The Role of Ligand-Ligand Interactions in Multimodal Ligand Conformational Equilibria and Surface Pattern Formation.

Multimodal chromatography uses multiple modes of interaction such as charge, hydrophobic, or hydrogen bonding to separate proteins. Recently, we used molecular dynamics (MD) simulations to show that ligands immobilized on surfaces can interact and associate with neighboring ligands to form hydrophobic and charge patches, which may have important implications for the nature of protein-surface interactions. Here, we study interfacial systems of increasing complexity-from a single immobilized multimodal ligand to high density surfaces-to better understand how ligand behavior is affected by the presence of a surface and the presence of other ligands in the vicinity, and how this behavior scales to larger systems. We find that tethering a ligand to a surface restricts its conformations to a subset of those observed in free solution, yet the ligand maintains flexibility in the plane of the surface and can form contacts with neighboring ligands. We find that although the formation of a contact between two neighboring ligands is slightly unfavorable, three neighboring ligands exhibit a preference for the formation of a fully connected cluster. To explore how these trends in ligand association extend to a larger surface with high density of ligands, we performed coarse-grained Monte Carlo (MC) simulations of a 132-ligand surface using ligand interactions parametrized based on free energies obtained from the three-ligand MD simulations. Despite their simplicity, the coarse-grained simulations qualitatively capture the cluster size distribution of ligands observed in detailed MD simulations. Quantitative differences between the two suggest opportunities for improvements in the coarse-grained energy function for efficient predictions of cluster and pattern formations. Our approach presents a promising route to the engineering of multimodal patterns for future chromatographic resin design.

Hydrophobicity of proteins and nanostructured solutes is governed by topographical and chemical context.

Hydrophobic interactions drive many important biomolecular self-assembly phenomena. However, characterizing hydrophobicity at the nanoscale has remained a challenge due to its nontrivial dependence on the chemistry and topography of biomolecular surfaces. Here we use molecular simulations coupled with enhanced sampling methods to systematically displace water molecules from the hydration shells of nanostructured solutes and calculate the free energetics of interfacial water density fluctuations, which quantify the extent of solute-water adhesion, and therefore solute hydrophobicity. In particular, we characterize the hydrophobicity of curved graphene sheets, self-assembled monolayers (SAMs) with chemical patterns, and mutants of the protein hydrophobin-II. We find that water density fluctuations are enhanced near concave nonpolar surfaces compared with those near flat or convex ones, suggesting that concave surfaces are more hydrophobic. We also find that patterned SAMs and protein mutants, having the same number of nonpolar and polar sites but different geometrical arrangements, can display significantly different strengths of adhesion with water. Specifically, hydroxyl groups reduce the hydrophobicity of methyl-terminated SAMs most effectively not when they are clustered together but when they are separated by one methyl group. Hydrophobin-II mutants show that a charged amino acid reduces the hydrophobicity of a large nonpolar patch when placed at its center, rather than at its edge. Our results highlight the power of water density fluctuations-based measures to characterize the hydrophobicity of nanoscale surfaces and caution against the use of additive approximations, such as the commonly used surface area models or hydropathy scales for characterizing biomolecular hydrophobicity and the associated driving forces of assembly.

Formation of Ligand Clusters on Multimodal Chromatographic Surfaces.

