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Time-resolved fluorescence spectroscopy in combination with differential scanning calorimetry (DSC) was used to study the chemical interactions that occur when l-phenylalanine is introduced to solutions containing phosphatidylcholine vesicles. Studies reported in this work address open questions about l-Phe's affinity for lipid vesicle bilayers, the effects of l-Phe partitioning on bilayer properties, l-Phe's solvation within a lipid bilayer, and the amount of l-Phe within that local solvation environment. DSC data show that l-Phe reduces the amount of heat necessary to melt saturated phosphatidylcholine bilayers from their gel to liquid-crystalline state but does not change the transition temperature ( T gel-lc ). Time-resolved emission shows only a single l-Phe lifetime at low temperatures corresponding to l-Phe remaining solvated in aqueous solution. At temperatures close to T gel-lc , a second, shorter lifetime appears that is assigned to l-Phe already embedded within the membrane that becomes hydrated as water starts to permeate the lipid bilayer. This new lifetime is attributed to a conformationally restricted rotamer in the bilayer's polar headgroup region and accounts for up to 30% of the emission amplitude. Results reported for dipalmitoylphosphatidylcholine (DPPC, 16:0) lipid vesicles prove to be general, with similar effects observed for dimyristoylphosphatidylcholine (DMPC, 14:0) and distearoylphosphatidylcholine (DSPC, 18:0) vesicles. Taken together, these results create a complete and compelling picture of how l-Phe associates with model biological membranes. Furthermore, this approach to examining amino acid partitioning into membranes and the resulting solvation forces points to new strategies for studying the structure and chemistry of membrane-soluble peptides and selected membrane proteins.

Time-resolved fluorescence and differential scanning calorimetry (DSC) were used to examine how two amino acids, L-phenylalanine (L-PA) and N-acetyl-DL-tryptophan (NAT), affect the temperature-dependent membrane affinity of two structurally similar coumarin solutes for 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) vesicles. The 7-aminocoumarin solutes, coumarin 151 (C151) and coumarin 152 (C152), differ in their substitution at amine position-C151 is a primary amine, and C152 is a tertiary amine-and both solutes show different tendencies to associate with lipid bilayers consistent with differences in their respective log-P-values. Adding L-PA to the DPPC vesicle solution did not change C151's propensity to remain freely solvated in aqueous solution, but C152 showed a greater tendency to partition into the hydrophobic bilayer interior at temperatures below DPPC's gel-liquid crystalline transition temperature (T gel-lc ). This finding is consistent with L-PA's ability to enhance membrane permeability by disrupting chain-chain interactions. Adding NAT to DPPC-vesicle-containing solutions changed C151 and C152 affinity for the DPPC membranes in unexpected ways. DSC data show that NAT interacts strongly with the lipid bilayer, lowering T gel-lc by up to 2°C at concentrations of 10 mM. These effects disappear when either C151 or C152 is added to solution at concentrations below 10 μM, and T gel-lc returns to a value consistent with unperturbed DPPC bilayers. Together with DSC results, fluorescence data imply that NAT promotes coumarin adsorption to the vesicle bilayer surface. NAT's effects diminish above T gel-lc and imply that unlike L-PA, NAT does not penetrate into the bilayer but instead remains adsorbed to the bilayer's exterior. Taken in their entirety, these discoveries suggest that amino acids-and by inference, polypeptides and proteins-change solute affinity for lipid bilayers with specific effects that depend on individualized amino-acid-lipid-bilayer interactions., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Time-resolved fluorescence measurements were used to quantify partitioning of three different 7-aminocoumarin derivatives into DPPC vesicle bilayers as a function of temperature. The coumarin derivatives were structurally equivalent except for the degree of substitution at the 7-amine position. Calculated log  P (octanol: water partitioning) coefficients, a common indicator that correlates with bioconcentration, predict that the primary amine (coumarin 151 or C151) would experience a ∼40-fold partition enrichment in polar organic environments (log  P C151 = 1.6) while the tertiary amine's (coumarin 152 or C152) concentration should be >500 times enhanced (log  P C152 = 2.7). Both values predict that partitioning into lipid membranes is energetically favorable. Time-resolved emission spectra from C151 in solutions containing DPPC vesicles showed that within detection limits, the solute remained in the aqueous buffer regardless of temperature and vesicle bilayer phase. C152 displayed a sharp uptake into DPPC bilayers as the temperature approached DPPC's gel-liquid crystalline transition temperature, consistent with previously reported results ([ J. Phys. Chem. B 2017, 121, 4061-4070]). The secondary amine, synthesized specifically for these studies and dubbed C151.5 with a measured log  P value of 1.9, partitioned into the bilayer's polar head group with no pronounced temperature dependence. These experiments illustrate the limitations of using a gross descriptor of preferential solvation to describe solute partitioning into complex, heterogeneous systems having nanometer-scale dimensions. From a broader perspective, results presented in this work illustrate the need for more chemically informed tools for predicting a solute tendency for where and how much it will bioconcentrate within a biological membrane.

