Johanna Brazard

The microscopic understanding of the early stage of crystallization remains a great challenge. The major difficulty in tackling this problem is the stochastic nature of crystallization, from which both experimental and theoretical approaches suffer. A traditional approach to studying crystal nucleation has been measuring the rate of nucleation and modeling the process using classical nucleation theory. Recently, there have been major breakthroughs in both experimental and theoretical approaches to advance understanding of crystal nucleation. The long-awaited moment of “seeing” the instant of crystal nucleation has been realized via microscopies such as in situ and cryo-transmission electron microscopy (TEM). Molecular dynamics (MD) simulations assisted by machine learning have been developed to access the molecular-level details of nucleation processes. Despite these advances, two major questions remain out of reach: what happens just before a crystal nucleus is formed, and when and how a certain polymorph is chosen. A technique that has been underused in probing the early stage of crystallization is optical spectroscopy. The advantages of optical spectroscopy that make it particularly suited for tackling this problem are its sensitivity to structural order and symmetry, its temporal resolution, and the relative ease of experiments and sample preparation. Thus, optical spectroscopy provides information that is complementary to the findings from microscopies and MD simulations. Collectively, we are at an exciting period in this field where we may finally understand the early stage of crystallization and rationally control polymorphism. This perspective highlights recent developments in in situ optical spectroscopy and opportunities awaiting us in the near future.

The formation of crystals has been a long-standing unresolved question while its understanding is crucial for rational design of the crystallization process and controlling polymorphism. Glycine is a well-studied model system as it has been known to form three polymorphs, depending on crystallization conditions. Recently, a new hypothesis has been suggested that glycine forms pre-nucleation aggregates, both in water and saltwater, before nucleating as a metastable β-glycine polymorph. Molecular dynamics simulations proposed the structure of the pre-nucleation aggregates in water as a linear hydrogen-bonded network of glycine molecules. Herein, polarized second harmonic scattering (pol-SHS) was used to test this hypothesis by probing the structural details of glycine aggregates in water and saltwater. Depolarization ratio extracted from a series of concentrations of glycine solutions strongly indicates the presence of aggregates both in water and saltwater and their structure evolves from linear (dipolar) networks to more branched (less dipolar) networks. Retardation ratio of pol-SHS data provided the molecular level details of how water organizes around a glycine molecule/aggregate in water or saltwater. In an aqueous solution, water reorganizes from azimuthal to radial pattern when glycine is dissolved. In saltwater, water organizes in a radial pattern around salt ions and the addition of glycine does not alter the structural organization of water. Glycine aggregates grow while the long-range molecular correlation of the sodium chloride–water networks is maintained. This study demonstrates the potency of pol-SHS in investigating the structure of molecular aggregates in solution in the context of understanding the crystal nucleation mechanism.

Stereochemistry controls oligourethane folding through backbone torsions, yielding helices and extended zig-zag structures formed by isotactic and syndiotactic motifs, respectively.

Liquid-liquid phase separation of biomolecules can be induced in vitro by selecting specific concentrations and environmental conditions. Phase diagrams relating the temperature, pressure, etc. to the concentrations of proteins and co-solutes aid in identifying the specific phase separation conditions for target proteins. However, it remains challenging to determining complete phase diagrams for protein liquid-liquid phase separation. Here, we combine optical microscopy and in situ Raman spectroscopy to quantify the temperature-dependent concentrations of the light and dense phases of human γD-crystallin and construct a complete phase diagram. The method can be extended to systems in the presence of co-solutes, which enables the quantitative description of the partitioning of a co-solute in the light and dense phase of proteins.

