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Over the past decades pulsed electron-electron double resonance (PELDOR), often called double electron-electron resonance (DEER), became one of the major spectroscopic tools for measurements of nanometer-scale distances and distance distributions in non-crystalline biological and chemical systems. The method is based on detecting the amplitude of the primary (3-pulse DEER) or refocused (4-pulse DEER) spin echo for the so-called “observer” spins when the other spins coupled to the former by a dipolar interaction are flipped by a “pump” pulse at another EPR frequency. While the timing of the pump pulse is varied in steps, the positions of the observer pulses are typically fixed. For such a detection scheme the total length of the observer pulse train and the electron spin memory time determine the amplitude of the detected echo signal. Usually, the distance range considerations in DEER experiments dictate the total length of the observer pulse train to exceed the phase memory time by a factor of few and this leads to a dramatic loss of the signal-to-noise ratio (SNR). While the acquisition of the DEER signal seems to be irrational under such conditions, it is currently the preferred way to conduct DEER because of an effective filtering out of all other unwanted interactions. Here we propose a novel albeit simple approach to improve DEER sensitivity and decrease data acquisition time by introducing the signal acquisition scheme based on RELaxation Optimized Acquisition (Length) Distribution (DEER-RELOAD). In DEER-RELOAD the dipolar phase evolution signal is acquired in multiple segments in which the observer pulses are fixed at the positions to optimize SNR just for that specific segment. The length of the segment is chosen to maximize the signal acquisition efficiency according the phase relaxation properties of the spin system. The total DEER trace is then obtained by “stitching” the multiple segments into a one continuous trace. The utility of the DEER-RELOAD acquisition scheme has been demonstrated on an example of the standard 4-pulse DEER sequence applied to two membrane protein complexes labeled with nitroxides. While theoretical gains from the DEER-RELOAD scheme increase with the number of stitched segments, in practice, even dividing the acquisition of the DEER trace into two segments may improve SNR by a factor of >3, as it has been demonstrated for one of these two membrane proteins.

vesicles, and peptide translocation across their membranes. First, we found that daptomycin binds to POPC:POPG GUVs in the presence of 0.5, 2.0, and 20 mM Ca 2þ . Second, we placed the vesicles in a solution containing carboxyfluorescein (CF) to test for influx. However, influx was never observed. Third, GUVs containing inner vesicles were observed to test for the translocation of daptomycin across the outer membrane of the GUVs, and onto the membrane of the inner vesicles. Again, translocation was never observed. However, in solutions containing 2 mM Ca 2þ a significant amount (about 50%) of membranes that had bound daptomycin collapsed shortly after binding occurred, resulting in the destruction of the GUVs. Furthermore, at high Ca 2þ concentrations (20 mM), the formation of daptomycin-rich clusters on the GUV membrane was observed. At lower concentrations of Ca 2þ (2 mM), daptomycin was distributed evenly on the GUV membrane. Lysette is a 22 amino acid peptide derived from staphylococcal d-lysin that forms an amphipathic a-helix when bound at membrane-water interfaces. We previously found that the experimentally determined DG of binding for lysette is more favorable than that predicted by the Wimley-White interfacial hydrophobicity scale by about 4 kcal/mol. Closer investigation of the amino acid composition of other peptides that are well described by the Wimley-White interfacial scale, such as melittin, led us to hypothesize that a preponderance of isoleucine (Ile) over leucine residues (Leu), as found in lysette, may be responsible for the deviation from the Wimley-White prediction. To test our hypothesis, we designed lysette variants lysette-I and lysette-L. In lysette-I, all Leu residues were replaced by Ile, and in lysette-L, all Ile were replaced by Leu. We also designed a melittin variant, iso-melittin, in which all Leu residues were replace by Ile. Peptide-lipid equilibrium dissociation constants and helicities of peptides bound to zwitterionic phosphatidylcholine (POPC) vesicles were determined by stopped-flow fluorescence and circular dichroism. If the hypothesis were correct, Lysette-I and iso-melittin should bind significantly better to zwitterionic bilayers than their Leu-rich counterparts, if the helicities of the lipid-bound peptides were not influenced by the Leu-Ile substitution. We found that the lysette-I bound significantly better to POPC vesicles than the lysette-L variant, as predicted. In the case of melittin, however, iso-melittin bound with roughly the same affinity to POPC bilayers as the original Leurich melittin. The results are discussed in terms of peptide structures in solution and when bound at the water-lipid interface, and the location of the lipid-bound states in the membrane.

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