Calcium-Dependent Association of Calmodulin with the C-Terminal Domain of the Tetraspanin Protein Peripherin/rds

Peripherin/rds (p/rds), an integral membrane protein from the transmembrane 4 (TMF4) superfamily, possesses a multi-functional C-terminal domain that plays crucial roles in rod outer segment (ROS) disk renewal and structure. Here, we report that the calcium binding protein calmodulin (CaM) binds to the C-terminal domain of p/rds. Fluorescence spectroscopy reveals Ca2+-dependent association of CaM with a polypeptide corresponding to the C-terminal domain of p/rds. The fluorescence anisotropy of the polypeptide upon CaM titration yields a dissociation constant (KD) of 320 ± 150 nM. The results of the fluorescence experiments were confirmed by GST-pull down analyses in which a GST-p/rds C-terminal domain fusion protein was shown to pull down CaM in a calcium-dependent manner. Moreover, molecular modeling and sequence predictions suggest that the CaM binding domain resides in a p/rds functional hot spot, between residues E314 and G329. Predictions were confirmed by peptide competition studies and a GST-p/rds C-terminal domain construct in which the putative Ca2+/CaM binding site was scrambled. This GST-polypeptide did not associate with Ca2+/CaM. This putative calmodulin domain is highly conserved between human, mouse, rat, and bovine p/rds. Finally, the binding of Ca2+/CaM inhibited fusion between ROS disk and ROS plasma membranes as well as p/rds C-terminal-domain-induced fusion in model membrane studies. These results offer a new mechanism for the modulation of p/rds function. © 2007 American Chemical Society

Cholesterol Dynamics in Membranes

Time-resolved fluorescence anisotropy of the sterol analogue, cholestatrienol, and 13C nuclear magnetic resonance (NMR) spin lattice relaxation time (T1c) measurements of [13C4] labeled cholesterol were exploited to determine the correlation times characterizing the major modes of motion of cholesterol in unsonicated phospholipid multilamellar liposomes. Two modes of motion were found to be important: (a) rotational diffusion and (b) time dependence of the orientation of the director for axial diffusion, or wobble. From the time-resolved fluorescence anisotropy decays of cholestatrienol in egg phosphatidylcholine (PC) bilayers, a value for tau perpendicular, the correlation time for wobble, of 0.9 x 10(-9) s and a value for S perpendicular, the order parameter characterizing the same motion, of 0.45 s were calculated. Both tau perpendicular and S perpendicular were relatively insensitive to temperature and cholesterol content of the membranes. The T1c measurements of [13C4] labeled cholesterol did not provide a quantitative determination of tau parallel, the correlation time for axial diffusion. T1c from the lipid hydrocarbon chains suggested a value for tau perpendicular similar to that for cholesterol. Steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in a variety of pure and mixed lipid multilamellar liposomes. Both the lipid headgroups and the lipid hydrocarbons chains contributed to the determination of the sterol environment in the membrane, as revealed by these fluorescence measurements. In particular, effects of the phosphatidylethanolamine (PE) headgroup and of multiple unsaturation in the lipid hydrocarbon chains were observed. However, while the steady-state anisotropy was sensitive to these factors, the time-resolved fluorescence analysis indicated that tau perpendicular was not strongly affected by the lipid composition of the membrane. S perpendicular may be increased by the presence of PE. Both steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in three biological membranes: bovine rod outer segment (ROS) disk membranes, human erythrocyte plasma membranes, and light rabbit muscle sarcoplasmic reticulum membranes. In the ROS disk membranes the value for S perpendicular was marginally higher than in the PC membranes, perhaps reflecting the influence of PE. The dramatic difference noted was in the value for tau perpendicular. In both the ROS disk membranes and the erythrocyte membranes, tau perpendicular was one-third to one-fifth of tau perpendicular in the phospholipid bilayers. This result may reveal an influence of membrane proteins on sterol behavior

Controlling Multivalent Binding through Surface Chemistry: Model Study on Streptavidin.

