Small-Molecule Analysis Based on DNA Strand Displacement Using a Bacteriorhodopsin Photoelectric Transducer: Taking ATP as an Example.

A uniformly oriented purple membrane (PM) monolayer containing photoactive bacteriorhodopsin has recently been applied as a sensitive photoelectric transducer to assay color proteins and microbes quantitatively. This study extends its application to detecting small molecules, using adenosine triphosphate (ATP) as an example. A reverse detection method is used, which employs AuNPs labeling and specific DNA strand displacement. A PM monolayer-coated electrode is first covalently conjugated with an ATP-specific nucleic acid aptamer and then hybridized with another gold nanoparticle-labeled nucleic acid strand with a sequence that is partially complementary to the ATP aptamer, in order to significantly minimize the photocurrent that is generated by the PM. The resulting ATP-sensing chip restores its photocurrent production in the presence of ATP, and the photocurrent recovers more effectively as the ATP concentration increases. Direct and single-step ATP detection is achieved in 15 min, with detection limits of 5 nM and a dynamic range of 5 nM-0.1 mM. The sensing chip exhibits high selectivity against other ATP analogs and is satisfactorily stable in storage. The ATP-sensing chip is used to assay bacterial populations and achieves a detection limit for Bacillus subtilis and Escherichia coli of 102 and 103 CFU/mL, respectively. The demonstration shows that a variety of small molecules can be simultaneously quantified using PM-based biosensors.

Benchmark and performance of long-range corrected time-dependent density functional tight binding (LC-TD-DFTB) on rhodopsins and light-harvesting complexes.

The chromophores of rhodopsins (Rh) and light-harvesting (LH) complexes still represent a major challenge for a quantum chemical description due to their size and complex electronic structure. Since gradient corrected and hybrid density functional approaches have been shown to fail for these systems, only range-separated functionals seem to be a promising alternative to the more time consuming post-Hartree-Fock approaches. For extended sampling of optical properties, however, even more approximate approaches are required. Recently, a long-range corrected (LC) functional has been implemented into the efficient density functional tight binding (DFTB) method, allowing to sample the excited states properties of chromophores embedded into proteins using quantum mechanical/molecular mechanical (QM/MM) with the time-dependent (TD) DFTB approach. In the present study, we assess the accuracy of LC-TD-DFT and LC-TD-DFTB for rhodopsins (bacteriorhodopsin (bR) and pharaonis phoborhodopsin (ppR)) and LH complexes (light-harvesting complex II (LH2) and Fenna-Matthews-Olson (FMO) complex). This benchmark study shows the improved description of the color tuning parameters compared to standard DFT functionals. In general, LC-TD-DFTB can exhibit a similar performance as the corresponding LC functionals, allowing a reliable description of excited states properties at significantly reduced cost. The two chromophores investigated here pose complementary challenges: while huge sensitivity to external field perturbation (color tuning) and charge transfer excitations are characteristic for the retinal chromophore, the multi-chromophoric character of the LH complexes emphasizes a correct description of inter-chromophore couplings, giving less importance to color tuning. None of the investigated functionals masters both systems simultaneously with satisfactory accuracy. LC-TD-DFTB, at the current stage, although showing a systematic improvement compared to TD-DFTB cannot be recommended for studying color tuning in retinal proteins, similar to some of the LC-DFT functionals, because the response to external fields is still too weak. For sampling of LH-spectra, however, LC-TD-DFTB is a viable tool, allowing to efficiently sample absorption energies, as shown for three different LH complexes. As the calculations indicate, geometry optimization may overestimate the importance of local minima, which may be averaged over when using trajectories. Fast quantum chemical approaches therefore may allow for a direct sampling of spectra in the near future.

Improved production of bacteriorhodopsin from Halobacterium salinarum through direct amino acid supplement in the basal medium.

Enhanced production and growth of Halobacterium salinarum are achieved by direct supplement of essential amino acids in the modified nutrient culture medium. As arginine (R) and aspartate (D) are the main amino acid sources for producing bacteriorhodopsin efficiently from Halobacterium salinarum, both individual and combined effects of these two amino acids (in different compositions) in the basal medium were studied. The BR production is enhanced by 83% on the eighth day (saturated) for all individual and combined amino acid supplements. Maximum production of 201 mg/l is observed for combined amino acid (R3D2)-supplemented culture which is 4.46-fold higher than the conventional culture growth from the basal medium. The obtained results suggest the efficient method to enhance BR production at low cost and thus, open up the possibility to utilize this potential biomolecule for various photonics applications which were earlier restricted due to the high cost of protein molecules.

