Biomedical Engineering Reference
In-Depth Information
the. library. design. results. in. a. high. percentage. of. folded. proteins. or. when. an. extremely.
high-throughput.selection.can.be.applied.
Binary.patterning.is.a.simple.but.surprisingly.effective.method.for.generating.diverse.col-
lections.of.protein.sequences.that.adopt.a.deined.three-dimensional.fold..Hydrophobic/
hydrophilic. interactions. are. dominant. drivers. of. protein. folding:. the. interiors. of. folded.
proteins.typically.contain.hydrophobic.amino.acids.that.pack.within.the.core.to.hide.from.
the.solvent..In.contrast,.surface.amino.acids.tend.to.be.polar.or.charged.and.interact.favor-
ably.with.water..A.library.designed.to.dictate.only.the.hydrophobic.or.hydrophilic.char-
acter.at.each.amino.acid.position.contained.proteins.of.diverse.primary.sequence,.most.of.
which.folded.into.the.desired.four-helix.bundle.structure.(Kamtekar.et.al..1993)..Many.of.
these.proteins.had.ordered,.native-like.cores.(Wei.et.al..2003a,b)..Screening.of.these.binary-
patterned.libraries.identiied.heme-binding.proteins.(Rojas.et.al..1997).that.bound.carbon.
monoxide.(Moffet.et.al..2001).and.proteins.with.peroxidase,.esterase,.and.lipase.enzymatic.
activity.(Das.and.Hecht.2007;.Patel.et.al..2009).
The.same.group.transformed.27.single-knockout.auxotrophic.
E. coli
.strains.with.a.library.
of.1.5.×.10
6
.patterned.helical.bundles.and.isolated.transformants.that.rescued.growth.of.four.
of.the.strains.on.minimal.media.(Fisher.et.al..2011)..Activity.could.not.be.measured.in.cell.
lysates.or.puriied.samples.of.these.proteins,.but.the.very.low.levels.of.activity.expected.
given. the. slow-growing. phenotype. would. likely. be. below. the. detection. limit. of. such.
assays..The.authors.go.to.admirable.lengths.to.rule.out.alternate.explanations,.including.
demonstrating.that.mutation.of.key.residues.in.the.synthetic.proteins.abolished.rescue.of.
growth..The.authors.then.showed.that.a.quadruple.knockout.could.be.rescued.by.cotrans-
formation.with.plasmids.encoding.four.selected.synthetic.proteins..While.the.complexity.of.
the.cell.prevents.ruling.out.all.alternative.explanations,.it.seems.likely.that.at.least.some.of.
these.simple,.102-residue.helix.bundle.proteins,.which.were.neither.designed.nor.evolved.
but. directly. selected. from. a. designed. library,. possess. minimal. enzymatic. activity.. This. is.
especially.remarkable.considering.the.complexity.of.the.enzymes.they.replace.in.this.study..
Given.the.tendencies.of.four-helix.bundles,.it.is.likely.that.the.selected.enzymes.bind.met-
als.or.other.cofactors.upon.which.they.rely.for.function..Targeted.mutagenesis.to.introduce.
cavities.into.a.selected.bundle.resulted.in.a.pocket.capable.of.binding.small.aromatic.mol-
ecules.(Das.et.al..2011)..Scaffolds.with.designed.cavities.of.this.type.may.allow.the.selection.
of.enzymes.with.substrate.binding.clefts.similar.to.those.observed.in.natural.enzymes.
An. alternative. strategy. is. to. select. from. a. less-restricted. library. with. an. extremely.
high-throughput.method..In.vitro.methods.are.capable.of.selecting.desired.proteins.from.
libraries. of. 10
12
. or. more. mutants.. One. of. these,. mRNA. display,. has. been. used. to. select.
ATP-binding.proteins.from.random.sequences.of.80.amino.acids.(Keefe.and.Szostak.2001).
and,.more.recently,.to.select.enzymes.capable.of.ligating.two.RNA.molecules.(Seelig.and.
Szostak. 2007).. In. the. latter. study,. the. starting. library. consisted. of. a. zinc-inger. protein.
scaffold.with.two.randomized.loops.of.12.and.9.amino.acids..Selection.from.this.random.
library.was.followed.by.mutagenesis.and.recombination,.inally.yielding.zinc-dependent.
enzymes.that.accelerate.the.reaction.by.as.much.as.2.×.10
6
-fold.
3.5 Catalytic Antibodies
Modern. transition. state. theory. and. computer. simulations. suggest. that. the. dominant.
factor.in.enzymatic.catalysis.is.the.lowering.of.the.activation.barrier.by.stabilization.of.the.
