Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System.

Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding.

Probing the interaction of lipids with the non-annular binding sites of the potassium channel KcsA by magic-angle spinning NMR.

The activity of the potassium channel KcsA is tightly regulated through the interactions of anionic lipids with high-affinity non-annular lipid binding sites located at the interface between the channel's subunits. Here we present solid-state phosphorous NMR studies that resolve the negatively charged lipid phosphatidylglycerol within the non-annular lipid-binding site. Perturbations in chemical shift observed upon the binding of phosphatidylglycerol are indicative of the interaction of positively charged sidechains within the non-annular binding site and the negatively charged lipid headgroup. Site directed mutagenesis studies have attributed these charge interactions to R64 and R89. Functionally the removal of the positive charges from R64 and R89 appears to act synergistically to reduce the probability of channel opening.

Stability and membrane orientation of the fukutin transmembrane domain: a combined multiscale molecular dynamics and circular dichroism study.

The N-terminal domain of fukutin-I has been implicated in the localization of the protein in the endoplasmic reticulum and Golgi Apparatus. It has been proposed to mediate this through its interaction with the thinner lipid bilayers found in these compartments. Here we have employed multiscale molecular dynamics simulations and circular dichroism spectroscopy to explore the structure, stability, and orientation of the short 36-residue N-terminus of fukutin-I (FK1TMD) in lipids with differing tail lengths. Our results show that FK1TMD adopts a stable helical conformation in phosphatidylcholine lipids when oriented with its principal axis perpendicular to the bilayer plane. The stability of the helix is largely insensitive to the lipid tail length, preventing hydrophobic mismatch by virtue of its mobility and ability to tilt within the lipid bilayers. This suggests that changes in FK1TMD tilt in response to bilayer properties may be implicated in the regulation of its trafficking. Coarse-grained simulations of the complex Golgi membrane suggest the N-terminal domain may induce the formation of microdomains in the surrounding membrane through its preferential interaction with 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol 4,5-bisphosphate lipids.

Bilayer lipid membranes from falling droplets.

We describe a system that provides a rapid and simple way of forming suspended lipid bilayers within a microfluidic platform from an aqueous droplet. Bilayer lipid membranes are created in a polymeric device by contacting monolayers formed at a two-phase liquid-liquid interface. Microdroplets, containing membrane proteins, are injected onto an electrode positioned above an aperture machined through a conical cavity that is filled with a lipid-alkane solution. The formation of the BLM depends solely on the device geometry and leads to spontaneous formation of lipid bilayers simply by dispensing droplets of buffer. When an aqueous droplet containing transmembrane proteins or proteoliposomes is injected, straightforward electrophysiology measurements are possible. This method is suitable for incorporation into lab-on-a-chip devices and allows for buffer exchange and electrical measurements.

Binding of anionic lipids to at least three nonannular sites on the potassium channel KcsA is required for channel opening.

In addition to the annular or boundary lipids that surround the transmembrane surface of the potassium channel KcsA from Streptomyces lividans, x-ray crystallographic studies have detected one anionic lipid molecule bound at each protein-protein interface in the homotetrameric structure, at sites referred to as nonannular sites. The binding constant for phosphatidylglycerol at the nonannular sites has been determined using fluorescence quenching methods with a mutant of KcsA lacking the normal three lipid-exposed Trp residues. Binding is weak, with a binding constant of 0.42 +/- 0.06 in units of mol fraction, implying that the nonannular sites will only be approximately 70% occupied in bilayers of 100% phosphatidylglycerol. However, the nonannular sites show high selectivity for anionic lipids over zwitterionic lipids, and it is suggested that a change in packing at the protein-protein interface leads to a closing of the nonannular binding site in the unbound state. Increasing the anionic lipid content of the membrane leads to a large increase in open channel probability, from approximately 2.5% in the presence of 25 mol % phosphatidylglycerol to approximately 62% in 100 mol % phosphatidylglycerol. The relationship between open channel probability and phosphatidylglycerol content shows cooperativity. The data are consistent with a model in which three or four of the four nonannular sites in the KcsA homotetramer have to be occupied by anionic lipid for the channel to open. The conductance of the open channel increases with increasing concentration of anionic lipid, an effect possibly due to effects of anionic lipid on the concentration of K(+) close to the membrane surface.

