Diverse crystalline protein scaffolds through metal-dependent polymorphism.

As protein crystals are increasingly finding diverse applications as scaffolds, controlled crystal polymorphism presents a facile strategy to form crystalline assemblies with controllable porosity with minimal to no protein engineering. Polymorphs of consensus tetratricopeptide repeat proteins with varying porosity were obtained through co-crystallization with metal salts, exploiting the innate metal ion geometric requirements. A single structurally exposed negative amino acid cluster was responsible for metal coordination, despite the abundance of negatively charged residues. Density functional theory calculations showed that while most of the crystals were the most thermodynamically stable assemblies, some were kinetically trapped states. Thus, crystalline porosity diversity is achieved and controlled with metal coordination, opening a new scope in the application of proteins as biocompatible protein-metal-organic frameworks (POFs). In addition, metal-dependent polymorphic crystals allow direct comparison of metal coordination preferences.

Multicomponent Amorphous Solid Forms of Telmisartan: Insights into Mechanochemical Activation and Physicochemical Attributes.

The present work aims to explore the new solid forms of telmisartan (TEL) with alpha-ketoglutaric acid (KGA) and glutamic acid (GA) as potential coformers using mechanochemical approach and their role in augmentation in physicochemical parameters over pure crystalline TEL. Mechanochemical synthesis was performed using 1:1 stoichiometric ratio of TEL and the selected coformers in the presence of catalytic amount of ethanol for 1 h. The ground product was characterized by PXRD, DSC, and FTIR. The new solid forms were evaluated for apparent solubility, intrinsic dissolution, and physical stability. Preliminary characterization revealed the amorphization of the mechanochemical product as an alternate outcome of cocrystallization screening. Mechanistic understanding of the amorphous phase highlights the formation of amorphous-mediated cocrystallization that involves three steps, viz., molecular recognition, intermediate amorphous phase, and product nucleation. The solubility curves of both multicomponent amorphous solid forms (TEL-KGA and TEL-GA) showed the spring-parachute effect and revealed significant augmentation in apparent solubility (8-10-folds), and intrinsic dissolution release (6-9-folds) as compared to the pure drug. Besides, surface anisotropy and differential elemental distributions in intrinsic dissolution compacts of both solid forms were confirmed by FESEM and EDX mapping. Therefore, amorphous phases prepared from mechanochemical synthesis can serve as a potential solid form for the investigation of a cocrystal through amorphous-mediated cocrystallization. This has greater implications in solubility kinetics wherein the rapid precipitation of the amorphous phase can be prevented by the metastable cocrystal phase and contribute to the significant augmentation in the physicochemical parameters.

Optimization of Calcium Fluoride Crystallization Process for Treatment of High-Concentration Fluoride-Containing Semiconductor Industry Wastewater.

This study utilized a fluidized bed reactor (FBR) for fluoride removal from high-concentration fluoride-ion-containing simulated semiconductor industry wastewater and recovered high-purity CaF2 crystals. The effects of hydraulic retention time (HRT), pH, Ca2+ to F- ratio, upflow velocity, seed size and seed bed height were investigated by performing lab-scale batch experiments. Considering fluoride removal and CaF2 crystallization efficiency, 5 h HRT, pH 6, seed height of 50 cm and [Ca2+]/[F-] ratio of 0.55 (mol/mol) were found to be optimum. The effect of the interaction between the important process parameters on fluoride removal was further analyzed using response surface methodology (RSM) experimental design. The results showed that all the individual parameters have a significant impact (p = 0.0001) on fluoride removal. SEM-EDX and FTIR analysis showed the composition of the crystals formed inside FBR. HR-XRD analysis confirmed that the crystalline structure of samples was mainly CaF2. The results clearly demonstrated the feasibility of silica seed material containing FBR for efficient removal and recovery of fluoride as high-purity calcium fluoride crystals.

A preparation of debranched waxy maize starch derivatives: Effect of drying temperatures on crystallization and digestibility.

