On-Orbit Performance of the Helioseismic and Magnetic Imager Instrument onboard the Solar Dynamics Observatory.

The Helioseismic and Magnetic Imager (HMI) instrument is a major component of NASA's Solar Dynamics Observatory (SDO) spacecraft. Since commencement of full regular science operations on 1 May 2010, HMI has operated with remarkable continuity, e.g. during the more than five years of the SDO prime mission that ended 30 September 2015, HMI collected 98.4% of all possible 45-second velocity maps; minimizing gaps in these full-disk Dopplergrams is crucial for helioseismology. HMI velocity, intensity, and magnetic-field measurements are used in numerous investigations, so understanding the quality of the data is important. This article describes the calibration measurements used to track the performance of the HMI instrument, and it details trends in important instrument parameters during the prime mission. Regular calibration sequences provide information used to improve and update the calibration of HMI data. The set-point temperature of the instrument front window and optical bench is adjusted regularly to maintain instrument focus, and changes in the temperature-control scheme have been made to improve stability in the observable quantities. The exposure time has been changed to compensate for a 20% decrease in instrument throughput. Measurements of the performance of the shutter and tuning mechanisms show that they are aging as expected and continue to perform according to specification. Parameters of the tunable optical-filter elements are regularly adjusted to account for drifts in the central wavelength. Frequent measurements of changing CCD-camera characteristics, such as gain and flat field, are used to calibrate the observations. Infrequent expected events such as eclipses, transits, and spacecraft off-points interrupt regular instrument operations and provide the opportunity to perform additional calibration. Onboard instrument anomalies are rare and seem to occur quite uniformly in time. The instrument continues to perform very well.

Interaction of synthetic HA2 influenza fusion peptide analog with model membranes.

The interaction of the synthetic 21 amino acid peptide (AcE4K) with 1-oleoyl-2-[caproyl-7-NBD]-sn-glycero-3-phosphocholine membranes is used as a model system for the pH-sensitive binding of fusion peptides to membranes. The sequence of AcE4K (Ac-GLFEAIAGFIENGWEGMIDGK) is based on the sequence of the hemagglutinin HA2 fusion peptide and has similar partitioning into phosphatidylcholine membranes as the viral peptide. pH-dependent partitioning in the membrane, circular dichroism, tryptophan fluorescence, change of membrane area, and membrane strength, are measured to characterize various key aspects of the peptide-membrane interaction. The experimental results show that the partitioning of AcE4K in the membrane is pH dependent. The bound peptide inserts in the membrane, which increases the overall membrane area in a pH-dependent manner, however the depth of insertion of the peptide in the membrane is independent of pH. This result suggests that the binding of the peptide to the membrane is driven by the protonation of its three glutamatic acids and the aspartic acid, which results in an increase of the number of bound molecules as the pH decreases from pH 7 to 4.5. The transition between the bound state and the free state is characterized by the Gibbs energy for peptide binding. This Gibbs energy for pH 5 is equal to -30.2 kJ/mol (-7.2 kcal/mol). Most of the change of the Gibbs energy during the binding of AcE4K is due to the enthalpy of binding -27.3 kJ/mol (-6.5 kcal/mol), while the entropy change is relatively small and is on the order of 6.4 J/mol.K (2.3 cal/mol.K). The energy barrier separating the bound and the free state, is characterized by the Gibbs energy of the transition state for peptide adsorption. This Gibbs energy is equal to 51.3 kJ/mol (12.3 kcal/mol). The insertion of the peptide into the membrane is coupled with work for creation of a vacancy for the peptide in the membrane. This work is calculated from the measured area occupied by a single peptide molecule (220 A(2)) and the membrane elasticity (190 mN/m), and is equal to 15.5 kJ/mol (3.7 kcal/mol). The comparison of the work for creating a vacancy and the Gibbs energy of the transition state shows that the work for creating a vacancy may have significant effect on the rate of peptide insertion and therefore plays an important role in peptide binding. Because the work for creating a vacancy depends on membrane elasticity and the elasticity of the membrane is dependent on membrane composition, this provides a tool for modulating the pH for membrane instability by changing membrane composition. The insertion of the peptide in the membrane does not affect the membrane permeability for water, which shows that the peptide does not perturb substantially the packing of the hydrocarbon region. However, the ability of the membrane to retain solutes in the presence of peptide is compromised, suggesting that the inserted peptide promotes formation of short living pores. The integrity of the membrane is substantially compromised below pH 4.8 (threshold pH), when large pores are formed and the membrane breaks down. The binding of the peptide in the pore region is reversible, and the pore size varies on the experimental conditions, which suggests that the peptide in the pore region does not form oligomers.

