Search Results (5)

X-ray crystallography remains the gold standard for high-resolution structural biology, yet obtaining diffraction-quality crystals continues to pose a major bottleneck due to inherently low success rates. This review advocates a paradigm shift from probabilistic screening to rational engineering, reframing crystallization as a controllable self-assembly process. We provide a comprehensive overview of strategies that connect fundamental physicochemical principles to practical applications, beginning with contact design, which involves the active engineering of crystal contacts through surface entropy reduction (SER), introduction of electrostatic patches. Complementing these molecular approaches, we discuss physicochemical strategies that exploit heterogeneous nucleation on functionalized surfaces and gold nanoparticles (AuNPs) to lower the energy barrier for crystal formation. We also address scaffold design, utilizing rigid fusion partners and polymer-forming chaperones to promote crystallization even from low-concentration solutions. Furthermore, we highlight principles for controlling the behavior of multi-component complexes, based on our experimental experience. Finally, we examine de novo lattice design, which leverages AI tools such as AlphaFold and RFdiffusion to program crystal lattices from first principles. Together, these strategies establish an integrated workflow that links thermodynamic stability with crystallizability. Full article

Industrial biotechnology offers a potential ecological solution for PET recycling under relatively mild reaction conditions via enzymatic degradation, particularly using the leaf branch compost cutinase (LCC) quadruple mutant ICCG. To improve the efficient downstream processing of this biocatalyst after heterologous gene expression with a suitable production host, protein crystallization can serve as an effective purification/capture step. Enhancing protein crystallization was achieved in recent studies by introducing electrostatic (and aromatic) interactions in two homologous alcohol dehydrogenases (Lb/LkADH) and an ene reductase (NspER1-L1,5) produced with Escherichia coli. In this study, ICCG, which is difficult to crystallize, was utilized for the application of crystal contact engineering strategies, resulting in ICCG mutant L50Y (ICCGY). Previously focused on the Lys-Glu interaction for the introduction of electrostatic interactions at crystal contacts, the applicability of the engineering strategy was extended here to an Arg-Glu interaction to increase crystallizability, as shown for ICCGY T110E. Furthermore, the rationale of the engineering approach is demonstrated by introducing Lys and Glu at non-crystal contacts or sites without potential interaction partners as negative controls. These resulting mutants crystallized comparably but not superior to the wild-type protein. As demonstrated by this study, crystal contact engineering emerges as a promising approach for rationally enhancing protein crystallization. This advancement could significantly streamline biotechnological downstream processing, offering a more efficient pathway for research and industry. Full article

Protein crystallization is an alternative to well-established but cost-intensive and time-consuming chromatography in biotechnological processes, with protein crystallization defined as an essential unit operation for isolating proteins, e.g., active pharmaceutical ingredients. Crystalline therapeutic proteins attract interest in formulation and delivery processes of biopharmaceuticals due to the high purity, concentration, and stability of the crystalline state. Although improving protein crystallization is mainly achieved by high-throughput screening of crystallization conditions, recent studies have established a rational protein engineering approach to enhance crystallization for two homologous alcohol dehydrogenases from Lactobacillus brevis (LbADH) and Lactobacillus kefiri (LkADH). As generalizing crystallization processes across a wide range of target proteins remains challenging, this study takes a further step by applying the successful crystal contact engineering strategies for LbADH/LkADH to a non-homologous protein, an NADH-binding derivative of the Nostoc sp. PCC 1720 ene reductase (NspER1-L1,5). Here, the focus lies on introducing electrostatic interactions at crystal contacts, specifically between lysine and glutamic acid. Out of the nine tested NspER1-L1,5 mutants produced in E. coli, six crystallized, while four mutants revealed an increased propensity to crystallize in static µL-batch crystallization compared to the wild type: Q204K, Q350K, D352K, and T354K. The best-performing mutant Q204K was selected for upscaling, crystallizing faster than the wild type in a stirred batch crystallizer. Even when spiked with E. coli cell lysate, the mutant maintained increased crystallizability compared to the wild type. The results of this study highlight the potential of crystal contact engineering as a reliable tool for improving protein crystallization as an alternative to chromatography, paving the way for more efficient biotechnological downstream processing. Full article

In this study, unconventional C—Hlg···H–C (Hlg = Cl, Br, and I) interactions involving sp, sp², and sp³ organic halides were investigated at the RI-MP2/aug-cc-pVQZ level of theory. Energy Decomposition Analyses (EDA) and Natural Bonding Orbital (NBO) studies showed that these intermolecular contacts are mainly supported by orbital and dispersion contributions, which counteracted the unfavorable/slightly favorable electrostatics due to the halogen–hydrogen σ-hole facing. In addition, the Bader’s Quantum Theory of Atoms in Molecules (QTAIM) and the Noncovalent Interaction plot (NCIplot) visual index methodologies were used to further characterize the interactions discussed herein. We expect that the results reported herein will be useful in the fields of supramolecular chemistry, crystal engineering, and rational drug design, where the fine tuning of noncovalent interactions is crucial to achieve molecular recognition or a specific solid-state architecture. Full article

Protein crystallization can serve as a purification step in biotechnological processes but is often limited by the non-crystallizability of proteins. Enabling or improving crystallization is mostly achieved by high-throughput screening of crystallization conditions and, more recently, by rational crystal contact engineering. Two selected rational crystal contact mutations, Q126K and T102E, were transferred from the alcohol dehydrogenases of Lactobacillus brevis (LbADH) to Lactobacillus kefir (LkADH). Proteins were expressed in E. coli and batch protein crystallization was performed in stirred crystallizers. Highly similar crystal packing of LkADH wild type compared to LbADH, which is necessary for the transfer of crystal contact engineering strategies, was achieved by aligning purification tag and crystallization conditions, as shown by X-ray diffraction. After comparing the crystal sizes after crystallization of LkADH mutants with the wild type, the mean protein crystal size of LkADH mutants was reduced by 40–70% in length with a concomitant increase in the total amount of crystals (higher number of nucleation events). Applying this measure to the LkADH variants studied results in an order of crystallizability T102E > Q126K > LkADH wild type, which corresponds to the results with LbADH mutants and shows, for the first time, the successful transfer of crystal contact engineering strategies. Full article