Elongation of repetitive DNA by DNA polymerase from a hyperthermophilic bacterium Thermus thermophilus

Short repetitive DNA sequences are believed to be one of the primordial genetic elements that served as a source of complex large DNA found in the genome of modern organisms. However, the mechanism of its expansion (increase in repeat number) during the course of evolution is unclear. We demonstrate that the DNA polymerase of the hyperthermophilic bacterium Thermus thermophilus can elongate oligoDNA with several tandem repeats to very long DNA in vitro. For instance, 48mer repetitive oligoDNA (TACATGTA)₆, which has 25% GC content and a palindromic sequence, can be elongated up to ~10 000 bases by DNA polymerase at 74°C without template DNA. OligoDNA having a different GC content or a quasi-palindromic sequence can also be elongated, but less efficiently. A spectroscopic thermal melting experiment with the oligoDNA showed that its hairpin–coil transition temperature was very close to the elongation reaction temperature (74°C), but was much higher than the temperature at which duplex oligoDNA can exist stably. Taken together, we conclude that repetitive oligoDNA with a palindromic or quasi-palindromic sequence is elongated extensively by a hyperthermophilic DNA polymerase through hairpin–coil transitions. We propose that such an elongation mechanism might have been a driving force to expand primordial short DNA.     

Membranes of Tetrahymena: III. The effect of temperature on membrane core structures and fatty acid composition of Tetrahymena cells

Membrane core structures as revealed by the freeze-etch electron microscopy and the fatty acid composition measured by gas-liquid chromatography have been analyzed in Tetrahymena cells exposed to low temperature for varying periods.When cells were grown to mid-log phase at the optimal growth temperature of 28 °C and then chilled to 10 °C, cell division was inhibited. However, within 16 h the cells adapted to the low temperature.Chilling effected drastic structural alterations in the cores of different membrane types (membranes of the pellicula, the alveolar sacs, the endoplasmic reticulum and the nuclei). In all cases, there was a segregation of smooth faces from particle-rich faces in the fracture planes. However, the native membrane state, i.e. like that of cells grown at 28 °C, reappeared when the cells adapted to the low temperature.The total lipids of Tetrahymena cells contained primarily even-numbered fatty acids ranging from C₁₂ to C₁₈, but we also detected appreciable amounts of C₂₀ acids; this has not been reported before. During the initial phase of chilling, when cell division is inhibited, about 50% of the saturated fatty acids were replaced by unsaturated fatty acids, primarily monoenoic, dienoic and trienoic acids.We conclude that the structural recovery of the membranes in chilled Tetrahymena cells is accomplished by a desaturation of membrane fatty acids. This is discussed with respect to membrane “fluidity”.     

Effect of Temperature on the Potential and Current Thresholds of Axon Membrane

The effect of temperature on the potential and current thresholds of the squid giant axon membrane was measured with gross external electrodes. A central segment of the axon, 0.8 mm long and in sea water, was isolated by flowing low conductance, isoosmotic sucrose solution on each side; both ends were depolarized in isoosmotic KCl. Measured biphasic square wave currents at five cycles per second were applied between one end of the nerve and the membrane of the central segment. The membrane potential was recorded between the central sea water and the other depolarized end. The recorded potentials are developed only across the membrane impedance. Threshold current values ranged from 3.2 µa at 267deg;C to 1 µa at 7.5°C. Threshold potential values ranged from 50 mv at 26°C to 6 mv at 7.5°C. The mean Q₁₀ of threshold current was 2.3 (SD = 0.2), while the Q₁₀ for threshold potentials was 2.0 (SD = 0.1).     

Effect of Calcium, Temperature, and Polarizing Currents upon Alternating Current Excitation of Space-Clamped Squid Axons

Alternating current threshold excitation of space-clamped squid giant axons was measured as a function of frequency, external calcium concentration, temperature (from 10° to 35°C), and hyper- and depolarizing steps. In normal axons there is usually an optimum frequency at about 120 Hz, at which the threshold is a minimum. The threshold rises at both lower and higher frequencies to give a resonance curve. Low calcium causes an increase in optimum frequency, a decrease in current threshold, and an increase in sharpness of tuning in both real axons and axons computed according to the Hodgkin-Huxley formulation; high calcium causes opposite effects. An increase in temperature causes an increase of optimum frequency, an increase in sharpness of tuning, and an increase in threshold current in both real and computed axons. The Q₁₀ for the effect of temperature upon optimum frequency is 1.8 in real and computed axons at moderate temperatures. Hyperpolarization causes (a) a decrease in optimum frequency, (b) a decrease in sharpness of tuning, and (c) an increase in threshold. Depolarization causes opposite effects.     

