Macromolecular crowding in biological media is an essential factor for cellular function. The interplay of intermolecular interactions at multiple time and length scales governs a fine-tuned system of reaction and transport processes, including particularly protein diffusion as a limiting or driving factor. Using quasielastic neutron backscattering, we probe the protein self-diffusion in crowded aqueous solutions of bovine serum albumin on nanosecond time and nanometer length scales employing the same protein as crowding agent. The measured diffusion coefficient DðφÞ strongly decreases with increasing protein volume fraction φ explored within 7% ≤ φ ≤ 30%. With an ellipsoidal protein model and an analytical framework involving colloid diffusion theory, we separate the rotational D r ðφÞ and translational D t ðφÞ contributions to DðφÞ. The resulting D t ðφÞ is described by short-time self-diffusion of effective spheres. Protein self-diffusion at biological volume fractions is found to be slowed down to 20% of the dilute limit solely due to hydrodynamic interactions. macromolecular crowding | quasi-elastic neutron scattering | globular proteins T he interior of biological cells is a medium with a macromolecular volume fraction of up to 40%. This crowding crucially affects reaction kinetics and equilibria in the cell (1, 2). Cellular function and structure thus cannot be understood without a systematic understanding of both phase behavior and transport processes in crowded media. Diffusion is the main transport process for systems at low Reynolds numbers, governing many dynamic processes in nature (3). From the perspective of a single tracer molecule, all other molecules act as obstacles. In vivo diffusion coefficients for globular proteins in living cells (4-7) are strongly decreased compared to the in vitro diffusion coefficient in dilute buffer solutions. Systematic measurements of the tracer diffusion of proteins dissolved in concentrated suspensions of crowding agents, i.e., other proteins or polymers, reveal a complex dependence of the slowing down on the combination of tracer molecule and crowding agent (8-10). Furthermore, macromolecular crowding is found to induce subdiffusive behavior in several cases (11,12), being suggested as a slower but more reliable diffusive search process inside the cell (13). This anomalous diffusion process has been found also in theory and simulations (12-15) suggesting a crossover from subdiffusive behavior at small times to diffusive behavior at larger times.Proteins are macromolecules generally with a nonspherical shape and a nonhomogeneous surface charge, showing specific interactions with ligands. Furthermore, proteins not only show global motions like translational and rotational diffusion but also internal and interdomain motions. Therefore, proteins pose a challenge to colloid theory (16,17). In a recent simulation study Ando and Skolnick (4) revealed that using an equivalent-sphere model for macromolecules is a reasonable approximation to describe diffusion. Moreover, ...

We have studied a series of samples of bovine serum albumin (BSA) solutions with protein concentration, c, ranging from 2 to 500 mg/mL and ionic strength, I, from 0 to 2 M by small-angle X-ray scattering (SAXS). The scattering intensity distribution was compared to simulations using an oblate ellipsoid form factor with radii of 17 x 42 x 42 A, combined with either a screened Coulomb, repulsive structure factor, SSC(q), or an attractive square-well structure factor, SSW(q). At pH = 7, BSA is negatively charged. At low ionic strength, I < 0.3 M, the total interaction exhibits a decrease of the repulsive interaction when compared to the salt-free solution, as the net surface charge is screened, and the data can be fitted by assuming an ellipsoid form factor and screened Coulomb interaction. At moderate ionic strength (0.3-0.5 M), the interaction is rather weak, and a hard-sphere structure factor has been used to simulate the data with a higher volume fraction. Upon further increase of the ionic strength (I >or= 1.0 M), the overall interaction potential was dominated by an additional attractive potential, and the data could be successfully fitted by an ellipsoid form factor and a square-well potential model. The fit parameters, well depth and well width, indicate that the attractive potential caused by a high salt concentration is weak and long-ranged. Although the long-range, attractive potential dominated the protein interaction, no gelation or precipitation was observed in any of the samples. This is explained by the increase of a short-range, repulsive interaction between protein molecules by forming a hydration layer with increasing salt concentration. The competition between long-range, attractive and short-range, repulsive interactions accounted for the stability of concentrated BSA solution at high ionic strength.

We present a real-time study of protein crystallization of bovine β-lactoglobulin in the presence of CdCl(2) using small-angle X-ray scattering and optical microscopy. From observing the crystallization kinetics, we propose the following multistep crystallization mechanism that is consistent with our data. In the first step, an intermediate phase is formed, followed by the nucleation of crystals within the intermediate phase. During this period, the number of crystals increases with time, but the crystal growth is slowed down by the surrounding dense intermediate phase due to the low mobility. In the next step, the intermediate phase is consumed by nucleation and slow growth, and the crystals are exposed to the dilute phase. In this stage, the number of crystals becomes nearly constant, whereas the crystals grow rapidly due to access to the free protein molecules in the dilute phase. This real-time study not only provides evidence for a two-step nucleation process for protein crystallization but also elucidates the role and the structural signature of the metastable intermediate phase in this process.

The effective interactions and phase behavior of protein solutions under strong electrostatic coupling conditions are difficult to understand due to the complex charge pattern and irregular geometry of protein surfaces. This distinguishes them from related systems such as DNA or conventional colloids. In this work, we discuss the question of universality of the reentrant condensation (RC) of proteins in solution induced by multivalent counterions, i.e., redissolution on adding further salts after phase separation, as recently discovered (Zhang et al., Phys Rev Lett 2008; 101:148101). The discussion is based on a systematic investigation of five different proteins with different charge patterns and five different multivalent counterions. Zeta potential measurements confirm the effective charge inversion of proteins in the reentrant regime via binding of multivalent counterions, which is supported by Monte Carlo simulations. Charge inversion by trivalent cations requires an overall negative net charge of the protein. Statistical analysis of a representative set of protein sequences reveals that, in theory, this effect could be possible for about half of all proteins. Our results can be exploited for the control of the phase behavior of proteins, in particular facilitating protein crystallization.

Tuning of protein surface charge is a fundamental mechanism in biological systems. Protein charge is regulated in a physiological context by pH and interaction with counterions. We report on charge inversion and the related reentrant condensation in solutions of globular proteins with different multivalent metal cations. In particular, we focus on the changes in phase behavior and charge regulation due to pH effects caused by hydrolysis of metal ions. For several proteins and metal salts, charge inversion as measured by electrophoretic light scattering is found to be a universal phenomenon, the extent of which is dependent on the specific protein-salt combination. Reentrant phase diagrams show a much narrower phase-separated regime for acidic salts such as AlCl3 and FeCl3 compared to neutral salts such as YCl3 or LaCl3. The differences between acidic and neutral salts can be explained by the interplay of pH effects and binding of the multivalent counterions. The experimental findings are reproduced with good agreement by an analytical model for protein charging taking into account ion condensation, metal ion hydrolysis, and interaction with charged amino acid side chains on the protein surface. Finally, the relationship of charge inversion and reentrant condensation is discussed, suggesting that pH variation in combination with multivalent cations provides control over both attractive and repulsive interactions between proteins.