Multimodal chromatography is a powerful tool which uses multiple modes of interaction, such as charge and hydrophobicity, to purify protein-based therapeutics. In this work, we performed molecular dynamics simulations of a series of multimodal cation-exchange ligands immobilized on a hydrophilic self-assembled monolayer surface at the commercially relevant surface density (1 ligand/nm2). We found that ligands that were flexible and terminated in a hydrophobic group had a propensity to aggregate on the surface, while less flexible ligands containing a hydrophobic group closer to the surface did not aggregate. For aggregating ligands, this resulted in the formation of a surface pattern that contained relatively large patches of hydrophobicity and charge whose sizes exceeded the length scale of the individual ligands. On the other hand, lowering the surface density to 1 ligand/3 nm2 reduced or eliminated this aggregation behavior. In addition, the introduction of a flexible linker (corresponding to the commercially available ligand) enhanced cluster formation and allowed aggregation to occur at lower surface densities. Further, the use of flexible linkers enabled hydrophobic groups to collapse to the surface, reducing their accessibility. Finally, we developed an approach for quantifying differences in the observed surface patterns by calculating distributions of the patch size and patch length. This clustering phenomenon is likely to play a key role in governing protein-surface interactions in multimodal chromatography. This new understanding of multimodal surfaces has important implications for developing improved predictive models and designing new classes of multimodal separation materials.

Arginine mutations in antibody complementarity-determining regions display context-dependent affinity/specificity trade-offs.

Antibodies commonly accumulate charged mutations in their complementarity-determining regions (CDRs) during affinity maturation to enhance electrostatic interactions. However, charged mutations can mediate non-specific interactions, and it is unclear to what extent CDRs can accumulate charged residues to increase antibody affinity without compromising specificity. This is especially concerning for positively charged CDR mutations that are linked to antibody polyspecificity. To better understand antibody affinity/specificity trade-offs, we have selected single-chain antibody fragments specific for the negatively charged and hydrophobic Alzheimer's amyloid ß peptide using weak and stringent selections for antibody specificity. Antibody variants isolated using weak selections for specificity were enriched in arginine CDR mutations and displayed low specificity. Alanine-scanning mutagenesis revealed that the affinities of these antibodies were strongly dependent on their arginine mutations. Antibody variants isolated using stringent selections for specificity were also enriched in arginine CDR mutations, but these antibodies possessed significant improvements in specificity. Importantly, the affinities of the most specific antibodies were much less dependent on their arginine mutations, suggesting that over-reliance on arginine for affinity leads to reduced specificity. Structural modeling and molecular simulations reveal unique hydrophobic environments near the arginine CDR mutations. The more specific antibodies contained arginine mutations in the most hydrophobic portions of the CDRs, whereas the less specific antibodies contained arginine mutations in more hydrophilic regions. These findings demonstrate that arginine mutations in antibody CDRs display context-dependent impacts on specificity and that affinity/specificity trade-offs are governed by the relative contribution of arginine CDR residues to the overall antibody affinity.

Pathways to dewetting in hydrophobic confinement.

Liquid water can become metastable with respect to its vapor in hydrophobic confinement. The resulting dewetting transitions are often impeded by large kinetic barriers. According to macroscopic theory, such barriers arise from the free energy required to nucleate a critical vapor tube that spans the region between two hydrophobic surfaces--tubes with smaller radii collapse, whereas larger ones grow to dry the entire confined region. Using extensive molecular simulations of water between two nanoscopic hydrophobic surfaces, in conjunction with advanced sampling techniques, here we show that for intersurface separations that thermodynamically favor dewetting, the barrier to dewetting does not correspond to the formation of a (classical) critical vapor tube. Instead, it corresponds to an abrupt transition from an isolated cavity adjacent to one of the confining surfaces to a gap-spanning vapor tube that is already larger than the critical vapor tube anticipated by macroscopic theory. Correspondingly, the barrier to dewetting is also smaller than the classical expectation. We show that the peculiar nature of water density fluctuations adjacent to extended hydrophobic surfaces--namely, the enhanced likelihood of observing low-density fluctuations relative to Gaussian statistics--facilitates this nonclassical behavior. By stabilizing isolated cavities relative to vapor tubes, enhanced water density fluctuations thus stabilize novel pathways, which circumvent the classical barriers and offer diminished resistance to dewetting. Our results thus suggest a key role for fluctuations in speeding up the kinetics of numerous phenomena ranging from Cassie-Wenzel transitions on superhydrophobic surfaces, to hydrophobically driven biomolecular folding and assembly.

