The dynamics of proteins in solution includes a variety of processes, such as backbone and side-chain fluctuations, interdomain motions, as well as global rotational and translational (i.e. center of mass) diffusion. Since protein dynamics is related to protein function and essential transport processes, a detailed mechanistic understanding and monitoring of protein dynamics in solution is highly desirable. The hierarchical character of protein dynamics requires experimental tools addressing a broad range of time- and length scales. We discuss how different techniques contribute to a comprehensive picture of protein dynamics, and focus in particular on results from neutron spectroscopy. We outline the underlying principles and review available instrumentation as well as related analysis frameworks.

During protein crystallization and purification, proteins are commonly found in concentrated salt solutions. The exact interplay of the hydration shell, the salt ions, and protein–protein interactions under these conditions is far from being understood on a fundamental level, despite the obvious practical relevance. We have studied a model globular protein (bovine serum albumin, BSA) in concentrated salt solutions by small-angle neutron scattering (SANS). The data are also compared to previous studies using SAXS. The SANS results for dilute protein solutions give an averaged volume of BSA of 91 700 ų, which is about 37% smaller than that determined by SAXS. The difference in volume corresponds to the contribution of a hydration shell with a hydration level of 0.30 g g⁻¹protein. The forward intensity I(0) determined from Guinier analysis is used to determine the second virial coefficient, A₂, which describes the overall protein interactions in solution. It is found that A₂ follows the reverse order of the Hofmeister series, i.e.(NH₄)₂SO₄ < Na₂SO₄ < NaOAc < NaCl < NaNO₃ < NaSCN. The dimensionless second virial coefficient B₂, corrected for the particle volume and molecular weight, has been calculated using different approaches, and shows that B₂ with corrections for hydration and the non-spherical shape of the protein describes the interactions better than those determined from the bare protein. SANS data are further analyzed in the full q-range using liquid theoretical approaches, which gives results consistent with the A₂ analysis and the experimental structure factor.

We discuss the phase behavior and in particular crystallization of a model globular protein (beta-lactoglobulin) in solution in the presence of multivalent electrolytes. It has been shown previously that negatively charged globular proteins at neutral pH in the presence of multivalent counterions undergo a “re-entrant condensation (RC)” phase behavior (Zhang et al., Phys. Rev. Lett., 2008, 101, 148101), i.e. a phase-separated regime occurs in between two critical salt concentrations, c* < c**, giving a metastable liquid–liquid phase separation (LLPS). Crystallization from the condensed regime has been observed to follow different mechanisms. Near c*, crystals grow following a classic nucleation and growth mechanism; near c**, the crystallization follows a two-step crystallization mechanism, i.e, crystal growth follows a metastable LLPS. In this paper, we focus on the two-step crystal growth near c**. SAXS measurements indicate that proteins form clusters in this regime and the cluster size increases approaching c**. Upon lowering the temperature, in situ SAXS studies indicate that the clusters can directly form both a dense liquid phase and protein crystals. During the crystal growth, the metastable dense liquid phase is dissolved. Based on our observations, we discuss a nucleation mechanism starting from clusters in the dilute phase from a metastable LLPS. These protein clusters behave as the building blocks for nucleation, while the dense phase acts as a reservoir ensuring constant protein concentration in the dilute phase during crystal growth.

While the interplay between liquid-liquid phase separation (LLPS) and glass formation in biological systems is highly relevant for their structure formation and thus function, the exact underlying mechanisms are not well known. The kinetic arrest originates from the slowdown at the molecular level, but how this propagates to the dynamics of microscopic phase domains is not clear. Since with diffusion, viscoelasticity, and hydrodynamics, distinctly different mechanisms are at play, the dynamics needs to be monitored on the relevant time and length scales and compared to theories of phase separation. Using x-ray photon correlation spectroscopy, we determine the LLPS dynamics of a model protein solution upon low temperature quenches and find distinctly different dynamical regimes. We observe that the early stage LLPS is driven by the curvature of the free energy and speeds up upon increasing quench depth. In contrast, the late stage dynamics slows down with increasing quench depth, fingerprinting a nearby glass transition. The dynamics observed shows a ballistic type of motion, implying that viscoelasticity plays an important role during LLPS. We explore possible explanations based on the Cahn-Hilliard theory with nontrivial mobility parameters and find that these can only partially explain our findings.