Notes 20101027 CHEM 731 Student Presentations

From SnOwy - Ed's Wiki Notebook

Contents 1 ZH Al Hussain 1.1 Caseins 1.2 Objectives 2 A Baeissa 2.1 ROP 2.2 Cysteine-free ROP 2.3 Experiment 3 S Martens 3.1 improving predicting protein/peptide 3.2 Alanine 3.3 Conclusions 4 RA Broom 4.1 Absolute and relative binding free energies 4.2 Forces 4.3 Dynamics, Energy 4.4 Free energy perturbation 5 M Pyne 5.1 FeFe-hydrogenase libraries 5.2 Gene Shuffling 5.3 Hydrogenase 5.4 Approach 5.5 Results 6 H Primmer 6.1 Bifunctional fusion proteins 6.2 Goals 7 H Stubbs 7.1 Amyloid fibrils 7.2 Approach 7.3 Conclusion

ZH Al Hussain

Caseins

• a major protein found in milk
• two components αs1-casein and β-caseins
• β-caseins (β-CN) -- most abundant
• 209aa
• flexible, amphiphilic protein
• micellization behaviour
• may sit on the air/water interface (I suppose hydrophobic tails point into air)

Objectives

• study behaviour at air-water interface with scanning cysteine substitutions
• mutagenesis with PCR
  • interesting -- the diagram indicates that there are 208aa
• characterization of cysteinylated β-cn -- micellization -- dynamic light scattering (DLS)
• demonstrated a sigmoid curve
• looks like a plateau-slope-plateau shape -- hydrodynamic diameter (nm) on temperature (°C)
• found recombinant wild-type and mutated β-cn have different diameter --
• cystein at either terminus (or both) -- important for properties of β-cn

A Baeissa

ROP

• repressor of primer -- regulates plasmid copy number in E. coli

Cysteine-free ROP

• four-helix bundle core mutant
• homodimeric four-helix bundle
• two identical chains of 63 aa
• monomers arranged in anti-parallel
• a monomer is two helices
• reduced cys residues in core cys38, cys53 -- prone to oxidation -- destabilized mutants
• these two residues appear to be the most conserved

Experiment

• remove cys residues in protein
• why? experimentally tractable
• packing and intereactions for cys38 and cys52 are important ...
• two conformations discussed, well-packed/less-packed
• cys-free ROP variant
• -- created Ser/Ser, Ala/Ala, Val/Val and Ala/Val variants (@38, 52)
• A/A, A/V ROP both stable (green fluorescent protein used)
• S/S, V/V ROP not correct
• wildtype (wt) was a control

S Martens

improving predicting protein/peptide

• computational
• CHARMM
• AMBER
• force fields
• quantum mechanical
• protein and peptide modelling -- still difficult, developing reference computations
• assessment of theoretical methods
• UV spectroscopy -- determine number of hydrogen bonds
• computational chemistry -- determine reasonable conformations with energetics
• experimentally derived spectra used as positive control
• gas phase folding of alanine

Alanine

• phenyl ring
• flexible chain
• leads to dispersive effects -- conformational diversity
• labelled two conformers A, B.
• formation of β hairpin h-bond

Conclusions

• these tests are new, efficient -- quantum chem methods
• should assist prediction of other peptides (isolated conditions)
• some conformers considered energetically favourable, but not as often seen experimentally
• done at 0K enthalpies -- may be correctly ordered if heat was more likely

RA Broom

Absolute and relative binding free energies

1. molecular dynamics
2. free energy changes
3. free energy perturbations

Forces

• calculate the force for each atom for all other atoms in the system
• know force, know acceleration -- in an integral with respect to time, know velocity
• able to artificially fold short peptides

Dynamics, Energy

• able to calculate energy
• free energy changes -- able to go from unfolded to transition to folded
• ΔG free energy change
• if there are two final states, we describe the difference between those as ΔΔG

Free energy perturbation

• these simulations occur in a statistical ensemble
• unable to get ΔG from just looking at free energy of unfolded and free energy of folded state
• this is an enthalpy term
• measuring ligand binding
• we apply the force fields for a particular ligand, and scale it from 1.0 to 0.0
• we simultaneously apply the forces for water and scale it from 0.0 to 1.0
• this simulates unbinding a ligand

M Pyne

FeFe-hydrogenase libraries

• gene-shuffling to create novel FeFe-hydrogenase libraries

1. directed evolution, gene shuffling
2. hydrogenase

Gene Shuffling

• in vitro -- mimics natural selection
• rounds of diversification, amplification, selection
• diverse library of proteins
• error prone PCR used
• improve enzyme activity, stability, selectivity, sensitivity

Hydrogenase

• metabolic enzymes
• reduction of protons
• hydrogen gas
• BioH₂ -- highly attractive energy carrier of the future -- 3× more energy than hydrocarbons
• hydrogenase maintains redox
• hydrogenase inhibited by oxygen
  • includes maturation
  • transcription too
  • activity
• can select recombinant variants with increased activity

Approach

• start with top two hydrogenases for activity
• DNAse I used -- creates non-specific fragments
• homologous pieces anneal
• starting point for gene shuffling

Results

• in one round of this technique, got 2.5 fold increase in activity

H Primmer

Bifunctional fusion proteins

• antiviral research
• designed to treat HIV-I
• human engineered antibody domain (m36)
  • neutralizes HIV-I gp120 on envelope
• m36 is a very small peptide that binds and neutralizes gp120 at time of gp120-CD4 binding!
• a full anti-body is actually too larget to fit into the interface
• m36 -- short serum half-life

Goals

1. increase half-life
2. allow m36 to recruit cell-mediated immune response (effector functions)

• fusion protein: increase binding / lifespan -- perhaps will be enough to offset size problem
• m36 maturation -- phage-display library, error prone PCR
• library tested for both gp120 and gp140 envelop proteins
• fusion protein has xCD4 + m36 + human-Fc domain for effector functions
• means that we no longer require host CD4 for this to work
• Fc does not increase binding, but does not inhibit (important)
• enhanced neutralization of HIV-I
• better binding
• allows effector function
• m36 maturation -- error-prone PCR -- four gp120 high-binding mutants

H Stubbs

Amyloid fibrils

• cross-β spine
• anti-parallel beta-sheet

N - - - - - * - - - - C
  C - - - - * - - - - N
N - - - - - * - - - - C
C - - - - - * - - N

• a ladder is represented by the amino acids shown as asterisks

Approach

• structural data used to design a water-soluble mimic
• globular characteristics undesirable
• peptide self-assembly mimic (PSAM) system
• N- and C- globular domains
• beta sheets in between
• added a number of β-hairpins
• started with a PSAM that had various ladders ...
  • YY/LF ladder => dimer
  • FL/FL ladder => dimer
  • YY/KR ladder => monomer
  • YY/LE ladder => monomer
• found that the hydrophobic ladders stick together
• formed head-to-tail dimers (steric interaction of heads)

N - - - - Y L - - - - C
  C - - - Y F - - - - N
N - - - - Y L - - - - C
C - - - - Y F - - N

• example YY/LF ladder

Conclusion

• hydrophobic ladders drive "β-sheet lamination"
• disease implication -- binary repeating ladder patterns are pathogenic
• unusual, don't often occur in nature