Single Molecule Force Spectroscopy and Molecular Dynamics Simulations as a Combined Platform for Probing Protein Face-Specific Binding.

Biomolecular interactions frequently occur in orientation-specific manner. For example, prior nuclear magnetic resonance spectroscopy experiments in our lab have suggested the presence of a group of strongly binding residues on a particular face of the protein ubiquitin for interactions with Capto MMC multimodal ligands ("Capto" ligands) (Srinivasan, K.; et al. Langmuir 2014, 30 (44), 13205-13216). We present a clear confirmation of those studies by performing single-molecule force spectroscopy (SMFS) measurements of unbinding complemented with molecular dynamics (MD) calculations of the adsorption free energy of ubiquitin in two distinct orientations with self-assembled monolayers (SAMs) functionalized with "Capto" ligands. These orientations were maintained in the SMFS experiments by tethering ubiquitin mutants to SAM surfaces through strategically located cysteines, thus exposing the desired faces of the protein. Analogous orientations were maintained in MD simulations using suitable constraining methods. Remarkably, despite differences between the finer details of experimental and simulation methodologies, they confirm a clear preference for the previously hypothesized binding face of ubiquitin. Furthermore, MD simulations provided significant insights into the mechanism of protein binding onto this multimodal surface. Because SMFS and MD simulations both directly probe protein-surface interactions, this work establishes a key link between experiments and simulations at molecular scale through the determination of protein face-specific binding energetics. Our approach may have direct applications in biophysical systems where face- or orientation-specific interactions are important, such as biomaterials, sensors, and biomanufacturing.

Water-mediated ion-ion interactions are enhanced at the water vapor-liquid interface.

There is overwhelming evidence that ions are present near the vapor-liquid interface of aqueous salt solutions. Charged groups can also be driven to interfaces by attaching them to hydrophobic moieties. Despite their importance in many self-assembly phenomena, how ion-ion interactions are affected by interfaces is not understood. We use molecular simulations to show that the effective forces between small ions change character dramatically near the water vapor-liquid interface. Specifically, the water-mediated attraction between oppositely charged ions is enhanced relative to that in bulk water. Further, the repulsion between like-charged ions is weaker than that expected from a continuum dielectric description and can even become attractive as the ions are drawn to the vapor side. We show that thermodynamics of ion association are governed by a delicate balance of ion hydration, interfacial tension, and restriction of capillary fluctuations at the interface, leading to nonintuitive phenomena, such as water-mediated like charge attraction. "Sticky" electrostatic interactions may have important consequences on biomolecular structure, assembly, and aggregation at soft liquid interfaces. We demonstrate this by studying an interfacially active model peptide that changes its structure from α-helical to a hairpin-turn-like one in response to charging of its ends.

Role of arginine in mediating protein-carbon nanotube interactions.

Arginine-rich proteins (e.g., lysozyme) or poly-L-arginine peptides have been suggested as solvating and dispersing agents for single-wall carbon nanotubes (CNTs) in water. In addition, protein structure-function in porous and hydrophobic materials is of broad interest. The amino acid residue, arginine (Arg(+)), has been implicated as an important mediator of protein/peptide-CNT interactions. To understand the structural and thermodynamic aspects of this interaction at the molecular level, we employ molecular dynamics (MD) simulations of the protein lysozyme in the interior of a CNT, as well as of free solutions of Arg(+) in the presence of a CNT. To dissect the Arg(+)-CNT interaction further, we also perform simulations of aqueous solutions of the guanidinium ion (Gdm(+)) and the norvaline (Nva) residue in the presence of a CNT. We show that the interactions of lysozyme with the CNT are mediated by the surface Arg(+) residues. The strong interaction of Arg(+) residue with the CNT is primarily driven by the favorable interactions of the Gdm(+) group with the CNT wall. The Gdm(+) group is not as well-hydrated on its flat sides, which binds to the CNT wall. This is consistent with a similar binding of Gdm(+) ions to a hydrophobic polymer. In contrast, the Nva residue, which lacks the Gdm(+) group, binds to the CNT weakly. We present details of the free energy of binding, molecular structure, and dynamics of these solutes on the CNT surface. Our results highlight the important role of Arg(+) residues in protein-CNT or protein-carbon-based material interactions. Such interactions could be manipulated precisely through protein engineering, thereby offering control over protein orientation and structure on CNTs, graphene, or other hydrophobic interfaces.