The impact of recrystallization conditions and drying temperatures on the crystallization and digestibility of native waxy maize (Zea mays L.) starch (NWMS) was explored. This study involved subjecting NWMS to concurrent debranching and crystallization at 50 °C for up to 7 days. Samples were collected by oven-drying at 40, 60, and 80 °C for 24 h. This simultaneous debranching and crystallization process increased the resistant starch (RS) content by approximately 48 % compared to the native starch. The drying temperatures significantly influenced the RS content, with samples dried at 60 °C exhibiting the lowest digestibility. X-ray diffraction (XRD) analysis revealed that most crystals demonstrated a characteristic A-type arrangement. Debranching and crystallization processes enhanced the crystallinity of the samples. The specific crystal arrangement (A- or B-type) depended on the crystallization conditions. A 15 min heating of NWMS in a boiling water bath increased the digestible fraction to over 90 %, while the samples subjected to debranching and crystallization showed an increase to only about 45 %. A linear correlation between starch fractions and enthalpy was also observed.

Mathematical and computational modeling of fats and triacylglycerides.

Fats and oils are found in many food products; however, their macroscopic properties are difficult to predict, especially when blending different fats or oils together. With difficulties in sourcing specific fats or oils, whether due to availability or pricing, food companies may be required to find alternative sources for these ingredients, with possible differences in ingredient performance. Mathematical and computational modeling of these ingredients can provide a quick way to predict their properties, avoiding costly trials or manufacturing problems, while, most importantly, keeping the consumers happy. This review covers a range of mathematical models for triacylglycerides (TAGs) and fats, namely, models for the prediction of melting point, solid fat content, and crystallization temperature and composition. There are a number of models that have been designed for both TAGs and fats and which have been shown to agree very well with empirical measurements, using both kinetic and thermodynamic approaches, with models for TAGs being used to, in turn, predict fat properties. The last section describes computational models to simulate the behavior of TAGs using molecular dynamics (MD). Simulation of TAGs using MD, however, is still at an early stage, although the most recent papers on this topic are bringing this area up to speed.

Efficient in situ screening of and data collection from microcrystals in crystallization plates.

A considerable bottleneck in serial crystallography at XFEL and synchrotron sources is the efficient production of large quantities of homogenous, well diffracting microcrystals. Efficient high-throughput screening of batch-grown microcrystals and the determination of ground-state structures from different conditions is thus of considerable value in the early stages of a project. Here, a highly sample-efficient methodology to measure serial crystallography data from microcrystals by raster scanning within standard in situ 96-well crystallization plates is described. Structures were determined from very small quantities of microcrystal suspension and the results were compared with those from other sample-delivery methods. The analysis of a two-dimensional batch crystallization screen using this method is also described as a useful guide for further optimization and the selection of appropriate conditions for scaling up microcrystallization.

The Enrichment of Docosahexaenoic Acid from Microalgal Oil by Urea Complexation via Rotary-evaporation Crystallization.

Urea complexation is a widely used method for enriching polyunsaturated fatty acids, and cooling is the traditional approach for urea crystallization. This study aimed to investigate the potential of rotary-evaporation under vacuum as an alternative method for urea crystallization in urea complexation to enrich docosahexaenoic acid (DHA). DHA-containing microalgal oil was converted to ethyl esters (EE) as the raw material. In comparison to cooling, rotary-evaporation crystallization, as a post-treatment method for urea complexation, led to higher DHA contents in the non-urea included fractions. The ratios of urea to EE converted from DHA-containing microalgal oil was found to be the primary factors influencing urea complexation when using rotary-evaporation crystallization. Through an orthogonal test, optimal process conditions were determined, including a urea/EE ratio of 2, an ethanol/urea ratio of 7, and a rotary-evaporation temperature of 75℃. Under these conditions, a concentrate containing more than 90% DHA could be obtained.

Crystallization of Ethylene Plant Hormone Receptor-Screening for Structure.

The plant hormone ethylene is a key regulator of plant growth, development, and stress adaptation. Many ethylene-related responses, such as abscission, seed germination, or ripening, are of great importance to global agriculture. Ethylene perception and response are mediated by a family of integral membrane receptors (ETRs), which form dimers and higher-order oligomers in their functional state as determined by the binding of Cu(I), a cofactor to their transmembrane helices in the ER-Golgi endomembrane system. The molecular structure and signaling mechanism of the membrane-integral sensor domain are still unknown. In this article, we report on the crystallization of transmembrane (TM) and membrane-adjacent domains of plant ethylene receptors by Lipidic Cubic Phase (LCP) technology using vapor diffusion in meso crystallization. The TM domain of ethylene receptors ETR1 and ETR2, which is expressed in E. coli in high quantities and purity, was successfully crystallized using the LCP approach with different lipids, lipid mixtures, and additives. From our extensive screening of 9216 conditions, crystals were obtained from identical crystallization conditions for ETR1 (aa 1-316) and ETR2 (aa 1-186), diffracting at a medium-high resolution of 2-4 Å. However, data quality was poor and not sufficient for data processing or further structure determination due to rotational blur and high mosaicity. Metal ion loading and inhibitory peptides were explored to improve crystallization. The addition of Zn(II) increased the number of well-formed crystals, while the addition of ripening inhibitory peptide NIP improved crystal morphology. However, despite these improvements, further optimization of crystallization conditions is needed to obtain well-diffracting, highly-ordered crystals for high-resolution structural determination. Overcoming these challenges will represent a major breakthrough in structurally determining plant ethylene receptors and promote an understanding of the molecular mechanisms of ethylene signaling.

