Hen Egg White Lysozyme Research Articles

JINXED: just in time crystallization for easy structure determination of biological macromolecules.

Macromolecular crystallography is a well established method in the field of structural biology and has led to the majority of known protein structures to date. After focusing on static structures, the method is now under development towards the investigation of protein dynamics through time-resolved methods. These experiments often require multiple handling steps of the sensitive protein crystals, e.g. for ligand-soaking and cryo-protection. These handling steps can cause significant crystal damage, and hence reduce data quality. Furthermore, in time-resolved experiments based on serial crystallography, which use micrometre-sized crystals for short diffusion times of ligands, certain crystal morphologies with small solvent channels can prevent sufficient ligand diffusion. Described here is a method that combines protein crystallization and data collection in a novel one-step process. Corresponding experiments were successfully performed as a proof-of-principle using hen egg-white lysozyme and crystallization times of only a few seconds. This method, called JINXED (Just IN time Crystallization for Easy structure Determination), promises high-quality data due to the avoidance of crystal handling and has the potential to enable time-resolved experiments with crystals containing small solvent channels by adding potential ligands to the crystallization buffer, simulating traditional co-crystallization approaches.

The Ionization of Polymeric Materials Accelerates Protein Deposition on Hydrogel Contact Lens Material.

Contact lens materials include polymers that are ionized in the ocular pH condition and are susceptible to protein deposition due to their surface characteristics. Herein, we investigated the effect of the electrostatic state of the contact lens material and protein on protein deposition level using hen egg white lysozyme (HEWL) and bovine serum albumin (BSA) as model proteins and etafilcon A and hilafilcon B as model contact lens materials. Only HEWL deposition on etafilcon A showed a statistically significant pH-dependency (p < 0.05); protein deposition increased with pH. HEWL showed a positive zeta potential at acidic pH, while BSA showed a negative zeta potential at basic pH. Only etafilcon A showed a statistically significant pH-dependent point of zero charge (PZC) (p < 0.05), implying that its surface charge became more negative under basic conditions. This pH-dependency of etafilcon A is attributed to the pH-responsive degree of ionization of its constituent methacrylic acid (MAA). The presence of MAA and its degree of ionization could accelerate protein deposition; more HEWL deposited as pH increased despite the weak positive surface charge of HEWL. The highly negatively charged etafilcon A surface attracted HEWL, even overwhelming weak positive charge of HEWL, increasing the deposition with pH.

Amyloid fibril-nanocellulose interactions and self-assembly

Amyloid fibrils from inexpensive food proteins and nanocellulose are renewable and biodegradable materials with broad ranging applications, such as water purification, bioplastics and biomaterials. To improve the mechanical properties of hybrid amyloid-nanocellulose materials, their colloidal interactions need to be understood and tuned. A combination of turbidity and zeta potential measurements, rheology and atomic force microscopy point to the importance of electrostatic interactions. These interactions lead to entropy-driven polyelectrolyte complexation for positively charged hen egg white lysozyme (HEWL) amyloids with negatively charged nanocellulose. The complexation increased the elasticity of the amyloid network by cross-linking individual fibrils. Scaling laws suggest different contributions to elasticity depending on nanocellulose morphology: cellulose nanocrystals induce amyloid bundling and network formation, while cellulose nanofibrils contribute to a second network. The contribution of the amyloids to the elasticity of the entire network structure is independent of nanocellulose morphology and agrees with theoretical scaling laws. Finally, strong and almost transparent hybrid amyloid-nanocellulose gels were prepared in a slow self-assembly started from repulsive co-dispersions above the isoelectric point of the amyloids, followed by dialysis to decrease the pH and induce amyloid-nanocellulose attraction and cross-linking. In summary, the gained knowledge on colloidal interactions provides an important basis for the design of functional biohybrid materials based on these two biopolymers.