Interactions of Multimodal Ligands with Proteins: Insights into Selectivity Using Molecular Dynamics Simulations.

Fundamental understanding of protein-ligand interactions is important to the development of efficient bioseparations in multimodal chromatography. Here we employ molecular dynamics (MD) simulations to investigate the interactions of three different proteins--ubiquitin, cytochrome C, and α-chymotrypsinogen A, sampling a range of charge from +1e to +9e--with two multimodal chromatographic ligands containing similar chemical moieties--aromatic, carboxyl, and amide--in different structural arrangements. We use a spherical harmonic expansion to analyze ligand and individual moiety density profiles around the proteins. We find that the Capto MMC ligand, which contains an additional aliphatic group, displays stronger interactions than Nuvia CPrime ligand with all three proteins. Studying the ligand densities at the moiety level suggests that hydrophobic interactions play a major role in determining the locations of high ligand densities. Finally, the greater structural flexibility of the Capto MMC ligand compared to that of the Nuvia cPrime ligand allows for stronger structural complementarity and enables stronger hydrophobic interactions. These subtle and not-so-subtle differences in binding affinities and modalities for multimodal ligands can result in significantly different binding behavior towards proteins with important implications for bioprocessing.

Binding, structure, and dynamics of hydrophobic polymers near patterned self-assembled monolayer surfaces.

We use molecular dynamics simulations to study the binding, conformations, and dynamics of a flexible 25-mer hydrophobic polymer near well-defined patterned self-assembled monolayers containing a hydrophobic strip (with -CH3 head-groups) having different widths in a hydrophilic (-OH) background. We show that the polymer binds favorably to hydrophobic strips of all widths, including the subnanometer ones comprising 3, 2, or even 1 row of -CH3 head-groups, with the binding strength varying from about 107 to 25 kJ/mol for the widest to the narrowest strip. Near wide hydrophobic patches containing 5 or more -CH3 rows, pancakelike conformations are dominant, whereas hairpinlike structures become preferred ones near the narrower strips. In the vicinity of the narrowest 1-row strip, the polymer folds into semiglobular conformations, thus maintaining sufficient contact with the strip while sequestering its hydrophobic groups away from water. We also show that the confinement makes the translational dynamics of the polymer anisotropic as well as conformational dependent. Our results may help to understand and manipulate the self-assembly and dynamics of soft matter, such as polymers, peptides, and proteins, at inhomogeneous patterned surfaces.

Structure and dynamics of single hydrophobic/ionic heteropolymers at the vapor-liquid interface of water.

We focus on the conformational stability, structure, and dynamics of hydrophobic/charged homopolymers and heteropolymers at the vapor-liquid interface of water using extensive molecular dynamics simulations. Hydrophobic polymers collapse into globular structures in bulk water but unfold and sample a broad range of conformations at the vapor-liquid interface of water. We show that adding a pair of charges to a hydrophobic polymer at the interface can dramatically change its conformations, stabilizing hairpinlike structures, with molecular details depending on the location of the charged pair in the sequence. The translational dynamics of homopolymers and heteropolymers are also different, whereas the homopolymers skate on the interface with low drag, the tendency of charged groups to remain hydrated pulls the heteropolymers toward the liquid side of the interface, thus pinning them, increasing drag, and slowing the translational dynamics. The conformational dynamics of heteropolymers are also slower than that of the homopolymer and depend on the location of the charged groups in the sequence. Conformational dynamics are most restricted for the end-charged heteropolymer and speed up as the charge pair is moved toward the center of the sequence. We rationalize these trends using the fundamental understanding of the effects of the interface on primitive pair-level interactions between two hydrophobic groups and between oppositely charged ions in its vicinity.