Solute partitioning across a variety of alkane/aqueous interfaces was examined as a function of solute and alkane solvent structure. Solutes include p-nitrophenol (PNP), 3,5-dimethyl-p-nitrophenol (3,5-DMPNP), and 2,6-dimethyl-p-nitrophenol (2,6-DMPNP), the latter two being isomers distinguished solely by the location of methyl substituents on the aromatic ring. The alkane solvents included cylohexane, methylcyclohexane, octane, and iso-octane (2,2,4-trimethylpentane). PNP partitioned preferentially into the water by factors as high as 160:1. The dimethyl isomers partitioned more equally between water and the different alkanes. 2,6-DMPNP showed a 3-fold greater affinity for the alkane phase than 3,5-DMPNP. Ab initio calculations were used to characterize the molecular and electronic structure of the three solutes and to quantify individual contributions to each solute's solvation energy in model aqueous and alkane phases. Differences between 2,6-DMPNP and 3,5-DMPNP partitioning are interpreted based on the ability of the methyl groups in 2,6-DMPNP to weaken hydrogen bonding between the phenol group and adjacent water molecules. This diminished solvation interaction reduces the barrier to solute migration into the nonpolar organic phase despite the fact that 2,6-DMPNP has a larger (calculated) permanent, ground-state dipole than 3,5-DMPNP.

Resonance enhanced second harmonic generation (SHG) coupled with novel solvatochromic surfactants has been used to explore solvation across liquid/liquid interfaces in the presence of excess surface charge in the aqueous phase. The surfactants--dubbed "molecular rulers"--consist of hydrophobic, solvatochromic chromophores connected to charged headgroups via variable length alkyl spacers. Interfacial dipolar width is monitored as a function of chromophore/headgroup separation. Data show that cationic and anionic surfactants of equivalent lengths sample very different environments across a water/cyclohexane interface. The effective excitation wavelengths of cationic surfactants are shifted persistently to higher energies than in bulk cyclohexane while anionic surfactants solvation converges smoothly from the aqueous to the organic limit with increasing spacer length. To further evaluate the effect of surface charge on interfacial solvation, SHG was used to probe the environment surrounding molecular ruler chromophores adsorbed to the aqueous liquid/vapor interface in the presence of densely packed monolayers of 1-octanol. These monolayer systems are shown to reproduce qualitatively many of the features associated with bulk water/alkane interfaces and are logistically easier to assemble. Changing the ionic strength of the underlying aqueous sub-phase suggests that the headgroup of cationic molecular ruler surfactants alters the electronic structure of the chromophore rather than properties of the surrounding nonpolar environment.

Molecular dynamics simulations have previously described how the physical properties across immiscible liquid-liquid interfaces should converge from aqueous to organic limits, but these predictions have largely gone untested, owing to difficulties associated with probing buried interfaces. X-ray and neutron scattering experiments have created detailed pictures of molecular structure at these boundaries, but such scattering studies cannot probe how surface-altered solvent structures affect interfacial solvating properties. Given that surface-mediated solvent properties control interfacial solute concentrations and reactivities, identifying the characteristic dimensions of interfacial solvation is essential for formulating predictive models of solution phase surface chemistry. Here we use specially synthesized solvatochromic surfactants that act as 'molecular rulers' and resonance-enhanced second-harmonic generation to measure the dipolar width of weakly and strongly associating liquid-liquid interfaces. Dipolar width describes the distance required for a dielectric environment to change from one phase to another. Our results show that polarity converges to a nonpolar limit on subnanometre length scales across a water-cyclohexane interface. However, polarity across the strongly associating, water-1-octanol interface is dominated by a nonpolar, alkane-like region. These data call into question the use of continuum descriptions of liquids to characterize interfacial solvation, and demonstrate that interfacial environments can vary in a non-additive manner from bulk solution limits.

Resonance-enhanced second harmonic generation (SHG) has been used to probe the solvatochromic behavior of two small, aromatic chromophores adsorbed to the aqueous/cyclohexane, liquid/liquid interface. SHG spectra of p-nitrophenol (PNP) and 2,6-dimethyl-PNP (dmPNP) indicate that these two chromophores sample markedly different environments. PNP sees a polar, waterlike environment, whereas solvent polarity surrounding dmPNP is dominated by the nonpolar, organic phase. Results suggest that subtle changes in solute structure can change the distribution of solutes across an interface and thus change a solute's local solvation environment.

Homologous series of solvatochromic neutral alcohols and ionic sulfates are synthesized and characterized. Each surfactant series consists of hydrophobic, p-nitroanisole-based chromophores attached to polar or ionic headgroups by n-alkyl spacers. UV absorption measurements show that the optical properties of surfactant chromophores closely track those of the parent chromophore. Interfacial tension measurements are used to calculate surface excess concentrations of ionic surfactants adsorbed to an aqueous-cyclohexane interface. With a hydrophobic chromophore, a hydrophilic headgroup, and a variable-length, alkyl spacer, these surfactants have the potential to function as molecular rulers: probes of molecular-scale variation in solvation forces across condensed-phase interfaces. Changing the separation between the hydrophobic, solvatochromic probe and the hydrophilic headgroup should enable different members of a homologous series to span different interfacial widths, thus exposing the chromophore to different chemical environments. This idea is explored by using surface-specific, nonlinear optical spectroscopy. Resonant second harmonic spectra of p-nitroanisole and the surfactant product 4a adsorbed to an aqueous-cyclohexane interface show the surfactant spectrum blue-shifted 9 nm relative to the spectrum of adsorbed p-nitroanisole. On the basis of chromophore solvatochromism, these results are consistent with a less polar environment surrounding the surfactant chromophore. Significant differences in interfacial solvation resulting from a approximately 5 A separation between the surfactant headgroup and chromophore support recently proposed models of molecularly sharp, microscopically flat aqueous-alkane interfaces.

The setting of aberration tolerances is considered for systems in which the emergent wave front has a nonuniform amplitude across a full circular aperture. Tolerances can be derived from the transfer function for a specified spatial frequency; no simple equivalent of Maréchal's tolerances can be derived to suit all cases well, although limits can be given.