Programming crystallization kinetics of self-assembled DNA crystals with 5-methylcytosine modification.

Self-assembled DNA crystals offer a precise chemical platform at the ångström-scale for DNA nanotechnology, holding enormous potential in material separation, catalysis, and DNA data storage. However, accurately controlling the crystallization kinetics of such DNA crystals remains challenging. Herein, we found that atomic-level 5-methylcytosine (5mC) modification can regulate the crystallization kinetics of DNA crystal by tuning the hybridization rates of DNA motifs. We discovered that by manipulating the axial and combination of 5mC modification on the sticky ends of DNA tensegrity triangle motifs, we can obtain a series of DNA crystals with controllable morphological features. Through DNA-PAINT and FRET-labeled DNA strand displacement experiments, we elucidate that atomic-level 5mC modification enhances the affinity constant of DNA hybridization at both the single-molecule and macroscopic scales. This enhancement can be harnessed for kinetic-driven control of the preferential growth direction of DNA crystals. The 5mC modification strategy can overcome the limitations of DNA sequence design imposed by limited nucleobase numbers in various DNA hybridization reactions. This strategy provides a new avenue for the manipulation of DNA crystal structure, valuable for the advancement of DNA and biomacromolecular crystallography.

Elastin recoil is driven by the hydrophobic effect.

Elastin is an extracellular matrix material found in all vertebrates. Its reversible elasticity, robustness, and low stiffness are essential for the function of arteries, lungs, and skin. It is among the most resilient elastic materials known: During a human lifetime, arterial elastin undergoes in excess of 2 × 109 stretching/contracting cycles without replacement, and slow oxidative hardening has been identified as a limiting factor on human lifespan. For over 50 y, the mechanism of entropic recoil has been controversial. Herein, we report a combined NMR and thermomechanical study that establishes the hydrophobic effect as the primary driver of elastin function. Water ordering at the solvent:protein interface was observed as a function of stretch using double quantum 2H NMR, and the most extensive thermodynamic analysis performed to date was obtained by measuring elastin length and volume as a function of force and temperature in normal water, heavy water and with cosolvents. When stretched, elastin's heat capacity increases, water is ordered proportional to the degree of stretching, the internal energy decreases, and heat is released in excess of the work performed. These properties show that recoil in elastin under physiological conditions is primarily driven by the hydrophobic effect rather than by configurational entropy as is the case for rubber. Consistent with this conclusion are decreases in the thermodynamic signatures when cosolvents that alter the hydrophobic effect are introduced. We propose that hydrophobic effect-driven recoil, as opposed to a configurational entropy mechanism where hardening from crystallization can occur, is the origin of elastin's unusual resilience.

Fragility and Tendency to Crystallization for Structurally Related Compounds.

The present study was designed to investigate the physical stability of three organic materials with similar chemical structures. The examined compounds revealed completely different crystallization tendencies in their supercooled liquid states and were classified into three distinct classes based on their tendency to crystallize. (S)-4-Benzyl-2-oxazolidinone easily crystallizes during cooling from the melt; (S)-4-Benzylthiazolidine-2-thione does not crystallize during cooling from the melt, but crystallizes easily during subsequent reheating above Tg; and (S)-4-Benzyloxazolidine-2-thione does not crystallize either during cooling from the melt or during reheating. Such different tendencies to crystallize are observed despite the very similar chemical structures of the compounds, which only differ in oxide or sulfur atoms in one of their rings. We also studied the isothermal crystallization kinetics of the materials that were shown to transform into a crystalline state. Molecular dynamics and thermal properties were thoroughly investigated using broadband dielectric spectroscopy, as well as conventional and temperature-modulated differential scanning calorimetry in the wide temperature range. It was found that all three glass formers have the same dynamic fragility (m = 93), calculated directly from dielectric structural relaxation times. This result verifies that dynamic fragility is not related to the tendency to crystallize. In addition, thermodynamic fragility predictions were also made using calorimetric data. It was found that the thermodynamic fragility evaluated based on the width of the glass transition, observed in the temperature dependence of heat capacity, correlates best with the tendency to crystallize.