Extended surfaces modulate hydrophobic interactions of neighboring solutes.

Interfaces are a most common motif in complex systems. To understand how the presence of interfaces affects hydrophobic phenomena, we use molecular simulations and theory to study hydration of solutes at interfaces. The solutes range in size from subnanometer to a few nanometers. The interfaces are self-assembled monolayers with a range of chemistries, from hydrophilic to hydrophobic. We show that the driving force for assembly in the vicinity of a hydrophobic surface is weaker than that in bulk water and decreases with increasing temperature, in contrast to that in the bulk. We explain these distinct features in terms of an interplay between interfacial fluctuations and excluded volume effects--the physics encoded in Lum-Chandler-Weeks theory [Lum K, Chandler D, Weeks JD (1999) J Phys Chem B 103:4570-4577]. Our results suggest a catalytic role for hydrophobic interfaces in the unfolding of proteins, for example, in the interior of chaperonins and in amyloid formation.

Trimethylamine N-oxide (TMAO) and tert-butyl alcohol (TBA) at hydrophobic interfaces: insights from molecular dynamics simulations.

TMAO, a potent osmolyte, and TBA, a denaturant, have similar molecular architecture but somewhat different chemistry. We employ extensive molecular dynamics simulations to quantify their behavior at vapor-water and octane-water interfaces. We show that interfacial structure-density and orientation-and their dependence on solution concentration are markedly different for the two molecules. TMAO molecules are moderately surface active and adopt orientations with their N-O vector approximately parallel to the aqueous interface. That is, not all methyl groups of TMAO at the interface point away from the water phase. In contrast, TBA molecules act as molecular amphiphiles, are highly surface active, and, at low concentrations, adopt orientations with their methyl groups pointing away and the C-O vector pointing directly into water. The behavior of TMAO at aqueous interfaces is only weakly dependent on its solution concentration, whereas that of TBA depends strongly on concentration. We show that this concentration dependence arises from their different hydrogen bonding capabilities-TMAO can only accept hydrogen bonds from water, whereas TBA can accept (donate) hydrogen bonds from (to) water or other TBA molecules. The ability to self-associate, particularly visible in TBA molecules in the interfacial layer, allows them to sample a broad range of orientations at higher concentrations. In light of the role of TMAO and TBA in biomolecular stability, our results provide a reference with which to compare their behavior near biological interfaces. Also, given the ubiquity of aqueous interfaces in biology, chemistry, and technology, our results may be useful in the design of interfacially active small molecules with the aim to control their orientations and interactions.

Comparative Analysis of Protein Surface Hydrophobicity Maps Determined by Sparse Sampling INDUS and Spatial Aggregation Propensity.

Protein surface hydrophobicity plays a central role in various biological processes such as protein folding and aggregation, as well as in the design and manufacturing of biotherapeutics. While the hydrophobicity of protein surface patches has been linked to their constituent residue hydropathies, recent research has shown that protein surface hydrophobicity is more complex and characterized by the response of water to these surfaces. In this work, we employ water density perturbations to map the surface hydrophobicity of a set of model proteins using sparse indirect umbrella sampling simulations (SSI). This technique is used to identify hydrophobic surface patches for the set of model proteins, and the results are compared to those obtained from the widely adopted spatial aggregation propensity (SAP) technique. While SAP-based calculations show agreement with SSI in some cases, there are several examples of disagreement. We identify four general classes of difference in behavior and study factors that contribute to these differences. We find that the SAP method can sometimes mask the effect of weakly nonpolar or isolated nonpolar residues that can lead to strong hydrophobic patches on the protein surface. In addition, hydrophobic patches identified by SAP can exhibit shifts in both position and strength on the SSI map. Our results demonstrate that the combination of topography and chemical context controls the hydrophobicity of a given patch above and beyond the intrinsic polarity of the residues present on the patch surface. The availability of more accurate protein hydrophobicity maps in concert with new classes of hydrophobic molecular descriptors may create significant opportunities for in silico prediction of protein behavior for a range of applications, such as protein design, biomanufacturability, and downstream bioprocessing.