Controlled delivery of proteins into bilayer lipid membranes on chip.

The study and the exploitation of membrane proteins for drug screening applications requires a controllable and reliable method for their delivery into an artificial suspended membrane platform based on lab-on-a-chip technology. In this work, a polymeric device for forming lipid bilayers suitable for electrophysiology studies and biosensor applications is presented. The chip supports a single bilayer and is configured for controlled protein delivery through on-chip microfluidics. In order to demonstrate the principle of protein delivery, the potassium channel KcsA was reconstituted into proteoliposomes, which were then fused with the suspended bilayer on-chip. Fusion of single proteoliposomes with the membrane was identified electrically. Single channel conductance measurements of KcsA in the on-chip bilayer were recorded and these were compared to previously published data obtained with a conventional planar bilayer system.

Enhanced imaging of lipid rich nanoparticles embedded in methylcellulose films for transmission electron microscopy using mixtures of heavy metals.

Synthetic and naturally occurring lipid-rich nanoparticles are of wide ranging importance in biomedicine. They include liposomes, bicelles, nanodiscs, exosomes and virus particles. The quantitative study of these particles requires methods for high-resolution visualization of the whole population. One powerful imaging method is cryo-EM of vitrified samples, but this is technically demanding, requires specialized equipment, provides low contrast and does not reveal all particles present in a population. Another approach is classical negative stain-EM, which is more accessible but is difficult to standardize for larger lipidic structures, which are prone to artifacts of structure collapse and contrast variability. A third method uses embedment in methylcellulose films containing uranyl acetate as a contrasting agent. Methylcellulose embedment has been widely used for contrasting and supporting cryosections but only sporadically for visualizing lipid rich vesicular structures such as endosomes and exosomes. Here we present a simple methylcellulose-based method for routine and comprehensive visualization of synthetic lipid rich nanoparticles preparations, such as liposomes, bicelles and nanodiscs. It combines a novel double-staining mixture of uranyl acetate (UA) and tungsten-based electron stains (namely phosphotungstic acid (PTA) or sodium silicotungstate (STA)) with methylcellulose embedment. While the methylcellulose supports the delicate lipid structures during drying, the addition of PTA or STA to UA provides significant enhancement in lipid structure display and contrast as compared to UA alone. This double staining method should aid routine structural evaluation and quantification of lipid rich nanoparticles structures.

Calcium release from ryanodine receptors in the nucleoplasmic reticulum.

Ca(2+) signals control DNA synthesis and repair, gene transcription, and other cell functions that occur within the nucleus. The nuclear envelope can store Ca(2+) and release it into the nucleus via either the inositol 1,4,5-trisphosphate receptor (InsP3R) or the ryanodine receptor (RyR). Furthermore, many cell types have a reticular network within their nuclei and InsP3Rs on this nucleoplasmic reticulum permit local subnuclear control of Ca(2+) signals and Ca(2+)-dependent intranuclear events. However, it is unknown whether RyR similarly is expressed on the nucleoplasmic reticulum and can control subnuclear Ca(2+) signals. Here we report that the type 1 RyR is expressed on intranuclear extensions of the sarcoplasmic reticulum of C2C12 cells, a skeletal muscle derived cell line. In addition, two-photon photorelease of caged Ca(2+) in the region of the nucleoplasmic reticulum evoked Ca(2+)-induced Ca(2+) release (CICR) within the nucleus, which could be suppressed by the RyR inhibitor dantrolene. These results show that intranuclear extensions of the nuclear envelope have functional RyR and provide a possible mechanism whereby cells expressing RyR can regulate Ca(2+) signals in discrete regions within the nucleus.

Nanoparticle suspensions enclosed in methylcellulose: a new approach for quantifying nanoparticles in transmission electron microscopy.

