Modulating Phase Behavior in Fatty Acid-Modified Elastin-like Polypeptides (FAMEs): Insights into the Impact of Lipid Length on Thermodynamics and Kinetics of Phase Separation.

Although post-translational lipidation is prevalent in eukaryotes, its impact on the liquid-liquid phase separation of disordered proteins is still poorly understood. Here, we examined the thermodynamic phase boundaries and kinetics of aqueous two-phase system (ATPS) formation for a library of elastin-like polypeptides modified with saturated fatty acids of different chain lengths. By systematically altering the physicochemical properties of the attached lipids, we were able to correlate the molecular properties of lipids to changes in the thermodynamic phase boundaries and the kinetic stability of droplets formed by these proteins. We discovered that increasing the chain length lowers the phase separation temperature in a sigmoidal manner due to alterations in the unfavorable interactions between protein and water and changes in the entropy of phase separation. Our kinetic studies unveiled remarkable sensitivity to lipid length, which we propose is due to the temperature-dependent interactions between lipids and the protein. Strikingly, we found that the addition of just a single methylene group is sufficient to allow tuning of these interactions as a function of temperature, with proteins modified with C7-C9 lipids exhibiting non-Arrhenius dependence in their phase separation, a behavior that is absent for both shorter and longer fatty acids. This work advances our theoretical understanding of protein-lipid interactions and opens avenues for the rational design of lipidated proteins in biomedical paradigms, where precise control over the phase separation is pivotal.

A stepwise mechanism for aqueous two-phase system formation in concentrated antibody solutions.

Aqueous two-phase system (ATPS) formation is the macroscopic completion of liquid-liquid phase separation (LLPS), a process by which aqueous solutions demix into 2 distinct phases. We report the temperature-dependent kinetics of ATPS formation for solutions containing a monoclonal antibody and polyethylene glycol. Measurements are made by capturing dark-field images of protein-rich droplet suspensions as a function of time along a linear temperature gradient. The rate constants for ATPS formation fall into 3 kinetically distinct categories that are directly visualized along the temperature gradient. In the metastable region, just below the phase separation temperature, Tph , ATPS formation is slow and has a large negative apparent activation energy. By contrast, ATPS formation proceeds more rapidly in the spinodal region, below the metastable temperature, Tmeta , and a small positive apparent activation energy is observed. These region-specific apparent activation energies suggest that ATPS formation involves 2 steps with opposite temperature dependencies. Droplet growth is the first step, which accelerates with decreasing temperature as the solution becomes increasingly supersaturated. The second step, however, involves droplet coalescence and is proportional to temperature. It becomes the rate-limiting step in the spinodal region. At even colder temperatures, below a gelation temperature, Tgel , the proteins assemble into a kinetically trapped gel state that arrests ATPS formation. The kinetics of ATPS formation near Tgel is associated with a remarkably fragile solid-like gel structure, which can form below either the metastable or the spinodal region of the phase diagram.

Zn2+ Binds to Phosphatidylserine and Induces Membrane Blebbing.

Herein, we show that Zn2+ binds to phosphatidylserine (PS) lipids in supported lipid bilayers (SLBs), forming a PS-Zn2+ complex with an equilibrium dissociation constant of ∼100 µM. Significantly, Zn2+ binding to SLBs containing more than 10 mol % PS induces extensive reordering of the bilayer. This reordering is manifest through bright spots of high fluorescence intensity that can be observed when the bilayer contains a dye-labeled lipid. Measurements using atomic force microscopy (AFM) reveal that these spots represent three-dimensional unilamellar blebs. Bleb formation is ion specific, inducible by exposing the bilayer to µM concentrations of Zn2+ but not Mg2+, Cu2+, Co2+, or Mn2+. Moreover, Ca2+ can induce some blebbing at mM concentrations but not nearly as effectively as Zn2+. The interactions of divalent metal cations with PS lipids were further investigated by a combination of vibrational sum frequency spectroscopy (VSFS) and surface pressure-area isotherm measurements. VSFS revealed that Zn2+ and Ca2+ were bound to the phosphate and carboxylate moieties on PS via contact ion pairing, dehydrating the lipid headgroup, whereas Mg2+ and Cu2+ were bound without perturbing the hydration of these functional groups. Additionally, Zn2+ was found to dramatically reduce the area per lipid in lipid monolayers, while Mg2+ and Cu2+ did not. Ca2+ could also reduce the area per lipid but only when significantly higher surface pressures were applied. These measurements suggest that Zn2+ caused lipid blebbing by decreasing the area per lipid on the side of the bilayer to which the salt was exposed. Such findings have implications for blebbing, fusion, oxidation, and related properties of PS-rich membranes in biological systems where Zn2+ concentrations are asymmetrically distributed.