Exploring the Antioxidant Potential of Hen Egg White Lysozyme N-acetylmuramide Glycan Hydrolase in Full Fat Mozzarella Cheese

To explore the antioxidant potential of hen egg white lysozyme N-acetylmuramide Glycan hydrolase in full fat Mozzarella cheese, an experiment was conducted where control sample was brined without the addition of lysozyme; while four samples were brined along with 0.02%;w/v (T1), 0.04%;w/v (T2), 0.06%;w/v (T3) and 0.08%;w/v (T4) lysozyme. The antioxidant potential of hen egg white lysozyme in full fat Mozzarella cheese samples were measured through several methods, including 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid assay (ABTS), 2, 2-diphenyl-1-picrylhydrazyl (DPPH) assay, superoxide radical scavenging activity (PMS-NADH system), free fatty acid analysis and lipid peroxidation inhibition assay in a linoleic acid emulsion system. Intensity scale was used to evaluate the sensory characteristics of cheese. The results showed the percentage inhibition for DPPH, ABTS and superoxide anion activity in control sample increased up to 30 days; but decreased afterwards till 40 days of storage However in case of lysozyme treated samples % inhibition increased throughout storage. Free fatty acid % in full fat Mozzarella cheese was affected by lysozyme and storage days. Lipid peroxidation inhibitory activity in α-linoleic acid model system of lysozyme added samples showed lower absorbance at 500 nm showing higher lipid peroxidation inhibition. Furthermore, it was observed that absorbance was decreased till 30 days of storage which increased further upto 40 days storage period. The study showed that addition of lysozyme improved the shelf life and sensory characteristics of full fat Mozzarella cheese.&#x0D; Keywords: Lysozyme, Antioxidants, N-acetylmuramide Glycan Hydrolase, Full fat Mozzarella cheese,

Mechanistic Insight into the Amyloid Fibrillation Inhibition of Hen Egg White Lysozyme by Three Different Bile Acids.

Amyloid aggregation of protein is linked to many neurodegenerative diseases. Identification of small molecules capable of targeting amyloidogenic proteins has gained significant importance. Introduction of hydrophobic and hydrogen bonding interactions through site-specific binding of small molecular ligand to protein can effectively modulate the protein aggregation pathway. Here, we investigate the possible roles of three different bile acids, cholic acid (CA), taurocholic acid (TCA), and lithocholic acid (LCA) with varying hydrophobic and hydrogen bonding properties in inhibiting protein fibrillation. Bile acids are an important class of steroid compounds that are synthesized in the liver from cholesterol. Increasing evidence suggests that altered taurine transport, cholesterol metabolism, and bile acid synthesis have strong implications in Alzheimer's disease. We find that the hydrophilic bile acids, CA and TCA (taurine conjugated form of CA), are substantially more efficient inhibitors of lysozyme fibrillation than the most hydrophobic secondary bile acid LCA. Although LCA binds more strongly with the protein and masks the Trp residues more prominently through hydrophobic interactions, the lesser extent of hydrogen bonding interactions at the active site has made LCA a relatively weaker inhibitor of HEWL aggregation than CA and TCA. The introduction of a greater number of hydrogen bonding channels by CA and TCA with several key amino acid residues which are prone to form oligomers and fibrils has weakened the protein's internal hydrogen bonding capabilities for undergoing amyloid aggregation.

Proteins and their functionalization for finding therapeutic avenues in cancer: Current status and future prospective.