Transport of ions across peritoneal membrane

The electrical conductance of ions across the peritoneal membrane of young buffalo (approximately 18-24 months old) has been recorded. Aqueous solutions of NaF, NaNO₃, NaCl, Na₂SO₄, KF, KNO₃, KCl, K₂SO₄, MgCl₂, CaCl₂, CrCl₃, MnCl₂, FeCl₃, CoCl₂, and CuCl₂ were used. The conductance values have been found to increase with increase in concentration as well as with temperature (15 to 35 °C) in these cases. The slope of plots of specific conductance, κ, versus concentration exhibits a decrease in its values at relatively higher concentrations compared to those in extremely dilute solutions. Also, such slopes keep on increasing with increase in temperature. In addition, the conductance also attains a maximum limiting value at higher concentrations in the said cases. This may be attributed to a progressive accumulation of ionic species within the membrane. The κ values of electrolytes follow the sequence for the anions: SO₄²⁻>Cl⁻>NO₃⁻>F⁻ while that for the cations: K⁺>Na⁺>Ca²⁺>Mn²⁺>Co²⁺>Cu²⁺>Mg²⁺>Cr³⁺>Fe³⁺. In addition, the diffusion of ions depends upon the charge on the membrane and its porosity. The membrane porosity in relation to the size of the hydrated species diffusing through the membrane appears to determine the above sequence. As the diffusional paths in the membrane become more difficult in aqueous solutions, the mobility of large hydrated ions gets impeded by the membrane framework and the interaction with the fixed charge groups on the membrane matrix. Consequently, the membrane pores reduce the conductance of small ions, which are much hydrated. An increase in conductance with increase in temperature may be due to the state of hydration, which implies that the energy of activation for the ionic transport across the membrane follows the sequence of crystallographic radii of ions accordingly. The Eyring's equation, κ=(RT/Nh)exp[−ΔH*/RT]exp[ΔS*/R], has been found suitable for explaining the temperature dependence of conductance in the said cases. This is apparent from the linear plots of log[κNh/RT] versus 1/T. The results indicate that the permeation of ions through the membrane giving negative values of ΔS* suggest that there may be formation of either covalent linkage between the penetrating ions and the membrane material or else the permeation may not be the rate-determining step. On the one hand, a high ΔS* value associated with the high value of energy of activation, E_a, for diffusion may suggest the existence of either a large zone of activation or loosening of more chain segments of the membrane. On the other hand, low value of ΔS* implies that converse is true in such cases, i.e., either a small zone of activation or no loosening of the membrane structure upon permeation.     

Replication fork regression in repetitive DNAs

Among several different types of repetitive sequences found in the human genome, this study has examined the telomeric repeat, necessary for the protection of chromosome termini, and the disease-associated triplet repeat (CTG)·(CAG)_n. Evidence suggests that replication of both types of repeats is problematic and that a contributing factor is the repetitive nature of the DNA itself. Here we have used electron microscopy to investigate DNA structures formed at replication forks on large model DNAs containing these repeat sequences, in an attempt to elucidate the contributory effect that these repetitive DNAs may have on their replication. Visualization of the DNA revealed that there is a high propensity for a paused replication fork to spontaneously regress when moving through repetitive DNAs, and that this results in a four-way chickenfoot intermediate that could present a significant block to replication in vivo, possibly leading to unwanted recombination events, amplifications or deletions.     

Effect of External Calcium and of Temperature on Contraction in Snake Muscle Fibers

The effect of external calcium and of temperature on the contractile responses has been studied in voltage clamped snake twitch muscle fibers. Increasing [Ca⁺⁺]_o from 0.2 to 7.0 mM raised contractile threshold by 15–20 mV, the latter coinciding with the appearance of delayed rectification. The duration of contracture, the rates of rise and decay of tension depended on the level of depolarization and [Ca⁺⁺]_o. The minimum duration of repolarization necessary to restore the contractile response was much shorter in high [Ca⁺⁺]_o. When the bathing solution was cooled to 10 from 20°C the time-course of contracture was markedly prolonged and the outward current was reduced without significant change in maximum tension. The threshold for contraction tended to be somewhat lower at the lower temperature. The contractile repriming was much slower at low temperature. However, reduction in temperature slowed the rate of recovery much less at low [Ca⁺⁺]_o than at normal [Ca⁺⁺]_o.     