Immobilization of Phosphatidylinositides Revealed by Bilayer Leaflet Decoupling.

Phosphatidylinositol 4,5-bisphosphate (PIP2) has a significantly lower mobile fraction than most other lipids in supported lipid bilayers (SLBs). Moreover, the fraction of mobile PIP2 continuously decreases with time. To explore this, a bilayer unzipping technique was designed to uncouple the two leaflets of the SLB. The results demonstrate that PIP2 molecules in the top leaflet are fully mobile, while the PIP2 molecules in the lower leaflet are immobilized on the oxide support. Over time, mobile PIP2 species flip from the top leaflet to the bottom leaflet and become trapped. It was found that PIP2 flipped between the leaflets through a defect-mediated process. The flipping could be completely inhibited when holes in the bilayer were backfilled with bovine serum albumin (BSA). Moreover, by switching from H2O to D2O, it was shown that the primary interaction between PIP2 and the underlying substrate was due to hydrogen bond formation, which outcompeted electrostatic repulsion. Using substrates with fewer surface silanol groups, like oxidized polydimethylsiloxane, led to a large increase in the mobile fraction of PIP2. Moreover, PIP2 immobilization also occurred when the bilayer was supported on a protein surface rather than glass. These results may help to explain the behavior of PIP2 on the inner leaflet of the plasma membrane, where it is involved in attaching the membrane to the underlying cytoskeleton.

Molecular Mechanism for the Interactions of Hofmeister Cations with Macromolecules in Aqueous Solution.

Ion identity and concentration influence the solubility of macromolecules. To date, substantial effort has been focused on obtaining a molecular level understanding of specific effects for anions. By contrast, the role of cations has received significantly less attention and the underlying mechanisms by which cations interact with macromolecules remain more elusive. To address this issue, the solubility of poly(N-isopropylacrylamide), a thermoresponsive polymer with an amide moiety on its side chain, was studied in aqueous solutions with a series of nine different cation chloride salts as a function of salt concentration. Phase transition temperature measurements were correlated to molecular dynamics simulations. The results showed that although all cations were on average depleted from the macromolecule/water interface, more strongly hydrated cations were able to locally accumulate around the amide oxygen. These weakly favorable interactions helped to partially offset the salting-out effect. Moreover, the cations approached the interface together with chloride counterions in solvent-shared ion pairs. Because ion pairing was concentration-dependent, the mitigation of the dominant salting-out effect became greater as the salt concentration was increased. Weakly hydrated cations showed less propensity for ion pairing and weaker affinity for the amide oxygen. As such, there was substantially less mitigation of the net salting-out effect for these ions, even at high salt concentrations.

Trimethylamine N-oxide stabilizes proteins via a distinct mechanism compared with betaine and glycine.

We report experimental and computational studies investigating the effects of three osmolytes, trimethylamine N-oxide (TMAO), betaine, and glycine, on the hydrophobic collapse of an elastin-like polypeptide (ELP). All three osmolytes stabilize collapsed conformations of the ELP and reduce the lower critical solution temperature (LSCT) linearly with osmolyte concentration. As expected from conventional preferential solvation arguments, betaine and glycine both increase the surface tension at the air-water interface. TMAO, however, reduces the surface tension. Atomically detailed molecular dynamics (MD) simulations suggest that TMAO also slightly accumulates at the polymer-water interface, whereas glycine and betaine are strongly depleted. To investigate alternative mechanisms for osmolyte effects, we performed FTIR experiments that characterized the impact of each cosolvent on the bulk water structure. These experiments showed that TMAO red-shifts the OH stretch of the IR spectrum via a mechanism that was very sensitive to the protonation state of the NO moiety. Glycine also caused a red shift in the OH stretch region, whereas betaine minimally impacted this region. Thus, the effects of osmolytes on the OH spectrum appear uncorrelated with their effects upon hydrophobic collapse. Similarly, MD simulations suggested that TMAO disrupts the water structure to the least extent, whereas glycine exerts the greatest influence on the water structure. These results suggest that TMAO stabilizes collapsed conformations via a mechanism that is distinct from glycine and betaine. In particular, we propose that TMAO stabilizes proteins by acting as a surfactant for the heterogeneous surfaces of folded proteins.