Efficient encapsulation of antisense oligonucleotides in lipid vesicles using ionizable aminolipids: formation of novel small multilamellar vesicle structures.

Typical methods used for encapsulating antisense oligodeoxynucleotides (ODN) and plasmid DNA in lipid vesicles result in very low encapsulation efficiencies or employ cationic lipids that exhibit unfavorable pharmacokinetic and toxicity characteristics when administered intravenously. In this study, we describe and characterize a novel formulation process that utilizes an ionizable aminolipid (1,2-dioleoyl-3-dimethylammonium propane, DODAP) and an ethanol-containing buffer system for encapsulating large quantities (0.15--0.25 g ODN/g lipid) of polyanionic ODN in lipid vesicles. This process requires the presence of up to 40% ethanol (v/v) and initial formulation at acidic pH values where the DODAP is positively charged. In addition, the presence of a poly(ethylene glycol)-lipid was required during the formulation process to prevent aggregation. The 'stabilized antisense-lipid particles' (SALP) formed are stable on adjustment of the external pH to neutral pH values and the formulation process allows encapsulation efficiencies of up to 70%. ODN encapsulation was confirmed by nuclease protection assays and (31)P NMR measurements. Cryo-electron microscopy indicated that the final particles consisted of a mixed population of unilamellar and small multilamellar vesicles (80--140 nm diameter), the relative proportion of which was dependent on the initial ODN to lipid ratio. Finally, SALP exhibited significantly enhanced circulation lifetimes in mice relative to free antisense ODN, cationic lipid/ODN complexes and SALP prepared with quaternary aminolipids. Given the small particle sizes and improved encapsulation efficiency, ODN to lipid ratios, and circulation times of this formulation compared to others, we believe SALP represent a viable candidate for systemic applications involving nucleic acid therapeutics.

Spontaneous entrapment of polynucleotides upon electrostatic interaction with ethanol-destabilized cationic liposomes.

This study describes the effect of ethanol and the presence of poly(ethylene) glycol (PEG) lipids on the interaction of nucleotide-based polyelectrolytes with cationic liposomes. It is shown that preformed large unilamellar vesicles (LUVs) containing a cationic lipid and a PEG coating can be induced to entrap polynucleotides such as antisense oligonucleotides and plasmid DNA in the presence of ethanol. The interaction of the cationic liposomes with the polynucleotides leads to the formation of multilamellar liposomes ranging in size from 70 to 120 nm, only slightly bigger than the parent LUVs from which they originated. The degree of lamellarity as well as the size and polydispersity of the liposomes formed increases with increasing polynucleotide-to-lipid ratio. A direct correlation between the entrapment efficiency and the membrane-destabilizing effect of ethanol was observed. Although the morphology of the liposomes is still preserved at the ethanol concentrations used for entrapment (25-40%, v/v), entrapped low-molecular-weight solutes leak rapidly. In addition, lipids can flip-flop across the membrane and exchange rapidly between liposomes. Furthermore, there are indications that the interaction of the polynucleotides with the cationic liposomes in ethanol leads to formation of polynucleotide-cationic lipid domains, which act as adhesion points between liposomes. It is suggested that the spreading of this contact area leads to expulsion of PEG-ceramide and triggers processes that result in the formation of multilamellar systems with internalized polynucleotides. The high entrapment efficiencies achieved at high polyelectrolyte-to-lipid ratios and the small size and neutral character of these novel liposomal systems are of utility for liposomal delivery of macromolecular drugs.

Stabilized plasmid-lipid particles for systemic gene therapy.

