5 research outputs found
Polar Interactions Trump Hydrophobicity in Stabilizing the Self-Inserting Membrane Protein Mistic
Canonical integral membrane proteins
are attached to lipid bilayers
through hydrophobic transmembrane helices, whose topogenesis requires
sophisticated insertion machineries. By contrast, membrane proteins
that, for evolutionary or functional reasons, cannot rely on these
machineries need to resort to driving forces other than hydrophobicity.
A striking example is the self-inserting Bacillus subtilis protein Mistic, which is involved in biofilm formation and has found
application as a fusion tag supporting the recombinant production
and bilayer insertion of other membrane proteins. Although this unusual
protein contains numerous polar and charged residues and lacks characteristic
membrane-interaction motifs, it is tightly bound to membranes in vivo
and membrane-mimetic systems in vitro. Therefore, we set out to quantify
the contributions from polar and nonpolar interactions to the coupled
folding and insertion of Mistic. To this end, we defined conditions
under which the protein can be unfolded completely and reversibly
from various detergent micelles by urea in a two-state equilibrium
and where the unfolded state is independent of the detergent used
for solubilizing the folded state. This enabled equilibrium unfolding
experiments previously used for soluble and β-barrel membrane
proteins, revealing that polar interactions with ionic and zwitterionic
headgroups and, presumably, the interfacial dipole potential stabilize
the protein much more efficiently than nonpolar interactions with
the micelle core. These findings unveil the forces that allow a protein
to tightly interact with a membrane-mimetic environment without major
hydrophobic contributions and rationalize the differential suitability
of detergents for the extraction and solubilization of Mistic-tagged
membrane proteins
Engineering Asymmetric Lipid Vesicles: Accurate and Convenient Control of the Outer Leaflet Lipid Composition
The asymmetric distribution
of lipids between the two bilayer leaflets
represents a typical feature of biological membranes. The loss of
this asymmetry, for example the exposure of negatively charged lipids
on the extracellular membrane leaflet of mammalian cells, is involved
in apoptosis and occurs in tumor cells. Thus, the controlled production
of asymmetric liposomes helps to better understand such crucial cellular
processes. Here, we present an approach that allows us to design asymmetric
model-membrane experiments on a rational basis and predict the fraction
of exchanged lipid. In addition, we developed a label-free and nondestructive
assay to quantify the asymmetric uptake of negatively charged lipids
in terms of the zeta potential. This significantly enhances the applicability,
impact, and predictive power of model membranes
The Mechanism of Denaturation and the Unfolded State of the α‑Helical Membrane-Associated Protein Mistic
<i>In vitro</i> protein-folding studies using chemical
denaturants such as urea are indispensible in elucidating the forces
and mechanisms determining the stability, structure, and dynamics
of water-soluble proteins. By contrast, α-helical membrane-associated
proteins largely evade such approaches because they are resilient
to extensive unfolding. We have used optical and NMR spectroscopy
to provide an atomistic-level dissection of the effects of urea on
the structure and dynamics of the α-helical membrane-associated
protein Mistic as well as its interactions with detergent and solvent
molecules. In the presence of the zwitterionic detergent lauryl dimethylamine
oxide, increasing concentrations of urea result in a complex sequence
of conformational changes that go beyond simple two-state unfolding.
Exploiting this finding, we report the first high-resolution structural
models of the urea denaturation process of an α-helical membrane-associated
protein and its completely unfolded state, which contains almost no
regular secondary structure but nevertheless retains a topology close
to that of the folded state
The Mechanism of Denaturation and the Unfolded State of the α‑Helical Membrane-Associated Protein Mistic
<i>In vitro</i> protein-folding studies using chemical
denaturants such as urea are indispensible in elucidating the forces
and mechanisms determining the stability, structure, and dynamics
of water-soluble proteins. By contrast, α-helical membrane-associated
proteins largely evade such approaches because they are resilient
to extensive unfolding. We have used optical and NMR spectroscopy
to provide an atomistic-level dissection of the effects of urea on
the structure and dynamics of the α-helical membrane-associated
protein Mistic as well as its interactions with detergent and solvent
molecules. In the presence of the zwitterionic detergent lauryl dimethylamine
oxide, increasing concentrations of urea result in a complex sequence
of conformational changes that go beyond simple two-state unfolding.
Exploiting this finding, we report the first high-resolution structural
models of the urea denaturation process of an α-helical membrane-associated
protein and its completely unfolded state, which contains almost no
regular secondary structure but nevertheless retains a topology close
to that of the folded state
Extensive sequence and structural evolution of Arginase 2 inhibitory antibodies enabled by an unbiased approach to affinity maturation.
Affinity maturation is a powerful technique in antibody engineering for the in vitro evolution of antigen binding interactions. Key to the success of this process is the expansion of sequence and combinatorial diversity to increase the structural repertoire from which superior binding variants may be selected. However, conventional strategies are often restrictive and only focus on small regions of the antibody at a time. In this study, we used a method that combined antibody chain shuffling and a staggered-extension process to produce unbiased libraries, which recombined beneficial mutations from all six complementarity-determining regions (CDRs) in the affinity maturation of an inhibitory antibody to Arginase 2 (ARG2). We made use of the vast display capacity of ribosome display to accommodate the sequence space required for the diverse library builds. Further diversity was introduced through pool maturation to optimize seven leads of interest simultaneously. This resulted in antibodies with substantial improvements in binding properties and inhibition potency. The extensive sequence changes resulting from this approach were translated into striking structural changes for parent and affinity-matured antibodies bound to ARG2, with a large reorientation of the binding paratope facilitating increases in contact surface and shape complementarity to the antigen. The considerable gains in therapeutic properties seen from extensive sequence and structural evolution of the parent ARG2 inhibitory antibody clearly illustrate the advantages of the unbiased approach developed, which was key to the identification of high-affinity antibodies with the desired inhibitory potency and specificity