Although multivalent binding to surfaces is an important tool in nanotechnology, quantitative information about the residual valency and orientation of surface-bound molecules is missing. To address these questions, we study streptavidin (SAv) binding to commonly used biotinylated surfaces such as supported lipid bilayers (SLBs) and self-assembled monolayers (SAMs). Stability and kinetics of SAv binding are characterized by quartz crystal microbalance with dissipation monitoring, while the residual valency of immobilized SAv is quantified using spectroscopic ellipsometry by monitoring binding of biotinylated probes. Purpose-designed SAv constructs having controlled valencies (mono-, di-, trivalent in terms of biotin-binding sites) are studied to rationalize the results obtained on regular (tetravalent) SAv. We find that divalent interaction of SAv with biotinylated surfaces is a strict requirement for stable immobilization, while monovalent attachment is reversible and, in the case of SLBs, leads to the extraction of biotinylated lipids from the bilayer. The surface density and lateral mobility of biotin, and the SAv surface coverage are all found to influence the average orientation and residual valency of SAv on a biotinylated surface. We demonstrate how the residual valency can be adjusted to one or two biotin binding sites per immobilized SAv by choosing appropriate surface chemistry. The obtained results provide means for the rational design of surface-confined supramolecular architectures involving specific biointeractions at tunable valency. This knowledge can be used for the development of well-defined bioactive coatings, biosensors and biomimetic model systems

Electrostatic considerations affecting the calculated HOMO-LUMO gap in protein molecules.

A detailed study of energy differences between the highest occupied and lowest unoccupied molecular orbitals (HOMO-LUMO gaps) in protein systems and water clusters is presented. Recent work questioning the applicability of Kohn-Sham density-functional theory to proteins and large water clusters (E. Rudberg, J. Phys.: Condens. Mat. 2012, 24, 072202) has demonstrated vanishing HOMO-LUMO gaps for these systems, which is generally attributed to the treatment of exchange in the functional used. The present work shows that the vanishing gap is, in fact, an electrostatic artefact of the method used to prepare the system. Practical solutions for ensuring the gap is maintained when the system size is increased are demonstrated. This work has important implications for the use of large-scale density-functional theory in biomolecular systems, particularly in the simulation of photoemission, optical absorption and electronic transport, all of which depend critically on differences between energies of molecular orbitals.Comment: 13 pages, 4 figure

Interaction of 3â-amino-5-cholestene with phospholipids in binary and ternary bilayer membranes

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Langmuir, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://doi.org/10.1021/la203589u.3β-Amino-5-cholestene (aminocholesterol) is a synthetic sterol whose properties in bilayer membranes have been examined. In fluid palmitoyl sphingomyelin (PSM) bilayers, aminocholesterol and cholesterol were equally effective in increasing acyl chain order, based on changes in diphenylhexatriene (DPH) anisotropy. In fluid 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers, aminocholesterol ordered acyl chains, but slightly less efficiently than cholesterol. Aminocholesterol eliminated the PSM and DPPC gel-to-liquid crystalline phase transition enthalpy linearly with concentration, and the enthalpy approached zero at 30 mol% sterol. Whereas cholesterol was able to increase the thermostability of ordered PSM domains in a fluid bilayer, aminocholesterol under equal conditions failed to do this, suggesting that its interaction with PSM was not as favorable as cholesterol’s. In ternary mixed bilayers, containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), PSM or DPPC, and cholesterol at proportions to contain a liquid-ordered phase (60:40 by mol of POPC and PSM or DPPC, and 30 mol% cholesterol), the average life-time of trans parinaric acid (tPA) was close to 20 ns. When cholesterol was replaced with aminocholesterol in such mixed bilayers, the average life-time of tPA was only marginally shorter (about 18 ns). This observation, together with acyl chain ordering data, clearly shows that aminocholesterol was able to form a liquid-ordered phase with saturated PSM or DPPC. We conclude that aminocholesterol should be a good sterol replacement in model membrane systems for which a partial positive charge is deemed beneficial