The structure of 'stabilized plasmid-lipid particles' (SPLP) and their properties as systemic gene therapy vectors has been investigated. We show that SPLP can be visualized employing cryo-electron microscopy to be homogeneous particles of diameter 72 +/- 5 nm consisting of a lipid bilayer surrounding a core of plasmid DNA. It is also shown that SPLP exhibit long circulation lifetimes (circulation half-life >6 h) following intravenous (i.v.) injection in a murine tumor model resulting in accumulation of up to 3% of the total injected dose and concomitant reporter gene expression at a distal (hind flank) tumor site. In contrast, i v. injection of naked plasmid DNA or plasmid DNA-cationic liposome complexes did not result in significant plasmid delivery to the tumor site or gene expression at that site. Furthermore, it is shown that high doses of SPLP corresponding to 175 microg plasmid per mouse are nontoxic as assayed by monitoring serum enzyme levels, whereas i.v. injection of complexes give rise to significant toxicity at dose levels above 20 microg plasmid per mouse. It is concluded that SPLP exhibit properties consistent with potential utility as a nontoxic systemic gene therapy vector.

Rossby waves on the Sun as revealed by solar 'hills'

It is a long-standing puzzle that the Sun's photosphere--its visible surface--rotates differentially, with the equatorial regions rotating faster than the poles. It has been suggested that waves analogous to terrestrial Rossby waves, and known as r-mode oscillations, could explain the Sun's differential rotation: Rossby waves are seen in the oceans as large-scale (hundreds of kilometres) variations of sea-surface height (5-cm-high waves), which propagate slowly either east or west (they could take tens of years to cross the Pacific Ocean). Calculations show that the solar r-mode oscillations have properties that should be strongly constrained by differential rotation. Here we report the detection of 100-m-high 'hills' in the photosphere, spaced uniformly over the Sun's surface with a spacing of (8.7 +/- 0.6) x 10(4) km. If convection under the photosphere is organized by the r-modes, the observed corrugated photosphere is a probable surface manifestation of these solar oscillations.

Encapsulation of plasmid DNA in stabilized plasmid-lipid particles composed of different cationic lipid concentration for optimal transfection activity.

In previous work (Wheeler et al. (1999) Gene Therapy 6, 271-281) we have shown that plasmid DNA can be entrapped in "stabilized plasmid lipid particles" (SPLP) using low levels (5-10 mol%) of cationic lipid, the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), and a polyethyleneglycol (PEG) coating for stabilization. The PEG moieties are attached to a ceramide anchor containing an arachidoyl acyl group (PEG-CerC20). However, these SPLP exhibit low transfection potencies in vitro as compared to plasmid/cationic lipid complexes formed with liposomes composed of cationic and neutral lipid at a 1:1 lipid ratio. The objective of this study was to construct SPLPs with increased cationic lipid contents that result in maximum transfection levels. A phosphate buffer detergent dialysis technique is described resulting in formation of SPLP containing 7-42.5 mol% DODAC with reproducible encapsulation efficiency of up to 80%. An octanoyl acyl group was used as anchor for the PEG moiety (PEG-CerC8) permitting a quick exchange out of the SPLP to further optimize the in vitro and in vivo transfection. We have demonstrated that this technique can be used to encapsulate either linearized DNA or supercoiled plasmids ranging from 3-20 kb. The SPLP formed could be isolated from empty vesicles by sucrose density gradient centrifugation, and exhibited a narrow size distribution of approximately 75 +/- 6 nm as determined by cryo-electron microscopy. The high plasmid-to-lipid ratio observed corresponded to one plasmid per particle. The SPLP consist of a lipid bilayer surrounding the plasmid DNA as visualized by cryo-electron microscopy. SPLP containing a range of DODAC concentrations were tested for in vitro and in vivo transfection. In vitro, in COS-7 cells transfection reached a maximum after 48 h. The transfection efficiency increased when the DODAC concentration in the SPLP was decreased from 42.5 to 24 mol% DODAC. Decreasing the cationic lipid concentration improved transfection in part due to decreased toxicity. In vivo studies using an intraperitoneal B16 tumor model and intraperitoneal administration of SPLP showed maximum transfection activity for SPLP containing 24 mol% DODAC. Gene expression observed in tumor cells was increased by approximately one magnitude as compared to cationic lipid/DNA complexes. The SPLP were stable and upon storage at 4 degrees C no significant change in the transfection activity was observed over a one-year period. Thus this phosphate buffer detergent dialysis technique can be used to generate SPLP formulations containing a wide range of cationic lipid concentrations to determine optimal SPLP composition for high transfection activity and low toxicity.

