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Membrane
proteins in biomineralization
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Structure
and Function of the diatom silicon transporters
Diatoms are single-celled eukaryotic algae that surround themselves
with an outer cell wall, or frustule, composed mostly of amorphous
silica. This 'glass house' seems to offer diatoms a considerable
evolutionary advantage, and they contribute to >20% of all of
the photosynthetic activity on Earth. Frustule biogenesis takes
place almost completely within a specialized cell body, the silica
deposition vesicle, but the details of this biomineralization process
are not well understood. One remarkable feature of the frustule is the
capacity of the diatoms to form complex silica
structures that are tightly controlled at the nanoscale (for examples,
see image on the right taken from Bradbury,
PLoS Biology).
The raw material for frustule synthesis is thought
to be silicic acid,
a soluble form of silica found at low concentrations in the
environment. Diatoms must scavenge silicic acid and bring it into the
cell interior where it can be concentrated to form frustule silica.
This is thought to require an active transport system, and a novel
family of integral membrane proteins have been identified that function
as silicic acid transporters.
Although experiments in
whole cells provide strong evidence for this transport function
they cannot describe the molecular basis for silicic acid recognition
and transport. The schematic below summarizes the current understanding
of the structure and function of these transporters.
I am currently conducting the first investigation of silicon
transport proteins in vitro.
This is intended to provide a detailed description of the
protein:silicic acid interaction at the molecular level. Understanding
this novel organic:inorganic interface might provide a platform for
future nanoscience applications or the creation of a synthetic diatom.
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Surface
display of biomineralizing proteins
Outer membrane protein A (OmpA)is a diffusion
channel that resides in the outer membrane of E. coli.
It contains structureless external loops which are suitable targets for
recombinant protein engineering. Working with Prof. Daniel Morse at
UCSB, I used OmpA as a scaffold to support the display of
silicatein-alpha, a silica biomineralisation enzyme derived
from a marine sponge, at the E. coli
cell surface. The displayed enzyme is catalytically active and able to
synthesise inorganic
materials and organic
polymers at the
surface of the cell.
The image on the right shows a crystalline titanium
compound formed at the surface of a bacterial cell via biocatalysis.
(a) Crystallinity is demonstrated using small-angle electron
diffraction; (b) TEM shows that the product accumulates at low levels
at the cell surface.
Membrane
protein folding and reconstitution
Integral
membrane proteins are important
components of many biological processes and comprise ~30 % of all
cellular proteins. Surprisingly little is known about how these
proteins fold and assemble to their final structure. This information
could potentially be obtained through biophysical approaches
(structural biology, enzyme
kinetics, fluorescence methods etc.),
but this has proven challenging for membrane proteins. Suitable in vitro
systems are difficult to
establish
because simple artificial solvents cannot replicate the complex
heterogenous
lipid environment that membrane proteins normally experience.
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Folding of bacteriorhodopsin
My recent work has focused on the light-activated proton pump
bacteriorhodpsin (structure shown at top right). bR can be
reversibly unfolded under equilibrium
conditions by using mixed micelles composed of stabilizing and
denaturing
detergents. Through a combined kinetic and thermodynamic analysis,
we showed that the free energy of unfolding was linear with
denaturant
concentration, and that folding could be considered as a simple
two-state
process. This allowed us to apply the analytical toolkit
developed
for soluble protein
folding studies to a membrane protein for the first time. This revealed
that
the folding transition state is globally close to the unfolded state
and that
the
spontaneous unfolding of bR in the absence of denaturant is very
rare. A recent paper
suggested that the kinetic stability was mediated by the retinal
chromophore.
Further work has investigated the structure of the folding transition
state by using a phi-value
analysis. By focusing on a
single
transmembrane helix, helix B, we demonstrated that this helix appears
to form early on the folding pathway. We expect to extend this analysis
to other helices in due course in order to build up a complete
description of transition state structure.
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Reconstitution
and folding of EmrE
The
small multidrug transporter EmrE has a monomer unit comprising
110 amino acids arranged in four transmembrane alpha-helices (top panel,
right). The
relative simplicity of this protein make it an attractive candidate for
understanding the basis of polyspecifc multidrug transport. We
demonstrated
that EmrE could be functionally reconstituted into
artificial lipid vesicles and that the lipid composition of the
vesicles influenced the function of EmrE in a predictable
manner; when the lipid lateral pressure was increased by introducing
non-lamellar lipids, substrate transport was dramatically impaired (bottom panel
on right). Further
work from the Booth group has
built upon these findings to
further explore lipid effects in multidrug transport.
A lis of all recent publications can be found here.
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Dr
Paul Curnow