Nanoparticles are of increasing importance in biomedicine but quantification is problematic because current methods depend on indirect measurements at low resolution. Here we describe a new high-resolution method for measuring and quantifying nanoparticles in suspension. It involves premixing nanoparticles in a hydrophilic support medium (methylcellulose) before introducing heavy metal stains for visualization in small air-dried droplets by transmission electron microscopy (TEM). The use of methylcellulose avoids artifacts of conventional negative stain-TEM by (1) restricting interactions between the nanoparticles, (2) inhibiting binding to the specimen support films and (3) reducing compression after drying. Methylcellulose embedment provides effective electron imaging of liposomes, nanodiscs and viruses as well as comprehensive visualization of nanoparticle populations in droplets of known size. These qualities facilitate unbiased sampling, rapid size measurement and estimation of nanoparticle numbers by means of ratio counting using a colloidal gold calibrant. Specimen preparation and quantification take minutes and require a few microliters of sample using only basic laboratory equipment and a standard TEM.

The role of lipids in mechanosensation.

The ability of proteins to sense membrane tension is pervasive in biology. A higher-resolution structure of the Escherichia coli small-conductance mechanosensitive channel MscS identifies alkyl chains inside pockets formed by the transmembrane helices (TMs). Purified MscS contains E. coli lipids, and fluorescence quenching demonstrates that phospholipid acyl chains exchange between bilayer and TM pockets. Molecular dynamics and biophysical analyses show that the volume of the pockets and thus the number of lipid acyl chains within them decreases upon channel opening. Phospholipids with one acyl chain per head group (lysolipids) displace normal phospholipids (with two acyl chains) from MscS pockets and trigger channel opening. We propose that the extent of acyl-chain interdigitation in these pockets determines the conformation of MscS. When interdigitation is perturbed by increased membrane tension or by lysolipids, the closed state becomes unstable, and the channel gates.

Fluorescence quenching methods to study lipid-protein interactions.

This unit describes how fluorescence quenching methods can be used to determine binding constants for phospholipids binding to intrinsic membrane proteins. Reconstitution of a Trp-containing intrinsic membrane protein with bromine-containing phospholipids leads to quenching of the Trp fluorescence of the protein; the extent of quenching depends on the strength of binding of the phospholipid to the protein. Protocols are included for the synthesis of bromine-containing phospholipids from phospholipids containing carbon-carbon double bonds in their fatty acyl chains and for the reconstitution of membrane proteins into bilayers containing bromine-containing phospholipids. Details are included on data analysis, including equations and software that can be used for fitting the fluorescence quenching data.

The interfacial lipid binding site on the potassium channel KcsA is specific for anionic phospholipids.

Lipid binding to the potassium channel KcsA from Streptomyces lividans has been studied using quenching of the fluorescence of Trp residues by brominated phospholipids. It is shown that binding of phospholipids to nonannular lipid binding sites on KcsA, located one each at the four protein-protein interfaces in the tetrameric structure, is specific for anionic phospholipids, zwitterionic phosphatidylcholine being unable to bind at the sites. The binding constant for phosphatidylglycerol of 3.0 +/- 0.7 mol fraction(-1) means that in a membrane containing approximately 20 mol% phosphatidylglycerol, as in the Escherichia coli inner membrane, the nonannular sites will be approximately 37% occupied by phosphatidylglycerol. The binding constant for phosphatidic acid is similar to that for phosphatidylglycerol but binding constants for phosphatidylserine and cardiolipin are about double those for phosphatidylglycerol. Binding to annular sites around the circumference of the KcsA tetramer are different on the extracellular and intracellular faces of the membrane. On the extracellular face of the membrane the binding constants for anionic lipids are similar to those for phosphatidylcholine, the lack of specificity being consistent with the lack of any marked clusters of charged residues on KcsA close to the membrane on the extracellular side. In contrast, binding to annular sites on the intracellular side of the membrane shows a distinct structural specificity, with binding of phosphatidic acid and phosphatidylglycerol being stronger than binding of phosphatidylcholine, whereas binding constants for phosphatidylserine and cardiolipin are similar to that for phosphatidylcholine. It is suggested that this pattern of binding follows from the pattern of charge distribution on KcsA on the intracellular side of the membrane.