Stabilized plasmid-lipid particles: pharmacokinetics and plasmid delivery to distal tumors following intravenous injection.

A previous study has shown that plasmid DNA can be encapsulated in lipid particles (SPLP, "stabilized plasmid lipid particles") of approximately 70 nm diameter composed of 1,2-dioleoyl-3-phosphatidyl-ethanolamine (DOPE), the cationic lipid N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC) and poly(ethylene glycol) conjugated to ceramide (PEG-Cer) using a detergent dialysis process (Wheeler et al. (1999) Gene Therapy 6, 271-281). In this work we evaluated the potential of these SPLPs as systemic gene therapy vectors, determining their pharmacokinetics and the biodistribution of the plasmid and lipid components. It is shown that the blood clearance and the biodistribution of the SPLPs can be modulated by varying the acyl chain length of the ceramide group used as lipid anchor for the PEG polymer. Circulation lifetimes observed for SPLPs with PEG-CerC14 and PEG-CerC20 were t(1/2) = approximately 1 and approximately 10 h, respectively. The SPLPs are stable while circulating in the blood and the encapsulated DNA is fully protected from degradation by serum nucleases. The accelerated clearance of SPLPs with PEG-CerC14 is accompanied by increased accumulation in liver and spleen as compared to PEG-CerC20 SPLPs. Delivery of intact plasmid to liver and spleen was detected. Significant accumulation (approximately 10% of injected dose) of the long circulating SPLPs with PEG-CerC20 in a distal tumor (Lewis lung tumor in the mouse flank) was observed following i.v. application and delivery of intact plasmid to tumor tissue at approximately 6% injected dose/g tissue is demonstrated.

Enhanced anti-inflammatory activity of a liposomal intercellular adhesion molecule-1 antisense oligodeoxynucleotide in an acute model of contact hypersensitivity.

The anti-inflammatory activity of free and liposome-encapsulated oligonucleotide targeted against intercellular adhesion molecule-1 mRNA was investigated in a delayed type hypersensitivity model of acute inflammation in mice. Contact hypersensitivity reactions to 2, 4-dinitrofluorobenzene were monitored by measuring ear thickness and cellular infiltration, both of which were observed to be maximal 24 h after ear challenge. A murine-specific phosphorothioate oligodeoxynucleotide and various control sequences were each passively encapsulated into 100-nm diameter large unilamellar vesicles composed of egg phosphatidylcholine and cholesterol. All formulations were administered as a single-bolus injection into the tail vein approximately 15 min after initiating ear inflammation. Oligodeoxynucleotide dose was varied from 5 to 50 mg/kg and the extent of inflammation was assessed 24 h later. Mice treated with free oligonucleotide, empty vesicles, or encapsulated control sequences showed no measurable effect on ear swelling or cellular infiltration compared with untreated controls. However, mice that received the active sequence encapsulated in lipid vesicles exhibited near baseline levels of ear thickness and leukocyte infiltration, similar to that observed in mice treated with a topical corticosteroid. These data demonstrate the utility of liposome-encapsulated intercellular adhesion molecule-1 antisense oligonucleotide as a novel anti-inflammatory therapeutic.

Gene transfer mediated by fusion protein hemagglutinin reconstituted in cationic lipid vesicles.

