Protein Engineering

Question 1

Structure

Answer 1

1. Define
2. Structure-function relationships (SARS-CoV-2, PCOs)
3. Rational mutagenesis (RUBISCO, ERF VIIs, hypertrophic cardiomyopathy)
4. Deep mutational scanning (RUBISCO)
5. Directed evolution (carbonic anhydrase)
6. Synthetic Biology
7. De novo protein design

Question 2

engineering

Answer 2

• teleological
• specific
• deliberate
• optimised

Question 3

protein engineering

Answer 3

modifying a protein sequence for better suitability

Question 4

evolution

Answer 4

molecular tinkering

Question 5

KM

Answer 5

ES stability

Question 6

KCAT

Answer 6

rate of S->P conversion

Question 7

KCAT/KM

Answer 7

• catalytic efficiency
• highly biologically relevant

Question 8

VMAX

Answer 8

maximum KCAT

Question 9

How do you use protein engineering to understand structure-function relationships

Answer 9

1) insert gene of interest into plasmid cloning vector
2) insert primer w/ desired mutated sequence; strand separation and synthesis
3) digest DNA
4) transform bacteria
5) generate daughter cells (contain mutant DNA)
7) centrifuge soluble protein
8) purify
9) Western blot (SDS-Page)
10) functional assay

Question 10

SARS CoV-2 3CL protease clinical variant P108S

Answer 10

• decreased clinical severity in Japanese cohort
• kinetic and biochemical assays showed weakened substrate binding (Kmx2)
• engineering recombinant protein allows correlation of different structural properties with clinical outcome

Question 11

proline changes

Answer 11

significant for both primary and secondary structure

Question 12

3CL protease

Answer 12

• necessary for viral replication
• therapeutic target

Question 13

PCOs

Answer 13

• H164D: non-functional (decreased iron interaction)

Question 14

rational mutagenesis

Answer 14

using detailed protein structural and functional knowledge to make educated engineering decisions

Question 15

RUBISCO

Answer 15

• 0.7Gt
• multi-domain recombinant productjon
• requires chaperones
• diverse amoungst photosynthetic organisms

Question 16

describe the multi-domain recombinant production of RUBISCO

Answer 16

different subunits nuclear (RbcS) and chloroplastic (RbcL)

Question 17

Describe the photosynthetic hierarchy

Answer 17

red algae > plants > green algae > cyanobacteria

Question 18

“red lineage RUBISCO”

Answer 18

• unknown chaperones
• R. sphaeroides: transformable
• G. monolis: high efficiency
• chimaeric recombinant protein has high selectivity and KCAT

Question 19

Rhodobacter sphaeroides

Answer 19

proteobacterium

Question 20

Griffithsia monolis

Answer 20

• resolved crystal structure
• loop 6 encloses AS
• highly conserved
• A331, V334

Question 21

Rational mutagenesis of RUBISCO in vivo

Answer 21

• proof-of-principle
• in tobacco
• increased photosynthetic rate (micromole /s/m2)
• increased plant height (cm)
• decreased growth (due to inefficient expression)
• apply to endogenous RUBISCOs

Question 22

4pco

Answer 22

• increased anaerobic gene expression
• not ideal due to multifunctional PCOs

Question 23

4pco + modified PCO4

Answer 23

• increased anaerobic gene expression
• mixed fertility

Question 24

rational mutagenesis to combat inherited disease

Answer 24

• e.g. hypertrophic cardiomyopathy
• R403Q in beta-myosin heavy chain (an important ESP)
• correct using ABE + gRNA + cardiac promotor
• decreased hypertrophy phenotype
• increased ejection fraction

Question 25

hypertrophic cardiomyopathy

Answer 25

- most common cause of sudden cardiac death in adolescents and young adults - commonly found inherited mutations in heart sarcomere genes

Question 26

ABE

Answer 26

- adenine base editor - uses AAV vector

Question 27

AAV

Answer 27

adeno-associated virus

Question 28

Next steps for rational mutagenesis to target hypertrophic cardiomyopathy

Answer 28

- animal models and clinical trials

Question 29

"ABE might be a

Answer 29

one-time therapy to permanently correct HCM-causing variants, and prevent disease onset"

Question 30

Describe base editing in CRISPR

Answer 30

1) Cas makes a nick 2) base editing enzymes

Question 31

base editing enzymes

Answer 31

cytidine/adenosine deaminase

Question 32

Describe prime editing in CRISPR

Answer 32

- Cas9 makes a nick - prime editing gRNA + primer BD conjugated to RT; binding - Fen1: flap repair

Question 33

General challenges for rational mutagenesis to combat inherited disease

Answer 33

- correlating in vitro with in vivo - missing technologies for rapid screening - off-target effects - safe, effective delivery methods - ethical concerns

Question 34

Which inherited diseases are currently being tackled using rational mutagenesis?

