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Despite being used for decades as stabilizers, amino acids (AAs) remain mysterious components of many medical and biological formulations. Hypotheses on their role vary ranging from hydrotropic to protein-specific effects (stabilization against misfolding). Here, we deduce that AAs possess a new and broad colloidal property by finding that stabilizing effect of the AAs is comparable on dispersion of various proteins, plasmid DNA, and non-biological nanoparticles. The interactions among colloidal particles in dispersion are carefully evaluated by the second osmotic virial coefficient (B_22) and the potential of mean force. We propose a theoretical framework that explains the stabilization as the effect of weakly interacting small molecules with patchy nanoscale colloids. We validate it through quantitative comparison with experimental data by comparing equilibrium dissociation constants for AA/proteins obtained either by fitting the B22 data with this theory or experimentally. We find excellent quantitative agreement (e.g. proline/lysozyme 1.18 and 2.28 M, respectively) and indeed that the interactions are very weak. The theory presented implies that (i) charged AAs will be effective only for proteins of opposite charge; (ii) short peptides composed of n AAs will be as or more effective than n separate AAs; (iii) any small molecule weakly interacting with nanoscale colloids that increases the solvation of the surface will have a stabilizing effect. The experimental evidences corroborate all three predictions. Much like the ionic strength of the solution is commonly reported, our results imply that the same should be done for the small molecules, as they also affect fundamentally colloidal properties. As an example, we show that AAs vary the cloud point of a lysozyme solution by as much as 4 K.

Protein-protein interactions (PPI) differ when measured in test tubes and cells due to the complexity of the intracellular environment. Free amino acids (AAs) and their derivatives constitute a significant fraction of the intracellular volume and mass. Recently, we have found that AAs have a general property of rendering protein dispersions more stable by reducing the net attractive part of PPI. Here, we study the effects on PPI of different AA derivatives, AA mixtures, and short peptides. We find that all the tested AA derivatives modulate PPI in solution as well as AAs. Furthermore, we show that the modulation effect is additive when AAs form mixtures or are bound into short peptides. Therefore, this study demonstrates the universal effect of a class of small molecules (i.e. AAs and their biological derivatives) on the modulation of PPI and provides insights into rationally designing biocompatible molecules for stabilizing protein interactions and consequently tuning protein functions., Comment: 13 pages, 5 figures

In concentrated macromolecular dispersions, far-from-ideal intermolecular interactions determine the dispersion behaviors including phase transition, crystallization, and liquid-liquid phase separation. Here, we present a novel versatile capillary-cell design for analytical ultracentrifugation-sedimentation equilibrium (AUC-SE), ideal for studying samples at high concentrations. Current setups for such studies are difficult and unreliable to handle, leading to a low experimental success rate. The design presented here is easy to use, robust, and reusable for samples in both aqueous and organic solvents while requiring no special tools or chemical modification of AUC cells. The key and unique feature is the fabrication of liquid reservoirs directly on the bottom window of AUC cells, which can be easily realized by laser ablation or mechanical drilling. The channel length and optical path length are therefore tunable. The success rate for assembling this new cell is close to 100%. We demonstrate the practicality of this cell by studying: 1) the equation of state and second virial coefficients of concentrated gold nanoparticle dispersions in water and bovine serum albumin (BSA) as well as lysozyme solution in aqueous buffers, 2) the gelation phase transition of DNA and BSA solutions, and 3) liquid-liquid phase separation of concentrated BSA/polyethylene glycol (PEG) droplets., Comment: 19 pages, 6 figures

Liquid liquid phase separation (LLPS) of proteins is an intracellular process that is widely used by cells for many purposes. In living cells (in vivo), LLPS occurs in complex and crowded environments. Amino acids (AAs) are vital components of such environments, occupying a significant fraction of the cellular volume. In this work, we studied the effects of proline and other proteinogenic AAs on the LLPS of proteins, both in test tubes (in vitro) and in cells (in vivo). The effects of proline on the protein-protein interaction (PPI) and LLPS of both bovine serum albumin (BSA, a folded protein) and the low-complexity domain of fused in sarcoma (FUS267, an intrinsically disordered protein) is first established in vitro. Then, the effects of proline and other proteinogenic AAs on the formation of stress granules (SGs) by LLPS in U2OS and HeLa cells are studied. We find that the presence of AAs renders the net interaction between proteins more repulsive (i.e. stabilizes protein solution), thus suppressing protein phase separation in vitro and in vivo. We also show that the formation of SGs is suppressed by AAs using both immunofluorescence and live-cell microscopy. Our study reveals an underappreciated role of cellular AAs in modulating intracellular phase separation. It may find biomedical applications, especially in the treatment of protein aggregation diseases., Comment: 15 pages, 4 figures