Ion Transport across Biological Membranes by Carborane-Capped Gold Nanoparticles

Carborane-capped gold nanoparticles (Au/carborane NPs, 2-3 nm) can act as artificial ion transporters across biological membranes. The particles themselves are large hydrophobic anions that have the ability to disperse in aqueous media and to partition over both sides of a phospholipid bilayer membrane. Their presence therefore causes a membrane potential that is determined by the relative concentrations of particles on each side of the membrane according to the Nernst equation. The particles tend to adsorb to both sides of the membrane and can flip across if changes in membrane potential require their repartitioning. Such changes can be made either with a potentiostat in an electrochemical cell or by competition with another partitioning ion, for example, potassium in the presence of its specific transporter valinomycin. Carborane-capped gold nanoparticles have a ligand shell full of voids, which stem from the packing of near spherical ligands on a near spherical metal core. These voids are normally filled with sodium or potassium ions, and the charge is overcompensated by excess electrons in the metal core. The anionic particles are therefore able to take up and release a certain payload of cations and to adjust their net charge accordingly. It is demonstrated by potential-dependent fluorescence spectroscopy that polarized phospholipid membranes of vesicles can be depolarized by ion transport mediated by the particles. It is also shown that the particles act as alkali-ion-specific transporters across free-standing membranes under potentiostatic control. Magnesium ions are not transported

Aggregatibacter Actinomycetemcomitans Leukotoxin Cytotoxicity Occurs Through Bilayer Destabilization

The Gram-negative bacterium, Aggregatibacter actinomycetemcomitans, is a common inhabitant of the human upper aerodigestive tract. The organism produces an RTX (Repeats in ToXin) toxin (LtxA) that kills human white blood cells. LtxA is believed to be a membrane-damaging toxin, but details of the cell surface interaction for this and several other RTX toxins have yet to be elucidated. Initial morphological studies suggested that LtxA was bending the target cell membrane. Because the ability of a membrane to bend is a function of its lipid composition, we assessed the proficiency of LtxA to release of a fluorescent dye from a panel of liposomes composed of various lipids. Liposomes composed of lipids that form nonlamellar phases were susceptible to LtxA-induced damage while liposomes composed of lipids that do not form non-bilayer structures were not. Differential scanning calorimetry demonstrated that the toxin decreased the temperature at which the lipid transitions from a bilayer to a nonlamellar phase, while 31P nuclear magnetic resonance studies showed that the LtxA-induced transition from a bilayer to an inverted hexagonal phase occurs through the formation of an isotropic intermediate phase. These results indicate that LtxA cytotoxicity occurs through a process of membrane destabilization. © 2012 Blackwell Publishing Ltd

Spatiotemporal structure of intracranial electric fields induced by transcranial electric stimulation in humans and nonhuman primates

Transcranial electric stimulation (TES) is an emerging technique, developed to non-invasively modulate brain function. However, the spatiotemporal distribution of the intracranial electric fields induced by TES remains poorly understood. In particular, it is unclear how much current actually reaches the brain, and how it distributes across the brain. Lack of this basic information precludes a firm mechanistic understanding of TES effects. In this study we directly measure the spatial and temporal characteristics of the electric field generated by TES using stereotactic EEG (s-EEG) electrode arrays implanted in cebus monkeys and surgical epilepsy patients. We found a small frequency dependent decrease (10%) in magnitudes of TES induced potentials and negligible phase shifts over space. Electric field strengths were strongest in superficial brain regions with maximum values of about 0.5 mV/mm. Our results provide crucial information of the underlying biophysics in TES applications in humans and the optimization and design of TES stimulation protocols. In addition, our findings have broad implications concerning electric field propagation in non-invasive recording techniques such as EEG/MEG

Domain formation in DODAB–cholesterol mixed systems monitored via nile red anisotropy

The effect of the cholesterol (Ch) on liposomes composed of the cationic lipid dioctadecyldimethylammonium bromide (DODAB) was assessed by studying both the steady-state and time-resolved fluorescence anisotropy of the dye Nile Red. The information obtained combined with analysis of the steady-state emission and luorescence lifetime of Nile Red (NR) for different cholesterol concentrations (5–50%) elucidated the presence of “condensed complexes” and cholesterol-rich domains in these mixed systems. The steady-state fluorescence spectra were decomposed into the sum of two lognormal emissions, emanating from two different states, and the effect of temperature on the anisotropy decay of Nile Red for different cholesterol concentrations was observed. At room temperature, the time-resolved anisotropy decays are indicative of NR being relatively immobile (manifest by a high r∞ value). At higher temperature, rotational times ca. 1 ns were obtained throughout and a trend in increasing hindrance was seen with increase of Ch content