Combination of bacteriolytic therapy with magnetic field for Ehrlich tumour treatment.

Referable to the limited response of the current available cancer treatment modalities, new effective cancer fighting treatments are needed. This work investigates the efficiency of intratumoural injection of Pseudomonas aeruginosa bacteria combined with a local tumour exposure to extremely low frequency square pulsed magnetic field (ELF SPMF) in the mouse Ehrlich tumour. 64 Ehrlich ascites tumour-implanted female albino BALB/C mice were equally split up into 4 groups. Group 1 (GP1) was the positive control group. Group 2 (GP2) received a single intratumoural injection of P. aeruginosa bacteria. Group 3 (GP3) was exposed to ELF SPMF for tumour local exposure. Group 4 (GP4) was treated with P. aeruginosa intratumoural injection followed by local exposure of the tumour to ELF SPMF. Treatment monitoring was evaluated using ultrastructural examination, flow cytometry analysis in addition to the measurement of tumour dielectric properties. Tumour cell apoptosis was obvious in GP2 and GP4, but, with higher severity and percentages in GP2. Tumour biophysical properties revealed a significant increase in static conductivity σS of GP2, and decreases in dielectric increment Δɛ´of both GP2 and GP4 compared to the GP1. Unfortunately, GP2 mice showed severe signs of toxicity. We advocate the utilization of the combination of P. aeruginosa and SPMF to yield the most effective antitumour agent with less bacteria-related toxicity.

Spin-Dependent Transport through Chiral Molecules Studied by Spin-Dependent Electrochemistry.

Molecular spintronics (spin + electronics), which aims to exploit both the spin degree of freedom and the electron charge in molecular devices, has recently received massive attention. Our recent experiments on molecular spintronics employ chiral molecules which have the unexpected property of acting as spin filters, by way of an effect we call "chiral-induced spin selectivity" (CISS). In this Account, we discuss new types of spin-dependent electrochemistry measurements and their use to probe the spin-dependent charge transport properties of nonmagnetic chiral conductive polymers and biomolecules, such as oligopeptides, L/D cysteine, cytochrome c, bacteriorhodopsin (bR), and oligopeptide-CdSe nanoparticles (NPs) hybrid structures. Spin-dependent electrochemical measurements were carried out by employing ferromagnetic electrodes modified with chiral molecules used as the working electrode. Redox probes were used either in solution or when directly attached to the ferromagnetic electrodes. During the electrochemical measurements, the ferromagnetic electrode was magnetized either with its magnetic moment pointing "UP" or "DOWN" using a permanent magnet (H = 0.5 T), placed underneath the chemically modified ferromagnetic electrodes. The spin polarization of the current was found to be in the range of 5-30%, even in the case of small chiral molecules. Chiral films of the l- and d-cysteine tethered with a redox-active dye, toludin blue O, show spin polarizarion that depends on the chirality. Because the nickel electrodes are susceptible to corrosion, we explored the effect of coating them with a thin gold overlayer. The effect of the gold layer on the spin polarization of the electrons ejected from the electrode was investigated. In addition, the role of the structure of the protein on the spin selective transport was also studied as a function of bias voltage and the effect of protein denaturation was revealed. In addition to "dark" measurements, we also describe photoelectrochemical measurements in which light is used to affect the spin selective electron transport through the chiral molecules. We describe how the excitation of a chromophore (such as CdSe nanoparticles), which is attached to a chiral working electrode, can flip the preferred spin orientation of the photocurrent, when measured under the identical conditions. Thus, chirality-induced spin polarization, when combined with light and magnetic field effects, opens new avenues for the study of the spin transport properties of chiral molecules and biomolecules and for creating new types of spintronic devices in which light and molecular chirality provide new functions and properties.

Exploring membrane protein structural features by oxidative labeling and mass spectrometry.