In this paper, we quantify weak protein protein interactions in solution using Cross-Interaction Chromatography (CIC) and Surface Plasmon Resonance (SPR) and demonstrate that they can be modulated by the addition of free amino acids. With CIC, we determined the second osmotic virial cross-interaction coefficient (B23) as a proxy for the interaction strength between two different proteins. We perform SPR experiments to establish the binding affinity between the same proteins. With CIC, we show that the amino acids proline, glutamine, and arginine render the protein cross-interactions more repulsive or equivalently less attractive. Specifically, we measured B23 between lysozyme (Lys) and bovine serum albumin (BSA) and between Lys and protein isolates (whey and canola). We find that B23 increases when amino acids are added to the solution even at millimolar concentrations, corresponding to protein ligand stoichiometric ratios as low as 1 to 1. With SPR, we show that the binding affinity between proteins can change by one order of magnitude when 10 mM of glutamine are added. In the case of Lys and one whey protein isolate it changes from the mM to the M, thus by three orders of magnitude. Interestingly, this efficient modulation of the protein cross-interactions does not alter the protein's secondary structure. The capacity of amino acids to modulate protein cross-interactions at mM concentrations is remarkable and may have an impact across fields in particular for specific applications in the food or pharmaceutical industries., Comment: 12 pages, 5 figures

We demonstrate that highly-ordered two-dimensional crystals of ligand-capped gold nanoparticles display a local photo-mechanical stiffness as high as that of solids such as graphite. In out-of equilibrium electron diffraction experiments, a strong temperature jump is induced in a thin film with a femtosecond laser pulse. The initial electronic excitation transfers energy to the underlying structural degrees of freedom, with a rate generally proportional to the stiffness of the material. With femtosecond small-angle electron diffraction, we observe the temporal evolution of the diffraction feature associated to the nearest-neighbor nanoparticle distance. The Debye-Waller decay for the octanethiol-capped nanoparticles supracrystal, in particular, is found to be unexpectedly fast, almost as fast as the stiffest solid known and observed by the same technique, i.e. graphite. Our observations unravel that local stiffness in a dense supramolecular assembly can be created by Van der Waals interactions up to a level comparable to crystalline systems characterized by covalent bonding.

In metal nanoparticles (NPs) supracrystals, the metallic core provides some key properties, e.g. magnetization, plasmonic response or conductivity, with the ligand molecules giving rise to others like solubility, assembly or interaction with biomolecules. The formation of these supracrystals depends on a complex interplay between many forces, some stemming from the core, some from the ligands. At present, there is no known approach to characterize the local order of ligand molecules or their dynamics with atomic spatial resolution. Here, we develop a methodology based on small-angle ultrafast electron diffraction combined with angular cross-correlation analysis to characterize a two-dimensional supracrystal of dodecanethiol-coated gold NPs. We retrieve the static arrangement of the ligands, showing that at equilibrium they order in a preferential orientation on the NPs surface and throughout the two-dimensional supracrystal. Upon light excitation, positional disorder is induced in the supracrystal, while its overall homogeneity is surprisingly found to transiently increase. This suggests that transient annealing of the supracrystal takes place within few picoseconds (ps). This methodology will enable the systematic investigation of the dynamical structural properties of nano-assembled materials containing light elements, relevant for biological applications.

The controlled self-assembly of thiol stabilized gold nanocrystals in a mediating solvent and confined within mesoporous alumina was probed in situ with small angle x-ray scattering. The evolution of the self-assembly process was controlled reversibly via regulated changes in the amount of solvent condensed from an undersaturated vapor. Analysis indicated that the nanoparticles self-assembled into cylindrical monolayers within the porous template. Nanoparticle nearest-neighbor separation within the monolayer increased and the ordering decreased with the controlled addition of solvent. The process was reversible with the removal of solvent. Isotropic clusters of nanoparticles were also observed to form temporarily during desorption of the liquid solvent and disappeared upon complete removal of liquid. Measurements of the absorption and desorption of the solvent showed strong hysteresis upon thermal cycling. In addition, the capillary filling transition for the solvent in the nanoparticle-doped pores was shifted to larger chemical potential, relative to the liquid/vapor coexistence, by a factor of 4 as compared to the expected value for the same system without nanoparticles., Comment: 9 pages, 9 figures, appeared in Phys. Rev. B