Nonadditive Ion Effects Drive Both Collapse and Swelling of Thermoresponsive Polymers in Water.

When a mixture of two salts in an aqueous solution contains a weakly and a strongly hydrated anion, their combined effect is nonadditive. Herein, we report such nonadditive effects on the lower critical solution temperature (LCST) of poly( N-isopropylacrylamide) (PNiPAM) for a fixed concentration of Na2SO4 and an increasing concentration of NaI. Using molecular dynamics simulations and vibrational sum frequency spectroscopy, we demonstrate that at low concentrations of the weakly hydrated anion (I-), the cations (Na+) preferentially partition to the counterion cloud around the strongly hydrated anion (SO42-), leaving I- more hydrated. However, upon further increase in the NaI concentration, this weakly hydrated anion is forced out of solution to the polymer/water interface by sulfate. Thus, the LCST behavior of PNiPAM involves competing roles for ion hydration and polymer-iodide interactions. This concept can be generally applied to mixtures containing both a strongly and a weakly hydrated anion from the Hofmeister series.

Counter Cations Affect Transport in Aqueous Hydroxide Solutions with Ion Specificity.

The anomalously high mobility of hydroxide and hydronium ions in aqueous solutions is related to proton transfer and structural diffusion. The role of counterions in these solutions, however, is often considered to be negligible. Herein, we explore the impact of alkali metal counter cations on hydroxide solvation and mobility. Impedance measurements demonstrate that hydroxide mobility is attenuated by lithium relative to sodium and potassium. These results are explained by ab initio molecular dynamics simulations and experimental vibrational hydration shell spectroscopy, which reveal substantially stronger ion pairing between OH- and Li+ than with other cations. Hydration shell spectra and theoretical vibrational frequency calculations together imply that lithium and sodium cations have different effects on the delocalization of water protons donating a hydrogen bond to hydroxide. Specifically, lithium leads to enhanced proton delocalization compared with sodium. However, proton delocalization and the overall diffusion process are not necessarily correlated.

Modulation of Cu2+ Binding to Sphingosine-1-Phosphate by Lipid Charge.

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that is thought to participate in the regulation of many physiological processes and may play a key role in several diseases. Herein, we found that Cu2+ binds tightly to supported lipid bilayers (SLBs) containing S1P. Specifically, we demonstrated via fluorescence assays that Cu2+-S1P binding was bivalent and sensitive to the concentration of S1P in the SLB. In fact, the apparent equilibrium dissociation constant, KDApp, tightened by a factor of 132 from 4.5 µM to 34 nM as the S1P density was increased from 5.0 to 20 mol %. A major driving force for this apparent tightening was the more negative surface potential with increasing S1P concentration. This potential remained unaltered upon Cu2+ binding at pH 7.4 because two protons were released for every Cu2+ that bound. At pH 5.4, however, Cu2+ could not outcompete protons for the amine and no binding occurred. Moreover, at pH 9.4, the amine was partially deprotonated before Cu2+ binding and the surface potential became more positive on binding. The results for Cu2+-S1P binding were reminiscent of those for Cu2+-phosphatidylserine binding, where a carboxylate group helped to deprotonate the amine. In the case of S1P, however, the phosphate needed to bear two negative charges to facilitate amine deprotonation in the presence of Cu2+.

Multistep Interactions between Ibuprofen and Lipid Membranes.

Ibuprofen (IBU) interacts with phosphatidylcholine membranes in three distinct steps as a function of concentration. In a first step (<10 µM), IBU electrostatically adsorbs to the lipid headgroups and gradually decreases the interfacial potential. This first step helps to facilitate the second step (10-300 µM), in which hydrophobic insertion of the drug occurs. The second step disrupts the packing of the lipid acyl chains and expands the area per lipid. In a final step, above 300 µM IBU, the lipid membrane begins to solubilize, resulting in a detergent-like effect. The results described herein were obtained by a combination of fluorescence binding assays, vibrational sum frequency spectroscopy, and Langmuir monolayer compression experiments. By introducing trimethylammonium-propane, phosphatidylglycerol, and phosphatidylethanolamine lipids as well as cholesterol, we demonstrated that both the chemistry of the lipid headgroups and the packing of lipid acyl chains can substantially influence the interactions between IBU and the membranes. Moreover, different membrane chemistries can alter particular steps in the binding interaction.

