Notes 20101103 CHEM 731 Designing Stability, Coupling, Salt Bridges
From SnOwy - Ed's Wiki Notebook
Coupling
- the change in one amino acid's location and contribution to stability is linked to other amino acids
- able to detect these with point substitutions studies
- with two amino acids, we are able to visualize it in a square free energy diagram
- with three amino acids, we use a cube
- eventually, we want to create a hypercube with |amino acid| dimensions
Designing Stability
Title: An artificial di-iron oxo-protein with phenol oxidase activity
- engineered protein that allows binding of iron near phenol group
- allows new target for catalytic activity
- criticism that iron has a baseline activity without the enzyme but baseline was not reported
- model of substrate interaction
- iron was coordinated with histadine
- significance: engineered the coordination of zinc? and iron in a protein -- results in a simple enzyme
- proof of concept, activity was lower than naturally occurring enzyme
- the phenol groups are easily oxidizable groups -- good target for proof of concept
- additional alcohols were also tested, but phenols are larger -- show greater magnitude of activity
- group has since created more metal coordinating proteins
Title: Engineering Novel Metalloproteins: Design of Metal-Binding Sites into Native Protein Scaffolds
- a large review-like article which discusses many different modifications
- heme binds easily to a number of protein structures
- modification of membrane channel protein to change specificity of ion allowed to pass
- sometimes the changes are as simple as changing the amino acids to histidines to coordinate calciums through a channel
- zinc binding motifs -- made out of cysteine -- may be a problem as iron can also bind there
- most of these changes were done by hand so far
- sites that bind with metals don't necessarily have to be varied
Catalytic Antibodies
Title: An aspartate and a water molecule mediate efficient acid-base catalysis in a tailored antibody pocket
- recall: we want the enzyme to bind best to the transition state of a reaction -- not the reactants nor products
- we can't actually make a transition state -- these things are transient!
- the challenge is to use something that looks reasonably like such a state and have an antibody bind to it
- background: all proteins that can bind a transition state will affect the kinetics
- does not necessarily create a good enzyme
- often, with these catalytic antibodies -- the model transition state is too close to either reactant or product
- turnover affected
- to begin, they started with a library of antibodies and sought those antibodies that looked to bind with the transition state
- point substitutions iteratively refine the catalysis
- some mutations -- substitution of alanine for glutamate -- reduce the number of waters allowed in a binding pocket
- the catalytic antibodies in this paper tend not to demonstrate induced fit
- take home: possible to create catalytic antibodies by designing binding to transition state analogue
Title: Kemp elimination catalysts by computational enzyme design
- ROSETTA assisted protein scaffold selection
- minimize energy
- redesign active site -- increase its stability, increase its binding with the transition state
- shape complementarity to the substrate
- 25% of the enzymes screened were TIM barrels to start -- refinement with ROSETTA leads to >70% TIM barrels selected
- two competing final designs both based on amino acid substitution
- could not change the catalytic residues -- can change neighbouring residues -- "second sphere effect"
Screening for Enzymes
Title: Protein building blocks preserved by recombination
- a logical continuation of the recombined library idea
- recall: recombination library with DNA cutting enzyme and random religation
- this work describes finding the determinants of allowed intradomain pieces to be swapped
- SCHEMA used to calculate which amino acid substitutions can be tolerated
- disruption of enzyme with many point mutations results as a toleration-death threshold
- authors propose that the tolerated, reduced activity enzymes should be kept in a library
- these have many many new mutations -- with this diversity, possible to screen for new functions
- the two proteins that were recombined were βlactamases
Cysteins
Title: SCHEMA Recombination of a Fungal Cellulase Uncovers a Single Mutation that Contributes Markedly to Stability
- now looking at stability
- SCHEMA proteins can be used to develop linear models
- oddly, the intradomain chunks of protein increase stability linearly;
- that is, the total stability is simply the sum of those of the chunks!
- synthesized 31 chimeras -- those that were predicted to be stable were
- of those, eight had activity assays -- cellulose assays used -- able to hydrolyze equal or better than parent enzymes
- single block of sequence contributes almost all of the stability
- when the cysteine is mutated to serine in this particular stretch, increased stability
- also increased secretion of enzyme by ten fold
- measured temperature of inactivation
- note: left the catalytic chunk intact
- some of the chimeras were expressed poorly or were just secreted infrequently -- these had to be modified before assay
- demonstrated cysteine was responsible for destabilizing --
- two parent strains had cysteine, one had serine
- reversing the cys or ser showed the predicted increased or decreased stability
- they conjecture that serine is better at making hydrogen bonds
- in the crystal structure, we see that the h-bond actually connects to a backbone carbonyl ...
- note: we often think that cysteine is bad for stability due to oxidizing activity
- Dr. Meiering suggests that we should check alanine activity
- and that we need to know if oxidation is actually happening -- interesting!
- note: cysteine for this family of proteins is often mutated in nature
Title: Directed evolution of proteins for heterologous expression
- screening for a apo-protein -- ensure that the protein has the correct bound ion
- same with co-proteins
- need to ensure that we have the correct signal peptides for localization
- proteins may fold together and aggregate
- use a weaker promoter
- fusion tag to increase folding perhaps
- need to directly screen for function we want to get desired property
- protein may not fold well but will interact with GFP!
Title: Multi-step microfluidic droplet processing: kinetic analysis of an in vitro translated enzyme
- micro fluid channels have
- a physical bend
- a voltage difference
- the proteins accumulate one droplet at a time and are fused
- can be used to feed single yeast cells a specific droplet of reactant
