Reliably distinguishing protein nanocrystals from amorphous precipitate by means of depolarized dynamic light scattering

Abstract

Crystallization of biological macromolecules such as proteins implies several prerequisites, for example, the presence of one or more initial nuclei, sufficient amounts of the crystallizing substance and the chemical potential to provide the free energy needed to force the process. The initiation of a crystallization process itself is a stochastic event, forming symmetrically assembled nuclei over kinetically preferred protein‐dense liquid clusters. The presence of a spatial repetitive orientation of macromolecules in the early stages of the crystallization process has so far proved undetectable. However, early identification of the occurrences of unit cells is the key to nanocrystal detection. The optical properties of a crystal lattice offer a potential signal with which to detect whether a transition from disordered to ordered particles occurs, one that has so far not been tested in nanocrystalline applications. The ability of a lattice to depolarize laser light depends on the different refractive indices along different crystal axes. In this study a unique experimental setup is used to detect nanocrystal formation by application of depolarized scattered light. The results demonstrate the successful detection of nano‐sized protein crystals at early stages of crystal growth, allowing an effective differentiation between protein‐dense liquid cluster formation and ordered nanocrystals. The results are further verified by complementary methods like X‐ray powder diffraction, second harmonic generation, ultraviolet two‐photon excited fluorescence and scanning electron microscopy.

Serial femtosecond crystallography: A revolution in structural biology

“…homogenous and non-aggregated) sample is ideal for crystallization 85 . In addition, DLS is also used in SFX experiments to determine nanocrystal size distribution and homogeneity across multiple conditions 86 . DLS measures the light scattered by particles in solution.…”

Multi-Step Concanavalin A Phase Separation and Early-Stage Nucleation Monitored Via Dynamic and Depolarized Light Scattering

“…Furthermore, the 3D response surface model for DDLS signals showed an intensity higher than 150 kHz at 16% of PEG8k 9 minutes after initiation of the reaction, which characterizes the presence of a periodic structure in the ConA clusters (Figure 2e). Previous studies describing real-time monitoring of lysozyme, thioredoxin, and thaumatin crystal nucleation, applying simultaneous DLS/DDLS measurements, also describe changes in the polarization plane of the scattered light (DDLS signals up to 150 kHz) and confirmed-after verification by powder diffraction and scanning electron microscopy-the presence of crystalline lattices [43]. Thus, the volume fraction conferred by 16% PEG8k corresponds to the boundary line located between ConA clusters and the crystalline phase.…”

“…The DLS/DDLS instrument (XtalConcepts GmbH, 22525 Hamburg, Germany) was described previously by Schubert et al in 2015 [43]. The autocorrelation functions (ACF) obtained for all assays were analyzed by applying the CONTIN algorithm [44] and the decay time constants of the DLS (translation diffusion, D t ) and DDLS data (rotational diffusion, D r ) were calculated independently via the Stokes−Einstein and Stokes−Einstein−Debye equations [43], respectively. The viscosity was considered and adjusted according to the percentage of CA solutions in all assays (Table 1) and the actual hydrodynamic radius was calculated accordingly.…”

“…Higher scattering intensities correspond to either a higher concentration of molecules or to larger particles undergoing Brownian motion, which may also contribute to a false-positive depolarization and to an overestimation of the detected DDLS signal, due to multiple scattering of photons in the solution. In this context, Schubert et al in 2015 [43] defined a scattering intensity threshold by applying the same DDLS device, considering that intensities >5000 kHz are indicative of a contribution of artificial multiple photon scattering. The DLS signals acquired over 10 minutes for the ConA clustering process were lower than the discussed threshold when applying 16% PEG8k and exceeded the threshold limit for 17.6% PEG8k at approximately 7 minutes due to the faster growth rate with a higher concentration of nanoparticles, as depicted in Figure 4b.…”

Assessment of microcrystal quality by transmission electron microscopy for efficient serial femtosecond crystallography

“…Various methods have been used to identify microcrystals with homogenous physical properties. Such methods include dynamic light scattering (Schubert, 2015), second-order nonlinear imaging of chiral crystals (Luft, 2015), as well as microfluidic technologies (Abdallah et al, 2015; Kupitz et al, 2014) and transmission electron microscopy (TEM) (Gomery et al, 2013; Redecke et al, 2013; Stevenson et al, 2014b). In the latter case, we previously showed that not only is TEM capable of identifying conditions amenable to microcrystal formation, but also is capable of characterizing the intrinsic properties of the microcrystals (I.E.…”