11 research outputs found
Effects of Membrane Potential and Sphingolipid Structures on Fusion of Semliki Forest Virus
Cells expressing the E1 and E2 envelope proteins of Semliki Forest virus (SFV) were fused to voltage-clamped planar lipid bilayer membranes at low pH. Formation and evolution of fusion pores were electrically monitored by capacitance measurements, and membrane continuity was tracked by video fluorescence microscopy by including rhodamine-phosphatidylethanolamine in the bilayer. Fusion occurred without leakage for a negative potential applied to the trans side of the planar membrane. When a positive potential was applied, leakage was severe, obscuring the observation of any fusion. E1-mediated cell-cell fusion occurred without leakage for negative intracellular potentials but with substantial leakage for zero membrane potential. Thus, negative membrane potentials are generally required for nonleaky fusion. With planar bilayers as the target, the first fusion pore that formed almost always enlarged; pore flickering was a rare event. Similar to other target membranes, fusion required cholesterol and sphingolipids in the planar membrane. Sphingosine did not support fusion, but both ceramide, with even a minimal acyl chain (C(2)-ceramide), and lysosphingomyelin (lyso-SM) promoted fusion with the same kinetics. Thus, unrelated modifications to different parts of sphingosine yielded sphingolipids that supported fusion to the same degree. Fusion studies of pyrene-labeled SFV with cholesterol-containing liposomes showed that C(2)-ceramide supported fusion while lyso-SM did not, apparently due to its positive curvature effects. A model is proposed in which the hydroxyls of C-1 and C-3 as well as N of C-2 of the sphingosine backbone must orient so as to form multiple hydrogen bonds to amino acids of SFV E1 for fusion to proceed
Osmolyte Effects on Monoclonal Antibody Stability and Concentration-Dependent Protein Interactions with Water and Common Osmolytes
Preferential interactions of proteins
with water and osmolytes
play a major role in controlling the thermodynamics of protein solutions.
While changes in protein stability and shifts in phase behavior are
often reported with the addition of osmolytes, the underlying protein
interactions with water and/or osmolytes are typically inferred rather
than measured directly. In this work, KirkwoodâBuff integrals
for proteinâwater interactions (<i>G</i><sub>12</sub>) and proteinâosmolyte interactions (<i>G</i><sub>23</sub>) were determined as a function of osmolyte concentration
from density measurements of antistreptavidin immunoglobulin gamma-1
(AS-IgG1) in ternary aqueous solutions for a set of common neutral
osmolytes: sucrose, trehalose, sorbitol, and polyÂ(ethylene glycol)
(PEG). For sucrose and PEG solutions, both proteinâwater and
proteinâosmolyte interactions depend strongly on osmolyte concentrations
(<i>c</i><sub>3</sub>). Strikingly, both osmolytes change
from being preferentially excluded to preferentially accumulated with
increasing <i>c</i><sub>3</sub>. In contrast, sorbitol and
trehalose solutions do not show large enough preferential interactions
to be detected by densimetry. <i>G</i><sub>12</sub> and <i>G</i><sub>23</sub> values are used to estimate the transfer
free energy for native AS-IgG1 (ÎÎŒ<sub>2</sub><sup><i>N</i></sup>) and compared
with existing models. AS-IgG1 unfolding via calorimetry shows a linear
increase in midpoint temperatures as a function of trehalose, sucrose,
and sorbitol concentrations, but the opposite behavior for PEG. Together,
the results highlight limitations of existing models and common assumptions
regarding the mechanisms of protein stabilization by osmolytes. Finally,
PEG preferential interactions destabilize the Fab regions of AS-IgG1
more so than the C<sub>H</sub>2 or C<sub>H</sub>3 domains, illustrating
preferential interactions can be specific to different protein domains
Specific-Ion Effects on the Aggregation Mechanisms and ProteinâProtein Interactions for Anti-streptavidin Immunoglobulin Gammaâ1
Non-native
protein aggregation is common in the biopharmaceutical
industry and potentially jeopardizes product shelf life, therapeutic
efficacy, and patient safety. The present article focuses on the relationship(s)
among proteinâprotein interactions, aggregate growth mechanisms,
aggregate morphologies, and specific-ion effects for an anti-streptavidin
(AS) immunoglobulin gamma 1 (IgG1). Aggregation mechanisms of AS-IgG1
were determined as a function of pH and NaCl concentration with sodium
acetate buffer and compared to previous work with sodium citrate.
Aggregate size and shape were determined using a combination of laser
light scattering and small-angle neutron or X-ray scattering. Proteinâprotein
interactions were quantified in terms of the proteinâprotein
KirkwoodâBuff integral (<i>G</i><sub>22</sub>) determined
from static light scattering and in terms of the protein effective
charge (<i>Z</i><sub>eff</sub>) measured using electrophoretic
light scattering. Changing from citrate to acetate resulted in significantly
different proteinâprotein interactions as a function of pH
for low NaCl concentrations when the protein displayed positive <i>Z</i><sub>eff</sub>. Overall, the results suggest that electrostatic
repulsions between proteins were lessened because of preferential
accumulation of citrate anions, compared to acetate anions, at the
protein surface. The predominant aggregation mechanisms correlated
well with <i>G</i><sub>22</sub>, indicating that ion-specific
effects beyond traditional mean-field descriptions of electrostatic
proteinâprotein interactions are important for predicting qualitative
shifts in protein aggregation state diagrams. Interestingly, while
solution conditions dictated which mechanisms predominated, aggregate
average molecular weight and size displayed a common scaling behavior
across both citrate- and acetate-based systems
Structural and thermodynamic effects of ANS binding to human interleukin-1 receptor antagonist
Although 8-anilinonaphthalene-1-sulfonic acid (ANS) is frequently used in protein folding studies, the structural and thermodynamic effects of its binding to proteins are not well understood. Using high-resolution two-dimensional NMR and human interleukin-1 receptor antagonist (IL-1ra) as a model protein, we obtained detailed information on ANSâprotein interactions in the absence and presence of urea. The effects of ambient to elevated temperatures on the affinity and specificity of ANS binding were assessed from experiments performed at 25°C and 37°C. Overall, the affinity of ANS was lower at 37°C compared to 25°C, but no significant change in the site specificity of binding was observed from the chemical shift perturbation data. The same site-specific binding was evident in the presence of 5.2 M urea, well within the unfolding transition region, and resulted in selective stabilization of the folded state. Based on the two-state denaturation mechanism, ANS-dependent changes in the protein stability were estimated from relative intensities of two amide resonances specific to the folded and unfolded states of IL-1ra. No evidence was found for any ANS-induced partially denatured or aggregated forms of IL-1ra throughout the experimental conditions, consistent with a cooperative and reversible denaturation process. The NMR results support earlier observations on the tendency of ANS to interact with solvent-exposed positively charged sites on proteins. Under denaturing conditions, ANS binding appears to be selective to structured states rather than unfolded conformations. Interestingly, the binding occurs within a previously identified aggregation-critical region in IL-1ra, thus providing an insight into ligand-dependent protein aggregation