Calcium Directly Regulates Phosphatidylinositol 4,5-Bisphosphate Headgroup Conformation and Recognition.

The orchestrated recognition of phosphoinositides and concomitant intracellular release of Ca2+ is pivotal to almost every aspect of cellular processes, including membrane homeostasis, cell division and growth, vesicle trafficking, as well as secretion. Although Ca2+ is known to directly impact phosphoinositide clustering, little is known about the molecular basis for this or its significance in cellular signaling. Here, we study the direct interaction of Ca2+ with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), the main lipid marker of the plasma membrane. Electrokinetic potential measurements of PI(4,5)P2 containing liposomes reveal that Ca2+ as well as Mg2+ reduce the zeta potential of liposomes to nearly background levels of pure phosphatidylcholine membranes. Strikingly, lipid recognition by the default PI(4,5)P2 lipid sensor, phospholipase C delta 1 pleckstrin homology domain (PLC δ1-PH), is completely inhibited in the presence of Ca2+, while Mg2+ has no effect with 100 nm liposomes and modest effect with giant unilamellar vesicles. Consistent with biochemical data, vibrational sum frequency spectroscopy and atomistic molecular dynamics simulations reveal how Ca2+ binding to the PI(4,5)P2 headgroup and carbonyl regions leads to confined lipid headgroup tilting and conformational rearrangements. We rationalize these findings by the ability of calcium to block a highly specific interaction between PLC δ1-PH and PI(4,5)P2, encoded within the conformational properties of the lipid itself. Our studies demonstrate the possibility that switchable phosphoinositide conformational states can serve as lipid recognition and controlled cell signaling mechanisms.

Guanidinium can both Cause and Prevent the Hydrophobic Collapse of Biomacromolecules.

A combination of Fourier transform infrared and phase transition measurements as well as molecular computer simulations, and thermodynamic modeling were performed to probe the mechanisms by which guanidinium (Gnd+) salts influence the stability of the collapsed versus uncollapsed state of an elastin-like polypeptide (ELP), an uncharged thermoresponsive polymer. We found that the cation's action was highly dependent upon the counteranion with which it was paired. Specifically, Gnd+ was depleted from the ELP/water interface and was found to stabilize the collapsed state of the macromolecule when paired with well-hydrated anions such as SO42-. Stabilization in this case occurred via an excluded volume (or depletion) effect, whereby SO42- was strongly partitioned away from the ELP/water interface. Intriguingly, at low salt concentrations, Gnd+ was also found to stabilize the collapsed state of the ELP when paired with SCN-, which is a strong binder for the ELP. In this case, the anion and cation were both found to be enriched in the collapsed state of the polymer. The collapsed state was favored because the Gnd+ cross-linked the polymer chains together. Moreover, the anion helped partition Gnd+ to the polymer surface. At higher salt concentrations (>1.5 M), GndSCN switched to stabilizing the uncollapsed state because a sufficient amount of Gnd+ and SCN- partitioned to the polymer surface to prevent cross-linking from occurring. Finally, in a third case, it was found that salts which interacted in an intermediate fashion with the polymer (e.g., GndCl) favored the uncollapsed conformation at all salt concentrations. These results provide a detailed, molecular-level, mechanistic picture of how Gnd+ influences the stability of polypeptides in three distinct physical regimes by varying the anion. It also helps explain the circumstances under which guanidinium salts can act as powerful and versatile protein denaturants.

Supported Lipid Bilayers with Phosphatidylethanolamine as the Major Component.

