Nickel-chelating lipids offer a convenient platform for reversible immobilization of histidine-tagged proteins to liposome surfaces. This interaction recently found utility as a model system for studying membrane remodeling triggered by protein crowding. Despite its wide array of utility, the molecular details of transient protein association to the lipid surfaces decorated with such chelator lipids remains poorly understood. In this study, we explore the kinetics of protein–liposome association across a wide concentration range using stopped-flow fluorescence. The fluorescence of histidine-tagged protein containing an intrinsic fluorophore (superfolder green fluorescent protein, SfGFP) was quenched upon binding to Ni–NTA-modified liposomes containing the quencher Dabsyl-PE lipids. Stopped-flow fluorescence reveals a complex, multiexponential binding behavior with a fast (k obs ∼ 10–20 s–1) phase and slower (k obs < 4 s–1) phase. Interestingly, the observed rates for the slower phase increase initially under low concentrations but start decreasing once a critical concentration is reached. Despite differences in the binding time scales, we observe that the trend of decreasing rates is reproducible irrespective of the chelator lipid doping level, protein surface charge, or lipid composition. Consideration of the protein footprint and membrane surface area occupancy leads us to conclude that the multiphasic binding behavior is reflective of protein binding via two distinct binding conformations. We propose that preliminary steps in protein association involve binding of a sterically occlusive side-on conformation followed by reorganization that leads to an end-on conformation with increased packing density. These results are important for the improvement of histidine-tag-based immobilization strategies and offer mechanistic insight into intermediates preceding membrane bending driven by protein crowding.

Serine incorporator 5 (SER5) is a protein that upon incorporation into virions inhibits HIV-1 infectivity by interfering with the ability of the Env glycoprotein to promote viral fusion. The mechanisms by which SER5 antagonizes HIV-1 fusion are not well understood. A recent study of SER5’s structure revealed a lipid-binding pocket, suggesting the ability to sequester lipids. This finding, along with the well-documented modulation of HIV-1 infectivity by viral lipids, especially cholesterol, prompted our examination of SER5′s effect on the general lipid order of the HIV-1 membrane. Pseudoviruses bearing the SER5-sensitive HXB2-Env and containing SER5 or SER2, a control protein that lacks antiviral activity, were analyzed using two distinct lipid-order probes. We show that SER5 incorporation does not noticeably affect the lipid order of pseudoviruses. Although viral cholesterol extraction reduces HIV-1 infectivity, SER5+ viruses are less sensitive to cholesterol extraction than the control samples. In contrast, the virus’ sensitivity to cholesterol oxidation was not affected by SER5 incorporation. The hydrolytic release of sphingomyelin-sequestered cholesterol had a minimal impact on the apparent resistance to cholesterol extraction. Based on these results, we propose that a subpopulation of more stable Env glycoproteins responsible for the residual infectivity of SER5+ viruses is less sensitive to the cholesterol content of the viral membrane.

Dynamic modulation of lipid membrane curvature can be achieved by a number of peripheral protein binding mechanisms such as hydrophobic insertion of amphipathic helices and membrane scaffolding. Recently, an alternative mechanism was proposed in which crowding of peripherally bound proteins induces membrane curvature through steric pressure generated by lateral collisions. This effect was enhanced using intrinsically disordered proteins that possess high hydrodynamic radii, prompting us to explore whether membrane bending can be triggered by the folding—unfolding transition of surface-bound proteins. We utilized histidine-tagged human serum albumin bound to Ni-NTA- DGS containing liposomes as our model system to test this hypothesis. We found that reduction of the disulfide bonds in the protein resulted in unfolding of HSA, which subsequently led to membrane tubule formation. The frequency of tubule formation was found to be significantly higher when the proteins were unfolded while being localized to a phase-separated domain as opposed to randomly distributed in fluid phase liposomes, indicating that the steric pressure generated from protein unfolding can drive membrane deformation. Our results are critical for the design of peripheral membrane protein-immobilization strategies and open new avenues for exploring mechanisms of membrane bending driven by conformational changes of peripheral membrane proteins.

GK2848, a hypothetical protein from the thermophilic organism Geobacillus kaustophilus, was cloned and overexpressed in Escherichia coli. The protein was purified to homogeneity using Ni-NTA affinity-column and gel-filtration chromatography. The purified protein was crystallized using the sitting-drop vapour-diffusion method. The crystals diffracted to a resolution of 2.70 Å and belonged to the orthorhombic space group P2 1 2 1 2. GK2848 bears sequence homology to carbonic anhydrases of various bacterial species, indicating that it belongs to the carbonic anhydrase family of proteins. A subsequent carbonic anhydrase activity assay of GK2848 using the Wilbur-Anderson method confirmed its function as a carbonic anhydrase. A preliminary structure solution was obtained by molecular replacement using MOLREP. Mutation and biochemical characterization of the protein are in progress. The structure and functional analysis of GK2848 might provide valuable information on a novel class of carbonic anhydrases, as none of its homologous structures have been characterized.