Characterizing hydrophobicity of interfaces by using cavity formation, solute binding, and water correlations.

Hydrophobicity is often characterized macroscopically by the droplet contact angle. Molecular signatures of hydrophobicity have, however, remained elusive. Successful theories predict a drying transition leading to a vapor-like region near large hard-sphere solutes and interfaces. Adding attractions wets the interface with local density increasing with attractions. Here we present extensive molecular simulation studies of hydration of realistic surfaces with a wide range of chemistries from hydrophobic (-CF(3), -CH(3)) to hydrophilic (-OH, -CONH(2)). We show that the water density near weakly attractive hydrophobic surfaces (e.g., -CF(3)) can be bulk-like or larger, and provides a poor quantification of surface hydrophobicity. In contrast, the probability of cavity formation or the free energy of binding of hydrophobic solutes to interfaces correlates quantitatively with the macroscopic wetting properties and serves as an excellent signature of hydrophobicity. Specifically, the probability of cavity formation is enhanced in the vicinity of hydrophobic surfaces, and water-water correlations correspondingly display characteristics similar to those near a vapor-liquid interface. Hydrophilic surfaces suppress cavity formation and reduce the water-water correlation length. Our results suggest a potentially robust approach for characterizing hydrophobicity of more complex and heterogeneous surfaces of proteins and biomolecules, and other nanoscopic objects.

Connecting Non-Gaussian Water Density Fluctuations to the Lengthscale Dependent Crossover in Hydrophobic Hydration.

We connect density fluctuations in liquid water to lengthscale dependent crossover in hydrophobic hydration. Specifically, we employ indirect umbrella sampling (INDUS) simulations to characterize density fluctuations in observation volumes of various sizes and shapes in water and as a function of temperature and salt concentration. Consistent with previous observations, density fluctuations are Gaussian in small molecular scale volumes, but they display non-Gaussian "low-density fat tails" in larger volumes. These non-Gaussian tails are indicative of the proximity of water to its liquid to vapor phase transition and have implications on biomolecular interactions and function. We show that the onset of non-Gaussian fluctuations in large volumes is accompanied by the formation of a cavity in the observation volume. We develop a model that uses the physics of cavity-water interface formation as a key ingredient and show that it captures the nature of non-Gaussian density fluctuations over a broad region in water and in salt solutions. We discuss the limitations of this model in the very low density region of the distribution. Our calculations provide new insights into the origins of non-Gaussian density fluctuations in water and their connections to lengthscale dependent crossover in hydrophobic hydration.

Bridging Gaussian Density Fluctuations from Microscopic to Macroscopic Volumes: Applications to Non-Polar Solute Hydration Thermodynamics.

The hydration of hydrophobic solutes is intimately related to the spontaneous formation of cavities in water through ambient density fluctuations. Information theory-based modeling and simulations have shown that water density fluctuations in small volumes are approximately Gaussian. For limiting cases of microscopic and macroscopic volumes, water density fluctuations are known exactly and are rigorously related to the density and isothermal compressibility of water. Here, we develop a theory-interpolated gaussian fluctuation theory (IGFT)-that builds an analytical bridge to describe water density fluctuations from microscopic to molecular scales. This theory requires no detailed information about the water structure beyond the effective size of a water molecule and quantities that are readily obtained from water's equation-of-state-namely, the density and compressibility. Using simulations, we show that IGFT provides a good description of density fluctuations near the mean, that is, it characterizes the variance of occupancy fluctuations over all solute sizes. Moreover, when combined with the information theory, IGFT reproduces the well-known signatures of hydrophobic hydration, such as entropy convergence and solubility minima, for atomic-scale solutes smaller than the crossover length scale beyond which the Gaussian assumption breaks down. We further show that near hydrophobic and hydrophilic self-assembled monolayer surfaces in contact with water, the normalized solvent density fluctuations within observation volumes depend similarly on size as observed in the bulk, suggesting the feasibility of a modified version of IGFT for interfacial systems. Our work highlights the utility of a density fluctuation-based approach toward understanding and quantifying the solvation of non-polar solutes in water and the forces that drive them toward surfaces with different hydrophobicities.

