The number of mutations (usually 1-10 nucleotides/kbp) can be manipulated, generating a library of mutants that is then screened to identify variants with the desired property.
- This approach contrasts with the targeted and specific nature of site-directed mutagenesis (SDM)
How do you screen the mutant library?
- Depends on targeted property
- New functional enzyme/immunological/functional assay → high throughput assay
- Biophysical property: heat stability, pH stability
How can the solubility of a mutant protein be screened effectively, especially when looking for enhanced solubility?
- Screen for enhanced solubility is to fuse the mutant protein to another protein that is intrinsically insoluble.
- This fusion partner should be easy to identify, such as having a distinct color or fluorescence (e.g. GFP)
DNA shuffling
- Gene first cut into pieces using exonuclease then regenerated using a DNA polymerase
- Polymerase introduces mutations similar to error-prone PCR
- DNA pieces get mixed → mutations from separate copies can be combined
- RESULT: library of mutants subject to similar screening methodologies
How does DNA shuffling mimic natural evolution, and what distinguishes it from site-directed mutagenesis (SDM) and random mutagenesis (RM)?
- DNA shuffling mimics natural evolution by recombining genetic material, similar to the process observed in some organisms.
- Unlike artificial methods such as SDM and RM, DNA shuffling involves the creation of genetic diversity through recombination, reflecting a more natural approach to evolving protein
Provide an example of DNA shuffling application and its goal in protein evolution.
- DNA shuffling has been employed in evolving a class C cephalosporinase, a ß-lactam degrading enzyme, derived from four different bacterial species.
- The objective was to produce an enzyme capable of degrading a chemically related ß-lactam antibiotic, moxalactam, showcasing the versatility of DNA shuffling in evolving enzymes for specific functions.
Describe the two strategies adopted in the evolution of the cephalosporinase enzyme and their outcomes.
- Strategy I: individual genes from four different species underwent shuffling, introducing point mutations. The best-selected variant showed an 8-fold increase in resistance to the new drug.
- Strategy II: gene fragments from all four species were mixed in the DNA shuffling experiment, resulting in “new” proteins that exhibited between 270-540 fold more resistance to the new drug. The most effective enzyme in this strategy had eight discrete DNA segments from three of the four genes and 33 additional point mutations.
How was the sequence of the most active cephalosporinase mutant determined, and what structural information was obtained?
- Modeled using the crystal structure of the class C cephalosporinase from Enterobacter cloacae.
- The chimeric protein, with 63% identity to the wild-type, contained portions from three starting genes (Enterobacter, Klebsiella, and Citrobacter) and 33 point mutations.
- The swapped sections corresponded to segments of discrete secondary structure.
What was the objective of engineering lysozyme stability, and how did the researchers aim to achieve this?
Enhance the heat stability of lysozyme, a 164 amino acid enzyme from the T4 phage. The researchers sought to introduce greater structural integrity by adding disulfide bonds to the protein structure.
Describe the approach taken to introduce greater stability to lysozyme and the impact on its melting temperature.
- Introduced disulfide bonds by adding cystines between residues, linking different parts of the lysozyme structure
- Various pairs of cystines were introduced either individually or in combination
- RESULT: proteins showed different melting temperatures, with a moderate change for single disulfide introductions and a large change in stability when all three were introduced.
Why was the introduction of disulfide bonds important, and what was the practical implication mentioned in the context of washing powder enzymes?
- Provided greater structural integrity and heat stability to lysozyme.
- While the stability of lysozyme might not be highly significant, in the case of enzymes used in washing powder, stability is vital for effective performance.
What is Tm? What is lysozyme’s Tm?
- Temperature at which 50% of the enzyme is inactivated during irreversible heat denaturation
- 41.9℃
How does the number of unfolded conformations impact the stability of a protein?
The greater the number of unfolded conformations of a protein, the higher the entropic cost of folding into its single native state. To increase stability, the number of possible unfolded conformations can be reduced.
What strategy is commonly employed to increase protein stability?
- Introduction of disulfide bonds [constrains the number of folded conformations, ensuring certain parts of the structure are consistently associated unless reduced]
- The longer the loop between two cysteine residues, the more restricted the unfolded polypeptide chain becomes. This increased restriction enhances the stabilization of the folded structure.
Describe the experiment conducted by Brian Mathews in Oregon
- AIM: identify potential sites for disulfide bond introduction in the T4 enzyme structure.
- METHOD: analysis of the structures of various enzymes with naturally occurring disulfide bonds.
- 3 candidate disulfide bonds were created using one of the existing cystine 97
- Single n double disulfide mutants made so that the effect of specific bonds could be compared
- Introduced mutagenesis of 5 more
- RESULT: all mutants were stable in their oxidized forms compared to WT
- REASON: longer the loop b/w cystine residues of mutants w single disulfide bonds → larger effect on stability
If you introduce a disulfide into a protein, what are you doing to its flexibility?
Decrease flexibility
Describe the dipole associated with an α-helix and its role in ligand binding.
- An α-helix has a dipole with a positive charge at the N-terminus and a negative charge at the C-terminus.
- Dipole often involved in ligand binding, contributing to the stability of the binding interaction.
In α-helices not involved in ligand binding, how do side chain charges contribute to stability, and what specific interactions are observed?
In α-helices not involved in ligand binding, negatively charged side chains are often found close to the N-terminal end, and positively charged side chains near the C-terminal end. These residues interact with the helix dipole, contributing to the overall stability of the α-helix.
How were mutations in T4 lysozyme used to investigate the impact of side chain charges on α-helix stability, and what were the observed results?
- Mutations were made in T4 lysozyme to test the impact of the positive charge at the N-terminal end of selected helices.
- The mutations Ser 38 to Asp and Asn 144 to Asp each contributed about 2°C to the heat stability, indicating that altering side chain charges near the N-terminal end can influence the overall stability of α-helices.
Why does glycine have more conformational freedom compared to other amino acids?
- Lack of steric hindrance with its side chain, which is a hydrogen atom
- This absence of a bulky side chain allows for increased flexibility.
How does glycine behave within a folded protein versus in an unfolded state, and what role does its conformational flexibility play?
- Folded protein → specific conformation
- Unfolded state → it provides a much greater degree of flexibility.
- This flexibility allows the protein to explore various conformations, even “incorrectly” folded patterns, which might be essential during the folding process.
How can mutations involving proline and glycine affect protein stability?
- Theoretically, mutating glycine residues out n proline residues in should increase protein stability
- IN REALITY: can only be done in situations where mutations do not affect conformation of main chain in folded structure / cause the loss of favorable contacts w neighbouring side chains