Oscillation and Repetitive Firing in Squid Axons : Comparison of experiments with computations

Space-clamped squid axons treated with low calcium and computed Hodgkin-Huxley (HH) axons were stimulated by steps of superthreshold current from 101 to 400% of the rheobasic value over a temperature range of 5–27°C. The natural frequency of sustained repetitive firing of real and computed axons depended weakly upon stimulus intensity and strongly upon temperature, with a Q₁₀ of 2.7 (experimental) and 2.6 (computed). For real axons, but not the computed axon, the intervals between the first two spikes were shorter than between subsequent spikes. Constant spike frequencies from 75 Hz at low intensities and temperatures to 330 Hz at high intensities and temperatures were soon achieved. Subthreshold and superthreshold responses were sometimes intermixed in a train of responses from a real axon responding to a constant step of current, but not predicted by HH. The time interval following a spike was always longer than that following a subthreshold oscillation in slightly decalcified real axons, as Huxley and FitzHugh also found for computed axons. There was a bias toward spikes at the beginning of the train and toward subthreshold responses later on. Some repeated patterns were found, every second, third, or fourth response being a spike. Neither the HH equations nor the computed or experimental threshold behaviors show a critical temperature to support a membrane phase transition.     

Contrasting the Chromosomal Organization of Repetitive DNAs in Two Gryllidae Crickets with Highly Divergent Karyotypes

A large percentage of eukaryotic genomes consist of repetitive DNA that plays an important role in the organization, size and evolution. In the case of crickets, chromosomal variability has been found using classical cytogenetics, but almost no information concerning the organization of their repetitive DNAs is available. To better understand the chromosomal organization and diversification of repetitive DNAs in crickets, we studied the chromosomes of two Gryllidae species with highly divergent karyotypes, i.e., 2n(♂) = 29,X0 (Gryllus assimilis) and 2n = 9, neo-X₁X₂Y (Eneoptera surinamensis). The analyses were performed using classical cytogenetic techniques, repetitive DNA mapping and genome-size estimation. Conserved characteristics were observed, such as the occurrence of a small number of clusters of rDNAs and U snDNAs, in contrast to the multiple clusters/dispersal of the H3 histone genes. The positions of U2 snDNA and 18S rDNA are also conserved, being intermingled within the largest autosome. The distribution and base-pair composition of the heterochromatin and repetitive DNA pools of these organisms differed, suggesting reorganization. Although the microsatellite arrays had a similar distribution pattern, being dispersed along entire chromosomes, as has been observed in some grasshopper species, a band-like pattern was also observed in the E. surinamensis chromosomes, putatively due to their amplification and clustering. In addition to these differences, the genome of E. surinamensis is approximately 2.5 times larger than that of G. assimilis, which we hypothesize is due to the amplification of repetitive DNAs. Finally, we discuss the possible involvement of repetitive DNAs in the differentiation of the neo-sex chromosomes of E. surinamensis, as has been reported in other eukaryotic groups. This study provided an opportunity to explore the evolutionary dynamics of repetitive DNAs in two non-model species and will contribute to the understanding of chromosomal evolution in a group about which little chromosomal and genomic information is known.     

Macromolecular rate theory explains the temperature dependence of membrane conductance kinetics