Answer 34

- Duchenne muscular dystrophy - cystic fibrosis - sickle cell anaemia

Question 35

Deep mutational scanning

Answer 35

- aka: site-saturation mutagenesis - replacing every aa in a protein w/ every alternative - easiest in prokaryotes - identifies "unpredictable" desirable variants for further testing

Question 36

Deep mutational scanning of RUBISCO

Answer 36

- mutated all R. rubrum residues - Delta-rpi E. coli model: screen function - change CO2 availability - screen for higher affinity - reveals hotspots for future engineering

Question 37

Directed evolution

Answer 37

- using strategies to randomly introduce mutations into exonic sequences, functionally screening outcomes, then iterating for optimisation - aka "automated mutagenesis" - easiest in prokaryotes

Question 38

directed evolution + biocatalysis

Answer 38

1) random mutations introduce in gene of interest via error-prone PCR 2) genes inserted into bacteria 3) enzymes tested and efficiency selected for

Question 39

directed evolution can be used for

Answer 39

- biocatalysts - designer Abs

Question 40

designer Abs

Answer 40

- requires Ab library - rheumatoid arthritis

Question 41

directed evolution and Ab selection

Answer 41

1) genetic info for Ab binding site instead into phage dna 2) selection for Abs w/ strong attachments 3) random mutations introduced to bound Abs 4) increased affinity and specificity

Question 42

phages

Answer 42

- efficient gene delivery and self amplification - high mutation rate

Question 43

Coupling biocatalysts and ab selection

Answer 43

- combining desirable function in the BOI with successful phenotype allows continuous evolution; fast!

Question 44

In the lagoon

Answer 44

- constant inflow of fresh bacterial cells w/ mutagenesis + accessory plasmids - phage infection results in mutagenesis and gene expression - if BOI is functional, it interacts with RNAP; allows next cycle of infection alongside increase in functional properties - if BOI is non-functional, it leaves the lagoon

Question 45

Directed evolution and carbonic anhydrase

Answer 45

- carbon capture and sequestration - optimise temperature sensitivity - 9 rounds formed a temperature sensitive + alkaline-tolerant variant - allows solvent reuse

Question 46

Directed evolution and PETase

Answer 46

- chemotrypsin-like - bacteria evolved on okastuc recycling facility - PET is C source

Question 47

HotPETase

Answer 47

hydrolyses semi-crystalline PET

Question 48

Describe the directed evolution of HotPETase

Answer 48

i) array enzyme libraries in 96 deep-well plates ii) carry out reactions iii) incubate PET disc w/ enzyme to release soluble products iv) quench and precipitate v) quantify soluble degradation product (MHET, TPA by UPLC)

Question 49

OrthoRep

Answer 49

- use yeast to develop bacterial O2-tolerant THI4, for use in plants

Question 50

THI4

Answer 50

- enzyme - forms thiamine precursor - most efficient in anaerobic bacteria

Question 51

How did OrthoRep work?

Answer 51

- cells selected for in thiamine-free media - THI4-V12A is able to grow

Question 52

Synthetic biology - generally

Answer 52

- design and construction of new biological parts/ modifying existing biological systems - protein engineering is a subset of this

Question 53

Synthetic biology - here

Answer 53

- adding unnatural amino acids to existing proteins - can be incorporated in vivo using amber stop codons/ engineered tRNAs

Question 54

de novo protein design

Answer 54

- using in silico strategies to generate a protein sequence that is complementary to a known POI - validate in vitro and in vivo

Question 55

RFdiffusion

Answer 55

- generates sequence for protein binding - produces recombinant protein w/ high affinity interactions - actual structures recapitulate predicted

Question 56

RFdiffusion + AlphaFold2

Answer 56

- generate and filter sequence for anti-venoms

Question 57

Antivenoms in vitro

Answer 57

- strong interaction - structure as expected

Question 58

Antivenoms in vivo

Answer 58

- cells and mice - protective effect

Question 59

AlphaFold 1

Answer 59

- "fed" physical + evolutionary info - low resolution - worse than experimental data

Question 60

AlphaFold 2

Answer 60

- "learns" physical and evolutionary info - high resolution - as good as experimental data

Question 61

AlphaFold Multimer

Answer 61

- uses a "linker" technique to predict protein interactions - also RosETTA fold

Question 62

AlphaFold

Answer 62

- combines simulation of physical interactions with evolutionary modelling - pairs of interacting molecules are conserved if important

Question 63

AlphaFold 3

Answer 63

- models individual atoms (not aas) - can predict protein complicies - not so good for proteins wit unknown evolutionary relatives

Question 64

AI-supported protein engineering

Answer 64

- AlphaFold Multimer - ePip1 - in vitro: insensitive - in vivo (N. benthamiana): increased resistance

Question 65

Pip1

Answer 65

- ETI - apoplastic immune protease inhibited by P. infestans EpiC2b