Phosphatidylethanolamine (PE) is notoriously difficult to incorporate into model membrane systems, such as fluid supported lipid bilayers (SLBs), at high concentrations because of its intrinsic negative curvature. Using fluorescence-based techniques, we demonstrate that having fewer sites of unsaturation in the lipid tails leads to high-quality SLBs because these lipids help to minimize the curvature. Moreover, shorter saturated chains can help maintain the membranes in the fluid phase. Using these two guidelines, we find that up to 70 mol % PE can be incorporated into SLBs at room temperature and up to 90 mol % PE can be incorporated at 37 °C. Curiously, conditions under which three-dimensional tubules project outward from the planar surface as well as conditions under which domain formation occurs can be found. We have employed these model membrane systems to explore the ability of Ni2+ to bind to PE. It was found that this transition metal ion binds 1000-fold tighter to PE than to phosphatidylcholine lipids. In the future, this platform could be exploited to monitor the binding of other transition metal ions or the binding of antimicrobial peptides. It could also be employed to explore the physical properties of PE-containing membranes, such as phase domain behavior and intermolecular hydrogen bonding.

Cu(2+) Binds to Phosphatidylethanolamine and Increases Oxidation in Lipid Membranes.

Herein, we demonstrate that Cu(2+) binds bivalently to phosphatidylethanolamine (PE), the second most abundant lipid in mammalian cells. The apparent equilibrium dissociation constant, K(DApp), for the Cu(2+)-PE complex at physiological pH is approximately 2 µM and is insensitive to the concentration of PE in the membrane. By contrast, at pH 10.0, where PE lipids bear a negative charge, K(DApp) decreases with increasing PE content and has a value of 150 nM for bilayers containing 70 mol % PE. The oxidation of double bonds in PE-containing bilayers can be monitored in the presence of Cu(2+). Strikingly, it was found that the oxidation rate is 8.2 times faster at pH 7.4 for bilayers containing 70 mol % PE than for pure phosphatidylcholine (PC) bilayers upon exposure of both to 70 µM Cu(2+) and 10 mM hydrogen peroxide. The rate of oxidation increases linearly with the PE content in the membrane. These results may help explain the high level of lipid oxidation in PE-containing membranes for neurodegenerative diseases and autism where the Cu(2+) concentration in the body is abnormally high.

Unquenchable Surface Potential Dramatically Enhances Cu(2+) Binding to Phosphatidylserine Lipids.

Herein, the apparent equilibrium dissociation constant, K(Dapp), between Cu(2+) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS), a negatively charged phospholipid, was measured as a function of PS concentrations in supported lipid bilayers (SLBs). The results indicated that K(Dapp) for Cu(2+) binding to PS-containing SLBs was enhanced by a factor of 17,000 from 110 nM to 6.4 pM as the PS density in the membrane was increased from 1.0 to 20 mol %. Although Cu(2+) bound bivalently to POPS at higher PS concentrations, this was not the dominant factor in increasing the binding affinity. Rather, the higher concentration of Cu(2+) within the double layer above the membrane was largely responsible for the tightening. Unlike the binding of other divalent metal ions such as Ca(2+) and Mg(2+) to PS, Cu(2+) binding does not alter the net negative charge on the membrane as the Cu(PS)2 complex forms. As such, the Cu(2+) concentration within the double layer region was greatly amplified relative to its concentration in bulk solution as the PS density was increased. This created a far larger enhancement to the apparent binding affinity than is observed by standard multivalent effects. These findings should help provide an understanding on the extent of Cu(2+)-PS binding in cell membranes, which may be relevant to biological processes such as amyloid-ß peptide toxicity and lipid oxidation.

Simultaneous Detection of Multiple Proteins that Bind to the Identical Ligand in Supported Lipid Bilayers.

Herein, we developed a new separation-based detection method that is capable of simultaneously identifying multiple competitively binding proteins for the same ligand on supported lipid bilayers (SLBs). This strategy used unlabeled target analyte proteins that bind to fluorescently tagged, lipid-conjugated ligands within the SLB. The protein-ligand binding complexes were then focused under an applied potential to different locations within the SLB based on each protein's size and charge. Both protein identity and relative surface concentration information could be obtained, simultaneously. Specifically, the competitive binding of streptavidin and goat anti-biotin for biotin-conjugated lipids was explored. It was found that streptavidin could inhibit the binding of goat anti-biotin antibodies for biotin-cap-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)(biotin-cap-NBD-PE) lipids and that streptavidin more effectively outcompeted the anti-biotin antibody at lower protein concentrations. Also, modulating the chemical composition of the membrane helped control the ultimate focusing position and separation of the streptavidin-bound biotin, anti-biotin-bound biotin, and free biotin-conjugated lipid bands. The assay developed herein provides a simple and convenient strategy for simultaneously monitoring target analytes that bind to the identical ligand and may ultimately be useful in developing assays that help overcome problems associated with cross-reactivity.