The factor Q₁₀ is used in neuroscience to adjust reaction rates of voltage-activated membrane conductances to different temperatures and is widely assumed to be constant. By performing an analysis of published data of the reaction rates of sodium, potassium, and calcium membrane conductances, we demonstrate that 1) Q₁₀ is temperature dependent, 2) this relationship is similar across conductances, and 3) there is a strong effect at low temperatures (<15°C). We show that macromolecular rate theory (MMRT) explains this temperature dependency. MMRT predicts the existence of optimal temperatures at which reaction rates decrease as temperature increases, a phenomenon that we also found in the published data sets. We tested the consequences of using MMRT-adjusted reaction rates in the Hodgkin-Huxley model of the squid’s giant axon. The MMRT-adjusted model reproduces the temperature dependence of the rising and falling times of the action potential. Furthermore, the model also reproduces these properties for different squid species that live in different climates. In a second example, we compare spiking patterns of biophysical models based on human pyramidal neurons from the Allen Cell Types database at room and physiological temperatures. The original models, calibrated at 34°C, failed to generate realistic spikes at room temperature in more than half of the tested models, while the MMRT produces realistic spiking in all conditions. In another example, we show that using the MMRT correction in hippocampal pyramidal cell models results in 100% differences in voltage responses. Finally, we show that the shape of the Q₁₀ function results in systematic errors in predicting reaction rates. We propose that the optimal temperature could be a thermodynamical barrier to avoid over excitation in neurons. While this study is centered on membrane conductances, our results have important consequences for all biochemical reactions involved in cell signaling.     

Temperature Characteristics of Excitation in Space-Clamped Squid Axons

Temperature characteristics of excitability in the squid giant axon were measured for the space-clamped axon with the double sucrose gap technique. Threshold strength-duration curves were obtained for square wave current pulses from 10 µsec to 10 msec and at temperatures from 5°C to 35°C. The threshold change of potential, at which an action potential separated from a subthreshold response, averaged 17 mv at 20°C with a Q¹⁰ of 1.15. The average threshold current density at rheobase was 12 µa/cm² at 20°C with a Q¹⁰ of 2.35 compared to 2.3 obtained previously. At short times the threshold charge was 1.5·10^-8 coul/cm². This was relatively independent of temperature and occasionally showed a minimum in the temperature range. At intermediate times and all temperatures the threshold currents were less than for both the single time constant model and the two factor excitation process as developed by Hill. FitzHugh has made computer investigations of the effect of temperature on the excitation of the squid axon membrane as represented by the Hodgkin-Huxley equations. These are in general in good agreement with our experimental results.     

Energy thresholds for ATP synthesis in chloroplasts

We have investigated the ATP synthesis associated with acid-base transitions in chloroplast lamellae under conditions which allow simultaneous control of the thermodynamic variables, ΔpH, membrane potential and ΔG_ATP. These variables have been directly imposed rather than simply inferred. Since the initiation of labeled P_i incorporation seems to measure accurately the initiation of net ATP synthesis, the following conclusions can be drawn: (1) The proton-motive force which is just sufficient for ATP synthesis provides almost exactly the required energy for ΔG_ATP if the efflux of three H⁺ is required for each ATP molecule formed. (2) The membrane potential and the ΔpH contribute to the proton-motive force in a precisely additive way. Thus, the threshold can be reached or exceeded by a ΔpH in the absence of a membrane potential, by a membrane potential in the absence of a ΔpH, or by any combination of membrane potential and ΔpH. With a large enough membrane potential, ATP synthesis occurs even against a small inverse ΔpH. In each instance the combined ΔpH and membrane potential necessary for initiation of ATP synthesis represent the same threshold proton-motive force.     

Melatonin Alters Fluid Phase Coexistence in POPC/DPPC/Cholesterol Membranes

The structure and biophysical properties of lipid membranes are important for cellular functions in health and disease. In Alzheimer’s disease, the neuronal membrane is a target for toxic amyloid-β (Aβ). Melatonin is an important pineal gland hormone that has been shown to protect against Aβ toxicity in cellular and animal studies, but the molecular mechanism of this protection is not fully understood. Melatonin is a small membrane-active molecule that has been shown to interact with model lipid membranes and alter the membrane biophysical properties, such as membrane molecular order and dynamics. This effect of melatonin has been previously studied in simple model bilayers with one or two lipid components. To make it more relevant to neuronal membranes, we used a more complex ternary lipid mixture as our membrane model. In this study, we used ²H-NMR to investigate the effect of melatonin on the phase behavior of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and cholesterol lipid membranes. We used deuterium-labeled POPC-d₃₁ and DPPC-d₆₂,separately to probe the changes in hydrocarbon chain order as a function of temperature and melatonin concentration. We find that POPC/DPPC/cholesterol at molar proportions of 3:3:2 is close to liquid-disordered/liquid-ordered phase separation and that melatonin can induce phase separation in these ternary mixtures by preferentially incorporating into the disordered phase and increasing its level of disorder. At 5 mol% melatonin, we observed phase separation in samples with POPC-d₃₁, but not with DPPC-d₆₂, whereas at 10 mol% melatonin, phase separation was observed in both samples with either POPC-d₃₁ or DPPC-d₆₂. These results indicate that melatonin can have a strong effect on membrane structure and physical properties, which may provide some clues to understanding how melatonin protects against Aβ, and that choice of chain perdeuteration is an important consideration from a technical point of view.     

