Although the effects of ethanol on protein receptors and lipid membranes have been studied extensively, ethanol's effect on vesicles fusing to lipid bilayers is not known. To determine the effect of alcohols on fusion rates, we utilized the nystatin/ergosterol fusion assay to measure fusion of liposomes to a planar lipid bilayer (BLM). The addition of ethanol excited fusion when applied on the cis (vesicle) side, and inhibited fusion on the trans side. Other short-chain alcohols followed a similar pattern. In general, the inhibitory effect of alcohols (trans) occurs at lower doses than the excitatory (cis) effect, with a decrease of 29% in fusion rates at the legal driving limit of 0.08% (w/v) ethanol (IC 50 ¼ 0.2% v/v, 34 mM). Similar inhibitory effects were observed with methanol, propanol, and butanol, with ethanol being the most potent. Significant variability was observed with different alcohols when applied to the cis side. Ethanol and propanol enhanced fusion, butanol also enhanced fusion but was less potent, and low doses of methanol mildly inhibited fusion. The inhibition by trans addition of alcohols implies that they alter the planar membrane structure and thereby increase the activation energy required for fusion, likely through an increase in membrane fluidity. The cis data are likely a combination of the above effect and a proportionally greater lowering of the vesicle lysis tension and hydration repulsive pressure that combine to enhance fusion. Alternate hypotheses are also discussed. The inhibitory effect of ethanol on liposome-membrane fusion is large enough to provide a possible biophysical explanation of compromised neuronal behavior.
CRISPR-Cas systems impart adaptive immunity against foreign genetic elements in bacteria and archaea. In CRISPR-Cas system, DNA interference involves a nuclease which is guided by RNA to complementary DNA sequences (protospacer) with a requirement that the protospacer be followed by a special protospacer adjacent motif (PAM). This system has been repurposed for various genome-engineering applications with Cas9-RNA (Class 2, Type II CRISPR-Cas system) providing an overwhelming bulk of these efforts, but specificity in DNA targeting continues to be a challenge. Recently a new nuclease called Cpf1 of class 2 type V CRISPR-Cas system was identified (PAM: 5'-YTN-3', Protospacer: 24 base-pairs) with robust genome-editing activity. AT-rich PAM, longer protospacer, staggered-cleavage at PAM-distal site are some key differences of Cpf1 from Cas9 (PAM: 5'-NGG-3', Protospacer: 20 base-pairs, blunt-cleavage at PAM-proximal site). But little is known about kinetics and mechanism of Cpf1-RNA DNA interaction and how mismatches influence this interaction and nucleolytic activity despite the importance of competition between dissociation, nucleolysis and product release in rapid and accurate targeting. We have used single-molecule fluorescence imaging and biochemical assays to characterize the DNA interrogation by three major Cpf1 orthologs. The fluorescent and radio labeling geometries were designed such that they reported on three major Cpf1 activities i.e. binding, nucleolysis and release of nucleolysed products. Single molecule techniques are ideal for developing a kinetic basis of Cpf1 specificity as they can detect wide-ranging interactions (transient to long-lived) and identify multiple DNA-targeting steps in real-time, which complemented with biochemical assays provide a comprehensive picture of Cpf1 activity and specificity. Our studies reveal important differences between Cpf1 family and Cas9 which can hold important implications for rapidly expanding genome-engineering field. Moreover, the understanding of underlying mechanism of how Cpf1-RNA recognizes, cuts or rejects DNA targets will aid in further development of improved versions of this enzyme.
equal concentrations. NLPs were mixed with a 200-fold molar excess of DHPC/DMPC bicelles (equimolar 6-and 14-carbon acyl chains) in a stopped-flow fluorometer. The rate of lipid transfer was monitored by the appearance of unquenched NBD fluorescence at 520 nm. The observed pseudo-first-order rate constant was surprisingly small (0.26/sec). NLPs did not react with DHPC alone below its critical micelle concentration (cmc). Above the cmc, the reaction was complete within the instrument dead time. Thus, the rate-limiting step is not the reaction of NLPs with DHPC monomers or micelles. Added MSP1E3D1 had no effect on the rate, ruling out free apolipoprotein involvement. The NLP-bicelle mixing rate showed a strong temperature dependence (activation energy ¼ 28 kcal/mol). Near or below the DMPC phase transition temperature, the kinetics were biphasic. The results suggest NLP-bicelle mixing kinetics may be mechanistically similar to lipid mixing via fusion pores.
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