Molecular-level understanding of the early stage of crystallization remains a significant challenge. For a wide range of fundamental research and applications, it is of great importance to understand the mechanism of crystal polymorph selections in different solvents or in the presence of additives. We studied glycine crystallization in aqueous solution with or without the addition of sodium chloride (NaCl), one crystal nucleation at a time, using the recently developed single-crystal nucleation spectroscopy (SCNS). It has been reported that glycine forms γ-glycine when salt is added to aqueous solution, and the mechanism has been considered as a classical nucleation where γ-glycine forms directly as a crystal nucleus. Our study shows that metastable polymorph β-glycine forms first in aqueous solution both with or without NaCl through a nonclassical nucleation pathway. NaCl can stabilize the metastable β-glycine over several hours and also prevent it from converting to α-glycine. Eventually γ-glycine nucleates on the surface of β-glycine and then grows while dissolving the mother β-glycine. The crystal habit of β-glycine suggests that the stabilization by NaCl occurs at its polar face. SCNS provides crucial information to accelerate the investigation of the early stage of crystallization toward the rational control of polymorphisms.

Ferredoxins are small iron-sulfur proteins that engage in electron transfer (ET) with oxidoreductases across all domains of life. In bacteria, ferredoxins that contain two [4Fe-4S] clusters differ with respect to their electric midpoint potential: while “Alvin”-type ferredoxins show individual potentials between -500 and -650 mV vs. SHE, clostridial ferredoxins perform ET at indistinguishable potentials of approximately -400 mV vs. SHE. In this work, the electron transfer complex between clostridial ferredoxin CpFd and [FeFe]-hydrogenases from Clostridium pasteurianum (CpI) and green algae Chlamydomonas reinhardtii (CrHydA) was investigated spectroscopically. [FeFe]-hydrogenases are oxidoreductases that catalyze hydrogen turnover in bacteria and algae. Introducing the non-canonical amino acid para-cyanophenylalanine (pCNF) near to one of the iron-sulfur clusters of CpFd allowed for a quantification of electric field changes via the vibrational Stark effect (VSE) by Fourier-transform infrared (FTIR) spectroscopy. Upon reduction with H2 or auto-oxidation under N2, in situ FTIR difference spectroscopy reports on protein structural changes. Our data reveal that the affinity between ferredoxin and its redox partners is modulated by redox-dependent protein-protein interactions (PPIs). Prompted by these findings, we discuss whether clostridial ferredoxins might act as two-electron redox partners in contact with hydrogenase or other oxidoreductases.

Carbamate is an emerging class of a polymer backbone for constructing sequence-defined, abiotic polymers. It is expected that new functional materials can be de novo designed by controlling the primary polycarbamate sequence. While amino acids have been actively studied as building blocks for protein folding and peptide self-assembly, carbamates have not been widely investigated from this perspective. Here, we combined infrared (IR), vibrational circular dichroism (VCD), and nuclear magnetic resonance (NMR) spectroscopy with density functional theory (DFT) calculations to understand the conformation of carbamate monomer units in a nonpolar, aprotic environment (chloroform). Compared with amino acid building blocks, carbamates are more rigid, presumably due to the extended delocalization of π-electrons on the backbones. Cis configurations of the amide bond can be energetically stable in carbamates, whereas peptides often assume trans configurations at low energies. This study lays an essential foundation for future developments of carbamate-based sequence-defined polymer material design.

Dynamic light scattering (DLS) is a widely applied technique in multiple scientific and industrial fields for the size characterization of nanoscale objects in solution. While DLS is typically applied to characterize systems under static conditions, the emerging interest in using DLS on temporally evolving systems stimulates the latent need to improve the time resolution of measurements. Herein, we present a DLS microscopy setup (micro-DLS) that can accurately characterize the size of particles from autocorrelation functions built from sub-100 ms time windows, several orders of magnitude faster than previously reported. The system first registers the arrival time of the scattered photons using a time-correlated single photon counting module, which allows the construction of the autocorrelation function for size characterization based on a time window of freely chosen position and width. The setup could characterize both monomodal (60 or 220 nm polystyrene particles; PS) and multimodal size distributions (e.g., mixture of 20 nm LUDOX and 80 nm PS) with high accuracy in a sub-100 ms time window. Notably, the width of the size distribution became narrower as a shorter time window was used. This was attributed to the ability of the system to resolve the sub-ensemble of the broad size distribution, as the broad distribution could be reconstructed by accumulating the distribution obtained by consecutive 80 ms time windows. A DLS system with high temporal resolution will accelerate the expansion of its application toward systems that evolve as a function of time beyond its conventional use on static systems.