Despite their biological importance, the structural characterization of integral membrane proteins (IMPs) by x-ray crystallography and NMR spectroscopy remains challenging. Hence, there is a need for complementary approaches that are capable of probing IMP conformational features in a robust fashion. Covalent labeling relies on the principle that solvent accessible regions can be modified by reactive species, whereas buried segments are protected. The readout of the labeling pattern is conducted by mass spectrometry. Hydroxyl radical (·OH) introduces oxidative modifications at amino acid side chains. In this article, the authors discuss the application of ·OH labeling for the structural interrogation of IMPs. Kyte-Doolittle hydropathy analyses are widely used for generating IMP topology models. The validation of these models by mutational techniques is labor intensive. ·OH labeling can readily distinguish transmembrane elements from solvent-exposed loops, thereby providing an alternative topology validation tool. For IMPs with published crystal structures, oxidative modifications can report on functionally relevant dynamic features that are invisible in the static x-ray data. The coupling of pulsed ·OH labeling with rapid mixing techniques represents a novel approach for studying IMP folding kinetics. In conclusion, ·OH labeling is a versatile tool that can provide insights into the structure and dynamics of IMPs.

Efficient production and purification of functional bacteriorhodopsin with a wheat-germ cell-free system and a combination of Fos-choline and CHAPS detergents.

Cell-free translation is one potential approach to the production of functional transmembrane proteins. We have now examined various detergents as supplements to a wheat-germ cell-free system in order to optimize the production and subsequent purification of a functional model transmembrane protein, bacteriorhodopsin. We found that Fos-choline and CHAPS detergents counteracted each other's inhibitory effects on cell-free translation activity and thereby allowed the efficient production and subsequent purification of functional bacteriorhodopsin in high yield.

Cell-free expression of soluble and membrane proteins in an array device for drug screening.

Enzymes and membrane protein receptors represent almost three-quarters of all current drug targets. As a result, it would be beneficial to have a platform to produce them in a high-throughput format for drug screening. We have developed a miniaturized fluid array device for cell-free protein synthesis, and the device was exploited to produce both soluble and membrane proteins. Two membrane-associated proteins, bacteriorhodopsin and ApoA lipoprotein, were coexpressed in an expression medium in the presence of lipids. Simultaneous expression of ApoA lipoprotein enhanced the solubility of bacteriorhodopsin and would facilitate functional studies. In addition, the device was employed to produce two enzymes, luciferase and beta-lactamase, both of which were demonstrated to be compatible with enzyme inhibition assays. Beta-lactamase, a drug target associated with antibiotic resistance, was further used to show the capability of the device for drug screening. Beta-lactamase was synthesized in the 96 units of the device and then assayed by a range of concentrations of four mock drug compounds without harvesting and purification. The inhibitory effects of these compounds on beta-lactamase were measured in a parallel format, and the degree in their drug effectiveness agreed well with the data in the literature. This work demonstrated the feasibility of the use of the fluid array device and cell-free protein expression for drug screening, with advantages in less reagent consumption, shorter analysis time, and higher throughput.

Mass spectrometry combined with oxidative labeling for exploring protein structure and folding.

This review discusses various mass spectrometry (MS)-based approaches for exploring structural aspects of proteins in solution. Electrospray ionization (ESI)-MS, in particular, has found fascinating applications in this area. For example, when used in conjunction with solution-phase hydrogen/deuterium exchange (HDX), ESI-MS is a highly sensitive tool for probing conformational dynamics. The main focus of this article is a technique that is complementary to HDX, that is, the covalent labeling of proteins by hydroxyl radicals. The reactivity of individual amino acid side chains with *OH is strongly affected by their degree of solvent exposure. Thus, analysis of the oxidative labeling pattern by peptide mapping and tandem mass spectrometry provides detailed structural information. A convenient method for *OH production is the photolysis of H(2)O(2) by a pulsed UV laser, resulting in oxidative labeling on the microsecond time scale. Selected examples demonstrate the use of this technique for structural studies on membrane proteins, and the combination with rapid mixing devices for characterizing the properties of short-lived protein (un)folding intermediates.

Production of functional bacteriorhodopsin by an Escherichia coli cell-free protein synthesis system supplemented with steroid detergent and lipid.