Repeatability of adaptation in experimental populations of different sizes

The degree to which evolutionary trajectories and outcomes are repeatable across independent populations depends on the relative contribution of selection, chance and history. Population size has been shown theoretically and empirically to affect the amount of variation that arises among independent populations adapting to the same environment. Here, we measure the contribution of selection, chance and history in different-sized experimental populations of the unicellular alga Chlamydomonas reinhardtii adapting to a high salt environment to determine which component of evolution is affected by population size. We find that adaptation to salt is repeatable at the fitness level in medium (N_e = 5 × 10⁴) and large (N_e = 4 × 10⁵) populations because of the large contribution of selection. Adaptation is not repeatable in small (N_e = 5 × 10³) populations because of large constraints from history. The threshold between stochastic and deterministic evolution in this case is therefore between effective population sizes of 10³ and 10⁴. Our results indicate that diversity across populations is more likely to be maintained if they are small. Experimental outcomes in large populations are likely to be robust and can inform our predictions about outcomes in similar situations.     

Oxygen consumption by the bivalve Donax vittatus (da Costa)

The acute oxygen consumption of Donax vittatus (da Costa) freshly collected at different times from a beach at Barrassie, Ayrshire, Scotland, has been measured at different temperatures. The logarithmic relationship between oxygen consumption and body weight showed a significant difference on only one occasion, and a common regression coefficient (b) of 0.865 could be used for regressions of oxygen consumption on weight. Over the temperature range 2.9–20 °C oxygen consumption rose with temperature. There was a linear decline of Q₁₀ with temperature in the range 2.9 –20 °C. Differences in values of the constant (a) in the regression equation suggest that there is some acclimation to temperature, resulting in rotation of the rate/temperature curve counterclockwise for warm-acclimated animals, and a reduction of Q₁₀ in cold-acclimated animals. The differences in oxygen consumption which result are small and appear to have little practical significance. High levels of metabolically-inactive materials such as stored glycogen reserves lead to a reduction in the weight-specific oxygen consumption. Spawning animals show an increased oxygen consumption.     

Permeability changes resulting from virus-cell fusion: temperature-dependence of the contributing processes

Summary A new assay for membrane fusion, using the fluorescent probe pyrene-sulphonyl-phosphatidyl ethanolamine, has been developed. Fusion between the envelope of Sendai virus and human erythrocytes or Lettre cells has a Q₁₀ of 4 at 37° C, increasing to 7 at 7 ° C; there is no lag to onset of fusion. Viral neuraminidase has a Q₁₀ of 2.3 between 37° C and 4° C. Its action limits the extent of fusion by causing the elution of virus; this effect is particularly marked at low temperature because of the difference in Q₁₀ for fusion and neuraminidase. The temperature-dependence of the initiation of permeability changes following the removal of inhibitory amounts of Ca²⁺ is 2; thus membrane fusion is the principal temperature-sensitive step during the permeabilization of cells by Sendai virus. A recovery process, by which cells become insensitive to the removal of Ca²⁺ and which therefore limits the extent of permeabilization, has a Q₁₀ of 7.4 between 37° C and 21° C. It is concluded that the lag to onset of permeability changes is not due to a lag in virus-cell membrane fusion, but to the gradual acquisition of a threshold level of membrane damage; the extent of permeabilization depends on the rate of fusion relative to the rates of neuraminidase and recovery.     

