Reentrant Phase Behavior in Protein Solutions Induced by Multivalent Salts: Strong Effect of Anions Cl- Versus NO3.

In this work, the effects of the two anions Cl- and NO3- on the phase behavior of bovine serum albumin (BSA) in solution with trivalent salts are compared systematically. In the presence of trivalent metal salts, negatively charged proteins such as BSA in solution undergo a reentrant condensation (RC) phase behavior, which has been established for several proteins with chlorides of trivalent salts. Here, we show that replacing Cl- by NO3- leads to a marked change in the phase behavior. The effect is investigated for the two different cations Y3+ and La3+. The salts are thus YCl3, Y(NO3)3, LaCl3, and La(NO3)3. The experimental phase behavior shows that while the chloride salts induce both liquid-liquid phase separation (LLPS) and RC, the nitrate salts also induce LLPS, but RC becomes partial with La(NO3)3 and disappears with Y(NO3)3. The observed phase behavior is rationalized by effective protein-protein interactions which are characterized using small-angle X-ray scattering. The results based on the reduced second virial coefficients B2/ B2HS and 1/ I( q → 0) demonstrate that the NO3- salts induce a stronger attraction than the Cl- salts. Overall, the effective attraction, the width of the condensed regime in the RC phase diagram, and the nature of LLPS follow the order LaCl3 < YCl3 < La(NO3)3 < Y(NO3)3. Despite the decisive role of cations in RC phase behavior, isothermal titration calorimetry measurements indicate that replacing anions does not significantly influence the cation binding to proteins. The experimental results observed are discussed based on an "enhanced Hofmeister effect" including electrostatic and hydrophobic interactions between protein-cation complexes.

Structural Evolution of Metastable Protein Aggregates in the Presence of Trivalent Salt Studied by (V)SANS and SAXS.

We present a study of the structural evolution of protein aggregates formed in solutions of a globular protein, ß-lactoglobulin (BLG), in the presence of YCl3. These aggregates are often observed before crystallization starts and they are metastable with respect to the crystalline phase. Here we focus on the characterization of the hierarchical structure of this intermediate phase and its temperature dependent structure evolution using a combination of (very) small angle neutron and X-ray scattering (VSANS, SANS, and SAXS). Results show that the hierarchical structure ranges from nanometer scale protein monomer, dimer and compact protein clusters to micrometer scale fractal protein aggregates. Upon cooling, the overall hierarchical structure is preserved, but the evolution of the internal structure within the aggregates is clearly visible: the monomer-monomer correlation peak reduces its intensity and disappears completely at lower temperatures, whereas the cluster-cluster correlation is enhanced. At a larger length scale, the fractal dimension of protein aggregates increases. The kinetics of the structure change during a temperature ramp was further investigated using time-resolved SAXS. The time dependent SAXS profiles show clear isosbestic points and the kinetics of the structural evolution can be well described using a two-state model. These dynamic properties of protein aggregates on a broad length scale may be essential for being the precursors of nucleation.

On the question of two-step nucleation in protein crystallization.

We report a real-time study on protein crystallization in the presence of multivalent salts using small angle X-ray scattering (SAXS) and optical microscopy, focusing particularly on the nucleation mechanism as well as on the role of the metastable intermediate phase (MIP). Using bovine beta-lactoglobulin as a model system in the presence of the divalent salt CdCl2, we have monitored the early stage of crystallization kinetics which demonstrates a two-step nucleation mechanism: protein aggregates form a MIP, which is followed by the nucleation of crystals within the MIP. Here we focus on characterizing and tuning the structure of the MIP using salt and the related effects on the two-step nucleation kinetics. The results suggest that increasing the salt concentration near the transition zone pseudo-c** enhances the energy barrier for both MIPs and crystal nucleation, leading to slow growth. The structural evolution of the MIP and its effect on subsequent nucleation is discussed based on the growth kinetics. The observed kinetics can be well described, using a rate-equation model based on a clear physical two-step picture. 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 MIPs in the nonclassical process of protein crystallization.

Real-time observation of nonclassical protein crystallization kinetics.

We present a real-time study of protein crystallization of bovine ß-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.