Cell-free expression has become a highly promising tool for the efficient production of membrane proteins. In this study, we used a dialysis-based Escherichia coli cell-free system for the production of a membrane protein actively integrated into liposomes. The membrane protein was the light-driven proton pump bacteriorhodopsin, consisting of seven transmembrane alpha-helices. The cell-free expression system in the dialysis mode was supplemented with a combination of a detergent and a natural lipid, phosphatidylcholine from egg yolk, in only the reaction mixture. By examining a variety of detergents, we found that the combination of a steroid detergent (digitonin, cholate, or CHAPS) and egg phosphatidylcholine yielded a large amount (0.3-0.7 mg/mL reaction mixture) of the fully functional bacteriorhodopsin. We also analyzed the process of functional expression in our system. The synthesized polypeptide was well protected from aggregation by the detergent-lipid mixed micelles and/or lipid disks, and was integrated into liposomes upon detergent removal by dialysis. This approach might be useful for the high yield production of functional membrane proteins.

Channelrhodopsin-1: a light-gated proton channel in green algae.

Phototaxis and photophobic responses of green algae are mediated by rhodopsins with microbial-type chromophores. We report a complementary DNA sequence in the green alga Chlamydomonas reinhardtii that encodes a microbial opsin-related protein, which we term Channelopsin-1. The hydrophobic core region of the protein shows homology to the light-activated proton pump bacteriorhodopsin. Expression of Channelopsin-1, or only the hydrophobic core, in Xenopus laevis oocytes in the presence of all-trans retinal produces a light-gated conductance that shows characteristics of a channel selectively permeable for protons. We suggest that Channelrhodopsins are involved in phototaxis of green algae.

Characterization of acquired resistance in lesion-mimic transgenic potato expressing bacterio-opsin.

The lesion-mimic mutants of certain plants display necrotic lesions resembling those of the hypersensitive response and activate local and systemic defense responses in the absence of pathogens. We have engineered a lesion-mimic phenotype in transgenic Russet Burbank potato plants through constitutive expression of a bacterio-opsin (bO) proton pump derived from Halobacterium halobium. Transgenic potato plants exhibiting a lesion-mimic phenotype had increased levels of salicylic acid and overexpressed several pathogenesis-related messenger RNAs, all hallmarks of systemic acquired resistance (SAR). The lesion-mimic plants also displayed enhanced resistance to the US1 isolate (A1 mating type) of a fungal pathogen, Phytophthora infestans, a causal agent of late blight disease. In contrast, little resistance was observed against the US8 isolate (A2 mating type) of this pathogen. Furthermore, a majority of the transgenic plants displaying the lesion-mimic phenotype had increased susceptibility to potato virus X. The tubers of these plants were not resistant to the bacterial pathogen Erwinia carotovora. These results indicate that expression of bO can result in the activation of defense responses in transgenic potato plants and show for the first time that bO expression can confer resistance to a pathogenic fungus. However, our results also demonstrate that like SAR, this "engineered" resistance is likely to be limited to certain pathogens and particular cultivars.

Mutation of arginine 134 to lysine alters the pK(a)s of key groups involved in proton pumping by bacteriorhodopsin.

Arginine 134 is located near the extracellular surface of bacteriorhodopsin (bR) and may interact with one or more nearby glutamate residues. In the bR mutant R134K, light-induced Schiff-base deprotonation (formation of the M intermediate) exhibits several kinetic components and has a complex pH dependence. The kinetics and pH dependence of M formation were analyzed using the following general guidelines for interpreting M formation: (1) The fastest component of M formation reflects the redistribution of the Schiff-base proton to D85, the usual proton acceptor, in response to the change in the proton affinities of the Schiff base and D85 early in the photocycle; (2) Two additional components of M formation reflect transitions between spectroscopically similar substates of M. By applying these guidelines, supplemented by information about the pK(a)s of D85 and the proton release group from acid (purple-to-blue) and alkaline titrations of the absorption spectra of the unphotolyzed R134K pigment, we explain the pH dependence of M formation as being due to titration of the counterion, D85, and of the proton release group. We calculate, in R134K, that the pKa of D85 is 4.6 in the unphotolyzed state, while the pKa of the proton release group is 8.0 in the unphotolyzed state but drops to approximately 5.8 in the M intermediate. The same value for the pKa of the proton release group in the M intermediate is obtained when we use photocurrent measurements to monitor proton release. The altered values of these pK(a)s relative to the corresponding values in wild-type bR suggest that D85 and the proton release group are coupled more weakly in R134K than in the wild type.

Bacteriorhodopsin can be refolded from two independently stable transmembrane helices and the complementary five-helix fragment.