Study of plasma confinement in the L-2M stellarator during the formation of an edge transport barrier

A plasma confinement mode characterized by the formation of an edge transport barrier (ETB) was discovered in the L-2M stellarator after boronization of the vacuum vessel wall. The transition into this mode is accompanied by a jump in the electron temperature by 100–200 eV at the plasma edge and a sharp increase in the gradient of the electron temperature T _e in this region. The threshold power for the transition into the ETB confinement mode with an increased electron temperature gradient is P _thr ^?Te = (60 ± 15)n _e [10¹⁹ m^?3] kW. The formation of the ETB manifests itself also in a substantial change in the electron density profile. A density peak with a steep gradient at the outer side forms at the plasma edge. The threshold power for the transition into the ETB confinement mode corresponding to a substantial increase in the plasma density gradient near r = a is P _thr ^?Te = (67 ± 9)n _e [10¹⁹ m^?3] kW, which agrees to within experimental error with the threshold power for the transition into the ETB confinement mode determined from the sharp increase in the gradient of the electron temperature T _e . The value of P _thr for the L-2M stellarator agrees to within 25% with that obtained from the tokamak scaling. In the ETB confinement mode, the plasma energy W and the energy confinement time τ _E determined from diamagnetic measurements increase by 20–30% as compared to those obtained from the stellarator scaling for the confinement mode without an ETB. When the heating power increases by a factor of 2–3 above the threshold value, the effects related to improved energy confinement disappear.     

Differential extraction and structural specificity of specialized ubiquinone molecules in secondary electron transfer in chromatophores from Rhodopseudomonas sphaeroides, Ga

In chromatophores from the facultative photosynthetic bacterium, Rhodopseudomonas sphaeroides, Ga, the function of ubiquinone-10 (UQ-10) at two specialized binding sites (Q_B and Q_Z) has been determined by kinetic criteria. These were the rate of rereduction of flash-oxidized [BChl]₂⁺ through the back reaction, or the binary pattern of cytochrome b₅₆₁ (for the Q_b site), and the rapid rate of rereduction of flash-oxidized cytochrome c, or the relative amplitude of the antimycin-sensitive Phase III ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{~ 1.5}\text{ms}$) of the carotenoid spectral shift induced by a single turnover flash at E_h ~ 100 mV (for the Q_Z site). The phenomenon associated with the two binding sites behaved differently on extraction of UQ from lyophilized chromatophores using isooctane. By this selective extraction procedure it has been possible to show that UQ-10 molecules are required at different concentrations in the membrane for specific redox events in secondary electron transfer. The reduction of cytochrome b occurs in particles which no longer show the phenomena associated with Q_Z, but still possess a large proportion of Q_b, while rapid rereduction of flash-oxidized cytochrome c requires an additional complement of UQ-10 (Q_Z). Extracted particles lacking Q_Z and a large amount of Q_B have been reconstituted with different UQ homologs (UQ-1, UQ-3, and UQ-10). Specific redox events have been studied in reconstituted particles. All UQ homologs act as secondary acceptors from the reaction center; UQ-3 and UQ-10, but not UQ-1, are also able to reconstitute the function of Q_Z as electron donor to cytochrome c. Only UQ-10, however, is able to restore normal rates of the overall cyclic electron transfer induced by a train of flashes, and maximal rates of the light-induced ATP synthesis. The results are interpreted in terms of Q-cycle mechanisms in which quinone and quinol at both the Q_Z and Q_b sites are in rapid equilibrium with the quinone pool.     

Overcoming hysteresis to attain reversible equilibrium folding for outer membrane phospholipase A in phospholipid bilayers

The free energy of unfolding of a membrane protein from lipids into water (ΔG^o_w,l) describes its equilibrium thermodynamic stability. Knowing this parameter gives insight into a membrane protein's sequence-structure-energy relationships. However, there are few measures of membrane protein stability because of the technical difficulties associated with unfolded and partially folded states. Here, we describe the experimental process that allowed us to measure the ΔG^o_w,l of the outer membrane phospholipase A into large unilamellar vesicles (LUVs) of 1,2-dilauroyl-sn-glycero-3-phosphocholine. To arrive at this reversible folding condition, we screened a large number of experimental variables: temperature, incubation time, salt concentration, pH, lipid composition and liposome morphology. The principal challenge we encountered under most conditions was hysteresis between folding and unfolding titrations. A second factor that compromised reversible folding was the observation that a fraction of the protein population tended to aggregate. We found that hysteresis could be completely eliminated on a feasible timescale by conducting experiments at acidic pH, by the slow dilution of the protein in the initial titration setup and by utilizing a low concentration of a detergent as a temporary “holdase” to solubilize the protein upon its initial dilution into folding conditions. We confirmed that the detergent did not disrupt the LUVs using fluorescence emission of lipid-sensitive dyes and light scattering. The results of our parameter search should be generally useful for efforts to measure ΔG^o_w,l for other membrane proteins.