Hemagglutinin, the membrane fusion protein of influenza virus, is known to mediate a low-pH-dependent fusion reaction between the viral envelope and the limiting membrane of the endosomal cell compartment following cellular uptake of the virus particles by receptor-mediated endocytosis. Here we exploited this activity of hemagglutinin to achieve efficient gene delivery to cultured cells. Hemagglutinin was reconstituted in the presence of the monocationic lipid dioleoyldimethylammonium chloride (DODAC) to permit plasmid binding to the virosome surface. Virosomes with 30 mol% DODAC exhibited a distinct binding capacity for plasmid without causing aggregation. The virosome fusion activity was not affected by the cationic lipid DODAC as demonstrated by low-pH-dependent lipid mixing with erythrocyte ghosts. Efficient cell transfection of BHK-21 cells was observed with virosomes containing 30 mol% DODAC and plasmid encoding for beta-galactosidase (pCMV beta-gal) associated to their surface. The transfection activity observed was dependent on the functional activity of hemagglutinin. Contrary to DNA/cationic lipid complexes the transfection was not dependent on the cationic lipid to DNA charge ratio. Importantly, transfection of BHK-21 cells with pCMV beta-gal by DODAC-containing virosomes did not show any significant signs of cytotoxicity that is commonly observed with DNA/cationic lipid complexes. Together with the high levels of expression of the transgene this highlights the potential of DODAC-containing virosomes as a novel approach in nonviral gene transfer.

Stabilized plasmid-lipid particles for regional gene therapy: formulation and transfection properties.

Previous work (Wheeler et al, Gene Therapy 1999; 6: 271-281) has shown that plasmid DNA can be entrapped in 'stabilized plasmid-lipid particles' (SPLP) containing the fusogenic lipid dioleoylphosphatidylethanolamine (DOPE), low levels (5-10 mol%) of cationic lipid, and stabilized by a polyethyleneglycol (PEG) coating. The PEG moieties are attached to a ceramide anchor containing an arachidoyl acyl group (PEG-CerC20). These SPLP exhibit low transfection potencies in vitro, due in part to the long residence time of the PEG-CerC20 on the SPLP surface. In this work we employed SPLP stabilized by PEG attached to ceramide containing an octanoyl acyl group (PEG-CerC8), which is able to quickly exchange out of the SPLP, to develop systems that give rise to optimized in vitro and in vivo (regional) transfection. A particular objective was to achieve cationic lipid contents that give rise to maximum transfection levels. It is shown that by performing the dialysis procedure in the presence of increasing concentrations of citrate, SPLP containing up to 30 mol% of the cationic lipid dioleoydimethylammonium chloride (DODAC) could be generated. The SPLP produced could be isolated from empty vesicles by sucrose density gradient centrifugation, and exhibited a narrow size distribution (62 +/- 8 nm, as determined by freeze-fracture electron microscopy) and a high plasmid-to-lipid ratio of 65 microg/micromol (corresponding to one plasmid per particle) regardless of the DODAC content. It was found that isolated SPLP containing 20-24 mol% DODAC resulted in optimum transfection of COS-7 and HepG2 cells in vitro, with luciferase expression levels comparable to those achieved for plasmid DNA-cationic lipid complexes. In vivo studies employing an intraperitoneal B16 tumor model and intraperitoneal administration of SPLP also demonstrated maximum luciferase expression for DODAC contents of 20-24 mol% and significantly improved gene expression in tumor tissue as compared with complexes. We conclude that SPLP stabilized by PEG-CerC8 and containing 20-24 mol% cationic lipid are attractive alternatives to plasmid DNA-cationic lipid complexes for regional gene therapy applications.

Stabilized plasmid-lipid particles: construction and characterization.

A detergent dialysis procedure is described which allows encapsulation of plasmid DNA within a lipid envelope, where the resulting particle is stabilized in aqueous media by the presence of a poly(ethyleneglycol) (PEG) coating. These 'stabilized plasmid-lipid particles' (SPLP) exhibit an average size of 70 nm in diameter, contain one plasmid per particle and fully protect the encapsulated plasmid from digestion by serum nucleases and E. coli DNase I. Encapsulation is a sensitive function of cationic lipid content, with maximum entrapment observed at dioleoyldimethylammonium chloride (DODAC) contents of 5 to 10 mol%. The formulation process results in plasmid-trapping efficiencies of up to 70% and permits inclusion of 'fusigenic' lipids such as dioleoylphosphatidylethanolamine (DOPE). The in vitro transfection capabilities of SPLP are demonstrated to be strongly dependent on the length of the acyl chain contained in the ceramide group used to anchor the PEG polymer to the surface of the SPLP. Shorter acyl chain lengths result in a PEG coating which can dissociate from the SPLP surface, transforming the SPLP from a stable particle to a transfection-competent entity. It is suggested that SPLP may have utility as systemic gene delivery systems for gene therapy protocols.