An NH moiety is not required for anion binding to amides in aqueous solution.

Herein, we use a combination of thermodynamic and spectroscopic measurements to investigate the interactions of Hofmeister anions with a thermoresponsive polymer, poly(N,N-diethylacrylamide) (PDEA). This amide-based polymer does not contain an NH moiety in its chemical structure and, thus, can serve as a model to test if anions bind to amides in the absence of an NH site. The lower critical solution temperature (LCST) of PDEA was measured as a function of the concentration for 11 sodium salts in aqueous solutions, and followed a direct Hofmeister series for the ability of anions to precipitate the polymer. More strongly hydrated anions (CO3(2-), SO4(2-), S2O3(2-), H2PO4(-), F(-), and Cl(-)) linearly decreased the LCST of the polymer with increasing the salt concentration. Weakly hydrated anions (SCN(-), ClO4(-), I(-), NO3(-), and Br(-)) increased the LCST at lower salt concentrations but salted the polymer out at higher salt concentrations. Proton nuclear magnetic resonance (NMR) was used to probe the mechanism of the salting-in effect and showed apparent binding between weakly hydrated anions (SCN(-) and I(-)) and the α protons of the polymer backbone. Additional experiments performed by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy found little change in the amide I band upon the addition of salt, which is consistent with very limited, if any, interactions between the salt ions and the carbonyl moiety of the amide. These results support a molecular mechanism for ion-specific effects on proteins and model amides that does not specifically require an NH group to interact with the anions for the salting-in effect to occur.

Monitoring phosphatidic acid formation in intact phosphatidylcholine bilayers upon phospholipase D catalysis.

We have monitored the production of the negatively charged lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid acid (POPA), in supported lipid bilayers via the enzymatic hydrolysis of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC), a zwitterionic lipid. Experiments were performed with phospholipase D (PLD) in a Ca(2+) dependent fashion. The strategy for doing this involved using membrane-bound streptavidin as a biomarker for the charge on the membrane. The focusing position of streptavidin in electrophoretic-electroosmotic focusing (EEF) experiments was monitored via a fluorescent tag on this protein. The negative charge increased during these experiments due to the formation of POPA lipids. This caused the focusing position of streptavidin to migrate toward the negatively charged electrode. With the use of a calibration curve, the amount of POPA generated during this assay could be read out from the intact membrane, an objective that has been otherwise difficult to achieve because of the lack of unique chromophores on PA lipids. On the basis of these results, other enzymatic reactions involving the change in membrane charge could also be monitored in a similar way. This would include phosphorylation, dephosphorylation, lipid biosynthesis, and additional phospholipase reactions.

Cations bind only weakly to amides in aqueous solutions.

We investigated salt interactions with butyramide as a simple mimic of cation interactions with protein backbones. The experiments were performed in aqueous metal chloride solutions using two spectroscopic techniques. In the first, which provided information about contact pair formation, the response of the amide I band to the nature and concentration of salt was monitored in bulk aqueous solutions via attenuated total reflection Fourier transform infrared spectroscopy. It was found that molar concentrations of well-hydrated metal cations (Ca(2+), Mg(2+), Li(+)) led to the rise of a peak assigned to metal cation-bound amides (1645 cm(-1)) and a decrease in the peak associated with purely water-bound amides (1620 cm(-1)). In a complementary set of experiments, the effect of cation identity and concentration was investigated at the air/butyramide/water interface via vibrational sum frequency spectroscopy. In these studies, metal ion-amide binding led to the ordering of the adjacent water layer. Such experiments were sensitive to the interfacial partitioning of cations in either a contact pair with the amide or as a solvent separated pair. In both experiments, the ordering of the interactions of the cations was: Ca(2+) > Mg(2+) > Li(+) > Na(+) ≈ K(+). This is a direct cationic Hofmeister series. Even for Ca(2+), however, the apparent equilibrium dissociation constant of the cation with the amide carbonyl oxygen was no tighter than ∼8.5 M. For Na(+) and K(+), no evidence was found for any binding. As such, the interactions of metal cations with amides are far weaker than the analogous binding of weakly hydrated anions.