Crystallization and In Situ Room Temperature Data Collection Using the Crystallization Facility at Harwell and Beamline VMXi, Diamond Light Source.

Protocols for robotic protein crystallization using the Crystallization Facility at Harwell and in situ room temperature data collection from crystallization plates at Diamond Light Source beamline VMXi are described. This approach enables high-quality room-temperature crystal structures to be determined from multiple crystals in a straightforward manner and provides very rapid feedback on the results of crystallization trials as well as enabling serial crystallography. The value of room temperature structures in understanding protein structure, ligand binding, and dynamics is becoming increasingly recognized in the structural biology community. This pipeline is accessible to users from all over the world with several available modes of access. Crystallization experiments that are set up can be imaged and viewed remotely with crystals identified automatically using a machine learning tool. Data are measured in a queue-based system with up to 60° rotation datasets from user-selected crystals in a plate. Data from all the crystals within a particular well or sample group are automatically merged using xia2.multiplex with the outputs straightforwardly accessed via a web browser interface.

Nucleic-acid-base photofunctional cocrystal for information security and antimicrobial applications.

Cocrystal engineering is an efficient and simple strategy to construct functional materials, especially for the exploitation of novel and multifunctional materials. Herein, we report two kinds of nucleic-acid-base cocrystal systems that imitate the strong hydrogen bond interactions constructed in the form of complementary base pairing. The two cocrystals studied exhibit different colors of phosphorescence from their monomeric counterparts and show the feature of rare high-temperature phosphorescence. Mechanistic studies reveal that the strong hydrogen bond network stabilizes the triplet state and suppresses non-radiative transitions, resulting in phosphorescence even at 425 K. Moreover, the isolation effects of the hydrogen bond network regulate the interactions between the phosphor groups, realizing the manipulation from aggregation to single-molecule phosphorescence. Benefiting from the long-lived triplet state with a high quantum yield, the generation of reactive oxygen species by energy transfer is also available to utilize for some applications such as in photodynamic therapy and broad-spectrum microbicidal effects. In vitro experiments show that the cocrystals efficiently kill bacteria on a tooth surface and significantly help prevent dental caries. This work not only provides deep insight into the relationship of the structure-properties of cocrystal systems, but also facilitates the design of multifunctional cocrystal materials and enriches their potential applications.

Understanding the bio-crystallization: An insight to therapeutic relevance.

In the realm of biomedical engineering and materials science, the synthesis of biomaterials plays a pivotal role in advancing therapeutic strategies for regeneration of tissues. The deliberate control of crystallization processes in biomaterial synthesis has emerged as a key avenue for tailoring the properties of these materials, enabling the design of innovative solutions for a wide array of medical applications. This review delves into the interplay between controlled crystallization and biomaterial synthesis, exploring its multifaceted applications in the therapeutic domains. The investigation encompasses a wide spectrum of matrices, ranging from small molecules to large biomolecules, highlighting their unique contributions in modulating crystallization processes. Furthermore, the review critically assesses the analytical techniques and methodologies employed to probe and characterize the depths of crystallization dynamics. Advanced imaging, spectroscopic, and computational tools are discussed in the context of unraveling the intricate mechanisms governing nucleation and crystallization processes within the organic matrix. Finally we delve in the applications of such advance material in therapeutics of hard and soft tissues.

The modulatory effects of large and small extracellular vesicles from normal human urine on calcium oxalate crystallization, growth, aggregation, adhesion on renal cells, and invasion through extracellular matrix: An in vitro study.

Urinary extracellular vesicles (uEVs) play important roles in physiologic condition and various renal/urological disorders. However, their roles in kidney stone disease remain unclear. This study aimed to examine modulatory effects of large and small uEVs derived from normal human urine on calcium oxalate (CaOx) crystals (the main component in kidney stones). After isolation, large uEVs, small uEVs and total urinary proteins (TUPs) with equal (protein equivalent) concentration were added into various crystal assays to compare with the control (without uEVs or TUPs). TUPs strongly inhibited CaOx crystallization, growth, aggregation and crystal-cell adhesion. Large uEVs had lesser degree of inhibition against crystallization, growth and crystal-cell adhesion, and comparable degree of aggregation inhibition compared with TUPs. Small uEVs had comparable inhibitory effects as of TUPs for all these crystal assays. However, TUPs and large uEVs slightly promoted CaOx invasion through extracellular matrix, whereas small uEVs did not affect this. Matching of the proteins reported in six uEVs datasets with those in the kidney stone modulator (StoneMod) database revealed that uEVs contained 18 known CaOx stone modulators (mainly inhibitors). These findings suggest that uEVs derived from normal human urine serve as CaOx stone inhibitors to prevent healthy individuals from kidney stone formation.

