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Biophysics of Proteins at Surfaces: Assembly, Activation, Signaling
Wednesday Speaker Abstracts
Oligomerization of Pore Forming Proteins in Membranes at the Single Molecule Level
Ana J. Garcia Saez
.
University of Tübingen, Tübingen, Germany.
Pore forming proteins share the ability to pierce holes in the host membranes. They are usually
synthesized in an inactive form which is monomeric and soluble. Activation includes membrane
binding, where they undergo a conformational change followed by oligomerization and pore
formation. The assembly pathway is a key step in the molecular mechanism of membrane
permeabilization, but the underlying principles remain poorly understood. Here, I will present
single molecule approaches that provide new insight into the assembly pathway of the apoptotic
protein Bax and the sea anemone toxin Equinatoxin II, as representative examples of pore
forming toxins.
Nanolipoprotein Particles Confined within Nanoporous Sol-gel or Bound to GUVs
Marjorie Longo
.
University of California Davis, USA.
Nanolipoprotein Particles (NLPs) are disc-shaped nanometer-sized lipid bilayer patches
stabilized by a belt of scaffold proteins. NLPs have an average thickness of 5 nm, with a
diameter ranging from 10-25 nm depending on the stoichiometric ratios and types of lipids and
scaffold proteins being used. This allows NLPs to be compatible with the pore size (5-50 nm) of
mesoporous silica. Therefore, we perform entrapment of NLPs using a quick, simple sol-gel
processing technique for TMOS that includes evaporation of the majority of the methanol after
the hydrolysis reactions. To ensure proper functioning of silica sol-gel entrapped NLPs, we have
investigated the phase behavior of the lipids in addition to the secondary structure, localization,
and environmental polarity of the scaffold proteins. Our results indicate that silica gel-entrapped
NLPs remain intact, with only slightly altered lipid and scaffold protein structure and dynamics.
We will briefly discuss the potential to entrap NLPs containing embedded integral membrane
proteins (IMPs) for various applications such as biosensing, affinity chromatography, high-
throughput drug screening, and bio-reaction engineering.
Further, scaffold proteins can bear polyhistadine tags, which are capable of chelating to Cu2+
metal ions. Lipid-phase specific, iminodiacetic acid (IDA) functionalized lipids are also capable
of chelating Cu2+, providing a mechanism for phase-targeted binding of NLPs. We investigate
this binding via fluorescence microscopy and characterize interaction with phase-separated
supported bilayers and giant unilamellar vesicles (GUVs). The thermodynamics (enthalpy of
lipid mixing and steric pressure of protein crowding) and morphology of binding are also
examined. Targeted binding of NLPs bearing functional IMPs and/or other biomolecules to
supported lipid bilayers has a variety of applications, including development of nano-array
technologies and biosensors.