Studying pressure denaturation of a protein by molecular dynamics simulations.

Many globular proteins unfold when subjected to several kilobars of hydrostatic pressure. This "unfolding-up-on-squeezing" is counter-intuitive in that one expects mechanical compression of proteins with increasing pressure. Molecular simulations have the potential to provide fundamental understanding of pressure effects on proteins. However, the slow kinetics of unfolding, especially at high pressures, eliminates the possibility of its direct observation by molecular dynamics (MD) simulations. Motivated by experimental results-that pressure denatured states are water-swollen, and theoretical results-that water transfer into hydrophobic contacts becomes favorable with increasing pressure, we employ a water insertion method to generate unfolded states of the protein Staphylococcal Nuclease (Snase). Structural characteristics of these unfolded states-their water-swollen nature, retention of secondary structure, and overall compactness-mimic those observed in experiments. Using conformations of folded and unfolded states, we calculate their partial molar volumes in MD simulations and estimate the pressure-dependent free energy of unfolding. The volume of unfolding of Snase is negative (approximately -60 mL/mol at 1 bar) and is relatively insensitive to pressure, leading to its unfolding in the pressure range of 1500-2000 bars. Interestingly, once the protein is sufficiently water swollen, the partial molar volume of the protein appears to be insensitive to further conformational expansion or unfolding. Specifically, water-swollen structures with relatively low radii of gyration have partial molar volume that are similar to that of significantly more unfolded states. We find that the compressibility change on unfolding is negligible, consistent with experiments. We also analyze hydration shell fluctuations to comment on the hydration contributions to protein compressibility. Our study demonstrates the utility of molecular simulations in estimating volumetric properties and pressure stability of proteins, and can be potentially extended for applications to protein complexes and assemblies.

Self-assembly of TMAO at hydrophobic interfaces and its effect on protein adsorption: insights from experiments and simulations.

We offer a novel process to render hydrophobic surfaces resistant to relatively small proteins during adsorption. This was accomplished by self-assembly of a well-known natural osmolyte, trimethylamine oxide (TMAO), a small amphiphilic molecule, on a hydrophobic alkanethiol surface. Measurements of lysozyme (LYS) adsorption on several homogeneous substrates formed from functionalized alkanethiol self-assembled monolayers (SAMs) in the presence and absence of TMAO, and direct interaction energy between the protein and functionalized surfaces, demonstrate the protein-resistant properties of a noncovalently adsorbed self-assembled TMAO layer. Molecular dynamics simulations clearly show that TMAO molecules concentrate near the CH(3)-SAM surface and are preferentially excluded from LYS. Interestingly, TMAO molecules adsorb strongly on a hydrophobic CH(3)-SAM surface, but a trade-off between hydrogen bonding with water, and hydrophobic interactions with the underlying substrate results in a nonintuitive orientation of TMAO molecules at the interface. Additionally, hydrophobic interactions, usually responsible for nonspecific adsorption of proteins, are weakly affected by TMAO. In addition to TMAO, other osmolytes (sucrose, taurine, and betaine) and a larger homologue of TMAO (N,N-dimethylheptylamine-N-oxide) were tested for protein resistance and only N,N-dimethylheptylamine-N-oxide exhibited resistance similar to TMAO. The principle of osmolyte exclusion from the protein backbone is responsible for the protein-resistant property of the surface. We speculate that this novel process of surface modification may have wide applications due to its simplicity, low cost, regenerability, and flexibility.