Streamlining regular liquid chromatography with MALDI-TOF MS and NMR spectroscopy using automatic full-contact splitless spotting interface and flash-tap fractioning collection.

BACKGROUND: High-resolution matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and nuclear magnetic resonance (NMR) spectroscopy are powerful tools to identify unknown psychoactive substances. However, in complex matrices, trace levels of unknown substances usually require additional fractionation and concentration. Specialized liquid chromatography systems are necessary for both techniques. The small flow rate of nano LC, typically paired with MALDI-TOF MS, often results in prolonged fractionation times. Conversely, the larger flow rate of semi-preparative LC, used for NMR analysis, can be time-consuming and labor-intensive when concentrating samples. To address these issues, we developed an integrated automatic system that integrated to regular LC. RESULT: Automatic spot collector (ASC) and automatic fraction collector (AFC) were present in this study. The ASC utilized in-line matrix mixing, full-contact spotting and real time heating (50 °C), achieving great capacity of 5 µL droplet on MALDI plate, high recovery (76-116%) and rapid evaporation in 2 min. The analytes were concentrated 4-8 times, forming even crystallization, reaching the detection limit at the concentration of 50 µg L-1 for 12 psychoactive substances in urine. The AFC utilizes flexible tubing which flash-tapped the microtube's upper rim (3 mm depth) instead of reaching the bottom. This method prevents sample loss and minimizes the robotic arm's movement, providing a high fractionating speed at 6 s 12 psychoactive compounds were fractionated in a single round analysis (recovery: 81%-114%). Methamphetamine and nitrazepam obtained from drug-laced coffee samples were successful analyzed with photodiode array (PDA) after one AFC round and NMR after five rounds. SIGNIFICANCE: The ASC device employed real-time heating, in-line matrix mixing, and full-contact spotting to facilitate the samples spotting onto the MALDI target plate, thereby enhancing detection sensitivity in low-concentration and complex samples. The AFC device utilized the novel flash-tapping method to achieve rapid fractionation and high recovery rate. These devices were assembled using commercially available components, making them affordable (400 USD) for most laboratories while still meeting the required performance for advanced commercialized systems.

Effect of microwave alone and microwave-assisted modification on the physicochemical properties of starch and its application in food.

Native starch has poor stability and usually requires modification to expand its industrial application range. Commonly used methods are physical, chemical, enzymatic and compound modification. Microwave radiation, as a kind of physical method, is promising due to its uniform energy radiation, greenness, safety, non-toxicity. It can meet the demand of consumers for safe food. Microwave-assisted modification with other methods can directly or indirectly affect the structure of starch granules to obtain modified starch with high degree of substitution and low viscosity, and the modification efficiency is greatly improved. This paper reviews the effect of microwave radiation on the physicochemical properties of starch, such as granule morphology, crystallization characteristics, and gelatinization characteristics, as well as the application of microwave radiation in starch modification and starch food processing. It provides theoretical references and suggestions for the research of microwave heating modified starch and the deep processing of starchy foods.

An Artificial Gut/Absorption Simulator: Understanding the Impact of Absorption on In Vitro Dissolution, Speciation, and Precipitation of Amorphous Solid Dispersions.