Contact hypersensitivity: a simple model for the characterization of disease-site targeting by liposomes.

A murine model of delayed-type hypersensitivity (DTH) is characterized with respect to liposome accumulation at a site of inflammation. Mice were sensitized by painting the abdominal region with a solution of 2,4-dinitrofluorobenzene (DNFB) and inflammation was induced 5 days later by challenging the ear with a dilute solution of DNFB. The inflammatory response was readily monitored by measuring ear thickness (edema) and radiolabeled leukocyte infiltration. Maximum ear swelling and cellular infiltration occurred 24 h after the epicutaneous challenge with the ear returning to normal size after approximately 72 h. We demonstrate that large unilamellar vesicles (LUV) accumulate at the site of inflammation to a level more than 20-fold higher than that measured in the untreated ear. Vesicle delivery to the ear correlated with increased vascular leakage resulting from endothelium remodeling in response to DNFB challenge, and was not a consequence of increased local tissue blood volume. Extravasation occurred only during the first 24 h after ear challenge; after this time the permeability of the endothelium to vesicles returned to normal. We further showed that LUV with a diameter of 120 nm exhibit maximum levels of accumulation, that a polyethylene glycol surface coating does not increase delivery, and that the process can be inhibited by the application of topical corticosteroids at the time of induction. These data and the inflammation model are discussed with respect to developing lipid-based drug delivery vehicles designed to accumulate at inflammatory disease sites.

New approach to study of spilled crude oils using high resolution GC-MS (SIM) and metastable reaction monitoring GC-MS-MS.

Polycyclic aromatic hydrocarbons (PAHs) and geochemical biomarkers are good environmental markers to study the origin and evolution of an oil spill. To have access to the greatest number of molecular ratios, no fractionation of oil into aliphatic and aromatic compounds is made. Three analytical MS approaches are tested to analyze markers in this total hydrocarbon fraction: classical quadrupole GC-MS, high resolution GC-MS (HR GC-MS) and metastable reaction monitoring GC-MS-MS (MRM GC-MS-MS). This analytical approach is used to follow the evolution of PAHs in petroleum polluted mangrove soils over 8 years by using molecular ratios between polycyclic aromatic hydrocarbons and tri- and tetracyclic terpanes.

Time-resolved surface charge change on the cytoplasmic side of bacteriorhodopsin.

The pH-sensitive dye 5-iodoacetamidofluorescein was covalently bound to a single cysteine residue introduced by site-directed mutagenesis in position 101 on the cytoplasmic surface or in position 130 on the extracellular surface of the proton pump bacteriorhodopsin. Using time-resolved absorption spectroscopy at 495 nm a transient increase was observed in the apparent pK of the dye attached at residue 101. At pH 7.3 the rise and decay times of this pK-change (approximately 2 ms and approximately 60 ms) correlate well with decay times observed for the M and O intermediates and with the proton uptake time. Interpreting the pK-increase of +0.18 pH-unit in terms of a transiently more negative surface charge density, we calculate a change of -0.80 elementary charge per bacteriorhodopsin at the cytoplasmic surface. It is likely that this charge change is due to the transient deprotonation of aspartate-96. With the label in position 130 on the extracellular surface no transient pK-shift was detected.

Rapid long-range proton diffusion along the surface of the purple membrane and delayed proton transfer into the bulk.

The pH-indicator dye fluorescein was covalently bound to the surface of the purple membrane at position 72 on the extracellular side of bacteriorhopsin and at positions 101, 105, 160, or 231 on the cytoplasmic side by reacting bromomethylfluorescein with the sulfhydryl groups of cysteines introduced by site-directed mutagenesis. At position 72, on the extracellular surface, the light-induced proton release was detected 71 +/- 4 microseconds after the flash (conditions: pH 7.3, 22 degrees C, and 150 mM KCl). On the cytoplasmic side with the dye at positions 101, 105, and 160, the corresponding values were 77, 76, and 74 +/- 5 microseconds, respectively. Under the same conditions, the proton release time in the bulk medium as detected by pyranine was around 880 microseconds--i.e., slower by a factor of more than 10. The fact that the proton that is released on the extracellular side is detected much faster on the cytoplasmic surface than in the aqueous bulk phase demonstrates that it is retained on the surface and migrates along the purple membrane to the other side. These findings have interesting implications for bioenergetics and support models of local proton coupling. From the small difference between the proton detection times by labels on opposite sides of the membrane, we estimate that at 22 degrees C the proton surface diffusion constant is greater than 3 x 10(-5) cm2/s. At 5 degrees C, the proton release detection time at position 72 equals the faster of the two main rise times of the M intermediate (deprotonation of the Schiff base). At higher temperatures this correlation is gradually lost, but the curved Arrhenius plot for the proton release time is tangential to the linear Arrhenius plot for the rise of M at low temperatures. These observations are compatible with kinetic coupling between Schiff base deprotonation and proton release.