This paper describes experimental tests of the hypothesis that bacteriorhodopsin (BR) can fold by the association of independently stable transmembrane helices. Peptides containing the first and second helical segments of BR were chemically synthesized. These two peptides and the complementary five-helix fragment of BR were reconstituted in three separate populations of native-lipid vesicles which were then mixed and fused to allow the fragments to interact. After addition of retinal, absorption spectroscopy of the reconstituted BR and X-ray diffraction of two-dimensional crystals of this material showed that the native structure of BR was regenerated. The first two helices of BR can therefore be considered as independent folding domains, and covalent connections in the loops connecting the helices to each other and to the rest of the molecule are not essential for the appropriate association of the helices.

Effect of bacteriorhodopsin on the orientation of the headgroup of 1,2-dimyristoyl-sn-glycero-3-phosphocholine in bilayers: a 31P- and 2H-NMR study.

Bacteriorhodopsin (BR), purified from the halophilic bacterium, Halobacterium halobium, has been separated from the endogenous purple membrane lipids and reconstituted by detergent dialysis into bilayers of the zwitterionic phospholipid, 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), which was selectively deuterated at the headgroup in the choline alpha- and beta-methylene segments and the choline gamma-methyl groups. Complexes of DMPC/BR contents from 67:1 to 222:1 (mol/mol) were produced under conditions to promote formation of large vesicles (mean diameters 600-700 nm). The magnitudes of the 2H-NMR quadrupole splittings recorded from the deuterium-labelled headgroup segments, and the 31P-NMR chemical shift anisotropy (CSA) of the phosphate group appeared to vary linearly with the BR content in the complexes over the range of DMPC/BR ratios studied. On increasing the proportion of BR in the DMPC-BR complexes, the 2H-NMR quadrupole splittings measured from the choline gamma-methyl groups and the beta-methylene segments and the 31P-NMR CSA increased in magnitude, while the 2H-NMR quadrupole splitting from the alpha-methylene segment decreased. Such opposing changes in the choline alpha- and beta-methylene segment quadrupole splittings are similar to those reported on increasing the proportion of positively charged amphiphile at the bilayer surface (Seelig et al. (1987) Biochemistry 26, 7535-7541). It is suggested that BR presents a net positive charge to the phosphocholine headgroups at the protein/lipid interface.

Monomer-oligomer equilibrium of bacteriorhodopsin in reconstituted proteoliposomes. A freeze-fracture electron microscope study.

An improved freeze-fracture electron microscope procedure has been developed and applied to the study of the association of bacteriorhodopsin in large proteoliposomes reconstituted by reverse-phase evaporation with egg lecithin. Due to the improved accuracy and resolution of this procedure, intramembrane particles, the diameter of which (4.5 nm) closely matched that of bacteriorhodopsin monomer, could be observed at high lipid to protein ratios (greater than or equal to 40 w/w). At lower lipid to protein ratios, larger particles (diameter 7.5 nm) progressively appeared, resulting in bimodal particle size distributions up to a lipid to protein ratio of 1, where the large particles were the sole species present. These large particles were interpreted as corresponding to bacteriorhodopsin oligomers. Because of the large size and homogeneity of proteoliposomes, accurate particle density measurements could be performed. These confirmed the occurrence of a lipid to protein ratio-dependent bacteriorhodopsin monomer-oligomer equilibrium and further allowed us to identify the oligomer as a trimer or a tetramer. In complementary experiments, it was found that the bacteriorhodopsin monomer and oligomer had identical visible CD spectra and light-induced proton pumping rates. However, a large increase of the proton passive leak rate of proteoliposomes was found to be associated with oligomer formation. The appearance of these oligomers may be important as the first step in the formation of two-dimensional crystals of bacteriorhodopsin.

The relationship between the chromophore moiety and the cation binding sites in bacteriorhodopsin.

Bleaching of the purple membrane strongly reduces the number of divalent cation binding sites as well as their affinities. Conversely, deionization of the bleached membrane drastically inhibits the chromophore regeneration. Proteolysis experiments using bromelain show that the bleached membrane has an additional cleavage site probably located at the fifth loop, whereas in the blue membrane, the C-terminal tail is no longer susceptible to proteolysis. It is suggested that there exists a close relationship between the retinal environment and one or more of the cation binding sites.

