Protein/lipid interactions in phospholipid monolayers containing the bacterial antenna protein B800-850

Studies on monomolecular layers of phospholipids containing the antenna protein B800-850 (LHCP) and in some cases additionally the reaction center of the photosynthetic bacterium Rhodopseudomonas sphaeroides are reported. Information on monolayer preparation as well as on protein /lipid and protein/protein interaction is obtained by means of fluorescence spectroscopy and microscopy at the air/water interface in combination with film balance experiments. It is shown that a homogeneous distribution of functional proteins can be achieved. This can be transformed into a regular pattern-like distribution by inducing a phospholipid phase transition. Although the LHCP preferentially partitions into the fluid lipid phase, it decreases the lateral pressure necessary to crystallize the lipid. This is probably due to an increase in order of the fluid phase. A pressure-induced conformation change of the LHCP is detected via a drastic change in fluorescence yield. A highly efficient energy transfer from LHCP to the reaction center is observed. This proves the quantitative reconstitution of both types of proteins and indicates protein aggregation also in the monolayer

Calcium Causes a Conformational Change in Lamin A Tail Domain that Promotes Farnesyl-Mediated Membrane Association

AbstractLamin proteins contribute to nuclear structure and function, primarily at the inner nuclear membrane. The posttranslational processing pathway of lamin A includes farnesylation of the C-terminus, likely to increase membrane association, and subsequent proteolytic cleavage of the C-terminus. Hutchinson Gilford progeria syndrome is a premature aging disorder wherein a mutant version of lamin A, Δ50 lamin A, retains its farnesylation. We report here that membrane association of farnesylated Δ50 lamin A tail domains requires calcium. Experimental evidence and molecular dynamics simulations collectively suggest that the farnesyl group is sequestered within a hydrophobic region in the tail domain in the absence of calcium. Calcium binds to the tail domain with an affinity KD ≈ 250 μM where it alters the structure of the Ig-fold and increases the solvent accessibility of the C-terminus. In 2 mM CaCl2, the affinity of the farnesylated protein to a synthetic membrane is KD ≈ 2 μM, as measured with surface plasmon resonance, but showed a combination of aggregation and binding. Membrane binding in the absence of calcium could not be detected. We suggest that a conformational change induced in Δ50 lamin A with divalent cations plays a regulatory role in the posttranslational processing of lamin A, which may be important in disease pathogenesis

Electrostatic Interactions and Binding Orientation of HIV-1 Matrix Studied by Neutron Reflectivity

AbstractThe N-terminal matrix (MA) domain of the HIV-1 Gag protein is responsible for binding to the plasma membrane of host cells during viral assembly. The putative membrane-binding interface of MA was previously mapped by means of mutagenesis and analysis of its trimeric crystal structure. However, the orientation of MA on membranes has not been directly determined by experimental measurements. We present neutron reflectivity measurements that resolve the one-dimensional scattering length density profile of MA bound to a biomimetic of the native viral membrane. A molecular refinement procedure was developed using atomic structures of MA to determine the orientation of the protein on the membrane. The orientation defines a lipid-binding interface consistent with previous mutagenesis results. The MA protein maintains this orientation without the presence of a myristate group, driven only by electrostatic interactions. Furthermore, MA is found to penetrate the membrane headgroup region peripherally such that only the side chains of specific Lys and Arg residues interact with the surface. The results suggest that electrostatic interactions are sufficient to favorably orient MA on viral membrane mimics. The spatial determination of the membrane-bound protein demonstrates the ability of neutron reflectivity to discern orientation and penetration under physiologically relevant conditions

Single wall carbon nanotubes enter cells by endocytosis and not membrane penetration

<p>Abstract</p> <p>Background</p> <p>Carbon nanotubes are increasingly being tested for use in cellular applications. Determining the mode of entry is essential to control and regulate specific interactions with cells, to understand toxicological effects of nanotubes, and to develop nanotube-based cellular technologies. We investigated cellular uptake of Pluronic copolymer-stabilized, purified ~145 nm long single wall carbon nanotubes (SWCNTs) through a series of complementary cellular, cell-mimetic, and in vitro model membrane experiments.</p> <p>Results</p> <p>SWCNTs localized within fluorescently labeled endosomes, and confocal Raman spectroscopy showed a dramatic reduction in SWCNT uptake into cells at 4°C compared with 37°C. These data suggest energy-dependent endocytosis, as shown previously. We also examined the possibility for non-specific physical penetration of SWCNTs through the plasma membrane. Electrochemical impedance spectroscopy and Langmuir monolayer film balance measurements showed that Pluronic-stabilized SWCNTs associated with membranes but did not possess sufficient insertion energy to penetrate through the membrane. SWCNTs associated with vesicles made from plasma membranes but did not rupture the vesicles.</p> <p>Conclusions</p> <p>These measurements, combined, demonstrate that Pluronic-stabilized SWCNTs only enter cells via energy-dependent endocytosis, and association of SWCNTs to membrane likely increases uptake.</p