Covalently bound pH-indicator dyes at selected extracellular or cytoplasmic sites in bacteriorhodopsin. 1. Proton migration along the surface of bacteriorhodopsin micelles and its delayed transfer from surface to bulk.

The kinetics of the light-induced release and uptake of protons was monitored with the optical pH-indicator fluorescein covalently bound to various sites on the extracellular and cytoplasmic surfaces of bacteriorhodopsin. Selective labeling was achieved by reacting (iodoacetamido)fluorescein with the single cysteine residues in bacteriorhodopsin introduced at the desired positions by site-directed mutagenesis. All measurements were performed with bacteriorhodopsin micelles in phospholipid/detergent mixtures in 150 mM KCl at 22 degrees C, pH 7.3. Neither the replacements by cysteine nor the subsequent labeling affected the absorption spectrum of bacteriorhodopsin and the rise times of the M intermediate. Only the decay of M was altered for some bacteriorhodopsin mutants with cysteine residues on the cytoplasmic side. The proton release time detected with fluorescein attached to the extracellular surface (the proton release side) at position 72 (in the loop connecting helices B and C) or 130 (DE loop) was 22 +/- 4 microseconds, clearly faster than that measured with pyranine in the aqueous bulk phase (125 +/- 10 microseconds for wild-type and all mutants studied). For bacteriorhodopsin mutants labeled at positions 35, 101, 160, 229, and 231 in the cytoplasmic loop region (the proton uptake side), the released proton was observed with a time of 61 +/- 4 microseconds. This was about 3-fold slower than the release time on the extracellular side, but still significantly faster than that measured with pyranine in the bulk phase. These results suggest that the released protons are retained on the micellar surface and move more rapidly along this surface to the cytoplasmic side than from the surface to the bulk medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Covalently bound pH-indicator dyes at selected extracellular or cytoplasmic sites in bacteriorhodopsin. 2. Rotational orientation of helices D and E and kinetic correlation between M formation and proton release in bacteriorhodopsin micelles.

The kinetics of the light-induced proton release in bacteriorhodopsin/lipid/detergent micelles was monitored with the optical pH-indicator fluorescein bound covalently to positions 127-134 (helices D and E and the DE loop) on the extracellular side of the protein (the proton release side). Single cysteine residues were introduced in these positions by site-directed mutagenesis, and fluorescein was attached to the sulfhydryl group by reaction with (iodoacetamido)fluorescein. Two characteristic proton release times (approximately 20 and 70 microseconds) were observed. The faster time constant was recorded when fluorescein was attached to positions 127, 130, 131, 132, and 134, while the slower time was observed with the indicator bound to positions 128, 129, and 133. The results are rationalized by assuming specific helical wheel orientations for helics D and E and by making a choice for the residues in the DE loop: (i) The fast time constants occur with fluorescein either attached to residues 130, 131, and 132 that form the DE loop or when pointing toward the interior of the protein with its aqueous proton channel [residues 127 (helix D) and 134 (helix E)]. (ii) The slower time constants are detected with fluorescein exposed to the exterior lipid/detergent phase when bound to residues 128, 129 (both helix D), and 133 (helix E). This interpretation is supported by measurements of the polarity of the label environment which indicate for fluorescein in group i a more hydrophilic environment and for group ii a more hydrophobic environment. The fastest proton release time (10 microseconds) was observed with fluorescein bound to position 127.(ABSTRACT TRUNCATED AT 250 WORDS)