Upon dissolution, amorphous solid dispersions (ASDs) of poorly water-soluble compounds can generate supersaturated solutions consisting of bound and free drug species that are in dynamic equilibrium with each other. Only free drug is available for absorption. Drug species bound to bile micelles, polymer excipients, and amorphous and crystalline precipitate can reduce the drug solute's activity to permeate, but they can also serve as reservoirs to replenish free drug in solution lost to absorption. However, with multiple processes of dissolution, absorption, and speciation occurring simultaneously, it may become challenging to understand which processes lead to an increase or decrease in drug solution concentration. Closed, nonsink dissolution testing methods used routinely, in the absence of drug removal, allow only for static equilibrium to exist and obscure the impact of each drug species on absorption. An artificial gut simulator (AGS) introduced recently consists of a hollow fiber-based absorption module and allows mass transfer of the drug from the dissolution media at a physiological rate after tuning the operating parameters. In the present work, ASDs of varying drug loadings were prepared with a BCS-II model compound, ketoconazole (KTZ), and hypromellose acetate succinate (HPMCAS) polymer. Simultaneous dissolution and absorption testing of the ASDs was conducted with the AGS, and simple analytical techniques were utilized to elucidate the impact of bound drug species on absorption. In all cases, a lower amount of crystalline precipitate was formed in the presence of absorption relative to the nonsink dissolution "control". However, formation of HPMCAS-bound drug species and crystalline precipitate significantly reduced KTZ absorption. Moreover, at high drug loading, inclusion of an absorption module was shown to enhance ASD dissolution. The rank ordering of the ASDs with respect to dissolution was significantly different when nonsink dissolution versus AGS was used, and this discrepancy could be mechanistically elucidated by understanding drug dissolution and speciation in the presence of absorption.

Racemization of the substrate and product by serine palmitoyltransferase from Sphingobacterium multivorum yields two enantiomers of the product from d-serine.

Serine palmitoyltransferase (SPT) catalyzes the pyridoxal-5'-phosphate (PLP)-dependent decarboxylative condensation of l-serine and palmitoyl-CoA to form 3-ketodihydrosphingosine (KDS). Although SPT was shown to synthesize corresponding products from amino acids other than l-serine, it is still arguable whether SPT catalyzes the reaction with d-serine, which is a question of biological importance. Using high substrate and enzyme concentrations, KDS was detected after the incubation of SPT from Sphingobacterium multivorum with d-serine and palmitoyl-CoA. Furthermore, the KDS comprised equal amounts of 2S and 2R isomers. 1H-NMR study showed a slow hydrogen-deuterium exchange at Cα of serine mediated by SPT. We further confirmed that SPT catalyzed the racemization of serine. The rate of the KDS formation from d-serine was comparable to those for the α-hydrogen exchange and the racemization reaction. The structure of the d-serine-soaked crystal (1.65 Å resolution) showed a distinct electron density of the PLP-l-serine aldimine, interpreted as the racemized product trapped in the active site. The structure of the α-methyl-d-serine-soaked crystal (1.70 Å resolution) showed the PLP-α-methyl-d-serine aldimine, mimicking the d-serine-SPT complex prior to racemization. Based on these enzymological and structural analyses, the synthesis of KDS from d-serine was explained as the result of the slow racemization to l-serine, followed by the reaction with palmitoyl-CoA, and SPT would not catalyze the direct condensation between d-serine and palmitoyl-CoA. It was also shown that the S. multivorum SPT catalyzed the racemization of the product KDS, which would explain the presence of (2R)-KDS in the reaction products.

Leishmania profilin interacts with actin through an unusual structural mechanism to control cytoskeletal dynamics in parasites.

Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Because of their complex life cycle involving both vertebrate and insect hosts, and >1 billion years of evolutionarily distance, the cell biology of trypanosomatid parasites exhibits pronounced differences to animal cells. For example, the actin cytoskeleton of trypanosomatids is divergent when compared with other eukaryotes. To understand how actin dynamics are regulated in trypanosomatid parasites, we focused on a central actin-binding protein profilin. Co-crystal structure of Leishmania major actin in complex with L. major profilin revealed that, although the overall folds of actin and profilin are conserved in eukaryotes, Leishmania profilin contains a unique α-helical insertion, which interacts with the target binding cleft of actin monomer. This insertion is conserved across the Trypanosomatidae family and is similar to the structure of WASP homology-2 (WH2) domain, a small actin-binding motif found in many other cytoskeletal regulators. The WH2-like motif contributes to actin monomer binding and enhances the actin nucleotide exchange activity of Leishmania profilin. Moreover, Leishmania profilin inhibited formin-catalyzed actin filament assembly in a mechanism that is dependent on the presence of the WH2-like motif. By generating profilin knockout and knockin Leishmania mexicana strains, we show that profilin is important for efficient endocytic sorting in parasites, and that the ability to bind actin monomers and proline-rich proteins, and the presence of a functional WH2-like motif, are important for the in vivo function of Leishmania profilin. Collectively, this study uncovers molecular principles by which profilin regulates actin dynamics in trypanosomatids.