Characterization of a halobacterial gene affecting bacterio-opsin gene expression.

A substantial number of spontaneous bacterio-opsin mutants of Halobacterium halobium are the result of insertion elements up to 1400 bp upstream of the bacterio-opsin (bop) gene. The nucleotide sequence of 1800 bp upstream of the bop gene has been determined. There is a 1118 bp open reading frame (ORF) located within this region which is transcribed and which coincides with the distribution of insertion elements upstream of the bop gene in Bop mutants. Therefore, we propose that there is a gene (brp gene) 526 bp upstream of the bop gene. This putative gene is transcribed in the opposite direction as the bop gene and could encode a protein of 37,500 D (359 amino acids) with a codon usage similar to bacterio-opsin. The 5' terminus of the brp transcript has been determined. The brp transcript and the bop mRNA are complementary for 13 residues near their 5' termini and both transcripts start at or near the initiating codon of the gene. Both transcripts could form similar hairpin loop structures at their 5' termini which contain possible ribosomal binding sites. The DNA sequences immediately upstream of the bop and the brp genes have significant homologies and there is a short complementary sequence. The role of the brp gene in bacterio-opsin gene expression is unclear.

Origin and behavior of deuteron spin echoes in selectively labeled amino acids, myoglobin microcrystals, and purple membranes.

We have obtained deuterium NMR spin-echo spectra of crystalline DL-[gamma-2H6]valine, [S-methyl-2H3]-methionine, cyanoferrimyoglobin from sperm whale (Physeter catodon), containing deuteriomethyl groups at methionine-55 and methionine-131, and [gamma-2H6]valine-labeled bacteriorhodopsin in the purple membrane of Halobacterium halobium R1. By using 90-tau-beta 90 degrees (XY) and 90-tau-beta 0 degrees (XX) pulse sequences and observing the dependence of the spin-echo amplitude upon the interpulse spacing tau, we have determined that the so-called "quadrupole echoes" obtained in these typical selectively deuterated condensed-phase biological systems are in fact strongly modulated by proton-deuteron and deuteron-deuteron dipolar interactions. The two amino acids and the protein crystals behaved as typical organic solids, with no evidence of "liquid-like" behavior, even in the presence of excess water (in the case of the ferrimyoglobin crystals). However, with the valine-labeled bacteriorhodopsin, the tau-dependence of XY echoes as a function of temperature emphasized the "solid-like" behavior of the membrane "matrix", while the basic nature of the spin-echo response for the narrow central component of the spectrum clearly indicated the very "fluid" or "mobile" nature of a series of residues that are shown elsewhere [Keniry, M., Gutowsky, H. S., & Oldfield, E. (1984) Nature (London) 307, 383-386] to arise from the membrane surface. Our results thus suggest that such NMR methods may yield useful information on side-chain dynamics complementary to that of line-shape and spin-lattice relaxation time analyses.

Charge asymmetry of the purple membrane measured by uranyl quenching of dansyl fluorescence.

Purple membrane was covalently labeled with 5-(dimethylamino) naphthalene-1-sulfonyl hydrazine (dansyl hydrazine) by carbodiimide coupling to the cytoplasmic surface (carboxyl-terminal tail: 0.7 mol/mol bacteriorhodopsin) or by periodate oxidation and dimethylaminoborane reduction at the extracellular surface (glycolipids: 1 mol/mol). In 2 mM acetate buffer, pH 5.6, micromolar concentrations of UO2 +(2) were found to quench the dansyl groups on the cytoplasmic surface (maximum = 26%), while little quenching was observed at the extracellular surface (maximum = 4%). Uranyl ion quenched dansyl hydrazine in free solution at much higher concentrations. Uranyl also bound tightly to unmodified purple membrane, (apparent dissociation constant = 0.8 microM) as measured by a centrifugation assay. The maximum stoichiometry was 10 mol/mol of bacteriorhodopsin, which is close to the amount of phospholipid phosphorus in purple membrane. The results were analyzed on the assumptions that UO2 +(2) binds in a 1:1 complex with phospholipid phosphate and that the dansyl distribution and quenching mechanisms are the same at both surfaces. This indicates a 9:1 ratio of phosphate between the cytoplasmic and extracellular surfaces. Thus, the surface change density of the cytoplasmic side of the membrane is more negative than -0.010 charges/A2.