Despite the remarkable advancement in the health care sector, cancer remains the second most fatal disease globally. The existing conventional cancer treatments primarily include chemotherapy, which has been associated with little to severe side effects, and radiotherapy, which is usually expensive. To overcome these problems, target-specific nanocarriers have been explored for delivering chemo drugs. However, recent reports on using a few proteins having anticancer activity and further use of them as drug carriers have generated tremendous attention for furthering the research towards cancer therapy. Biomolecules, especially proteins, have emerged as suitable alternatives in cancer treatment due to multiple favourable properties including biocompatibility, biodegradability, and structural flexibility for easy surface functionalization. Several in vitro and in vivo studies have reported that various proteins derived from animal, plant, and bacterial species, demonstrated strong cytotoxic and antiproliferative properties against malignant cells in native and their different structural conformations. Moreover, surface tunable properties of these proteins help to bind a range of anticancer drugs and target ligands, thus making them efficient delivery agents in cancer therapy. Here, we discuss various proteins obtained from common exogenous sources and how they transform into effective anticancer agents. We also comprehensively discuss the tumor-killing mechanisms of different dietary proteins such as bovine α-lactalbumin, hen egg-white lysozyme, and their conjugates. We also articulate how protein nanostructures can be used as carriers for delivering cancer drugs and theranostics, and strategies to be adopted for improving their in vivo delivery and targeting. We further discuss the FDA-approved protein-based anticancer formulations along with those in different phases of clinical trials.

Tailoring of hydroxyapatite nanoparticle surfaces of varying morphologies to facilitate counterion diffusion and subsequent protein denaturation

Rapid advances in nanotechnology have led to the synthesis and development of various nanomaterials with complex structures and appropriate surface functionalization in recent years. Specifically designed and functionalized nanoparticles (NPs) are increasingly researched and hold great potential in biomedical applications (for example, imaging, diagnostics and therapeutics). Yet, the surface functionalization and biodegradability of NPs play a significant role in their application. Understanding the interactions occurring at the interface between the NPs and the biological components is thus crucial for predicting the fate of the NPs. In this work we study the effect of trilithium citrate functionalization of the hydroxyapatite NPs (HAp NPs) with and without cysteamine modification and their subsequent interaction with hen egg white lysozyme and corroborate the conformational changes of the protein with effective diffusion of the lithium (Li+) counter ion.

Textile Functionalization by Porous Protein Crystal Conjugation and Guest Molecule Loading

Protein crystals are versatile nanostructured materials that can be readily engineered for applications in nanomedicine and nanobiotechnology. Despite their versatility, the small size of typical individual protein crystals (less than one cubic mm) presents challenges for macroscale applications. One way to overcome this limitation is by immobilizing protein crystals onto larger substrates. Cotton is composed primarily of cellulose, the most common natural fiber in the world, and is routinely used in numerous material applications including textiles, explosives, paper, and bookbinding. Here, two types of protein crystals were conjugated to the cellulosic substrate of cotton fabric using a 1,1′-carbonyldiimidazole/aldehyde mediated coupling protocol. The efficacy of this attachment was assessed via accelerated laundering and quantified by fluorescence imaging. The ability to load guest molecules of varying sizes into the scaffold structure of the conjugated protein crystals was also assessed. This work demonstrates the potential to create multifunctional textiles by incorporating diverse protein crystal scaffolds that can be infused with a multiplicity of useful guest molecules. Cargo molecule loading and release kinetics will depend on the size of the guest molecules as well as the protein crystal solvent channel geometry. Here, we demonstrate the loading of a small molecule dye into the small pores of hen egg white lysozyme crystals and a model enzyme into the 13-nm pores delimited by “CJ” crystals composed of an isoprenoid-binding protein from Campylerbacter jejuni.

Structural Characterization of Nanobodies during Germline Maturation.

Camelid heavy-chain antibody variable domains (VHH), nanobodies, are the smallest-known functional antibody fragments with high therapeutic potential. In this study, we investigate a VHH binding to hen egg-white lysozyme (HEL). We structurally and dynamically characterized the conformational diversity of four VHH variants to elucidate the antigen-binding process. For two of these antibodies, not only are the dissociation constants known, but also the experimentally determined crystal structures of the VHH in complex with HEL are available. We performed well-tempered metadynamics simulations in combination with molecular dynamics simulations to capture a broad conformational space and to reconstruct the thermodynamics and kinetics of conformational transitions in the antigen-binding site, the paratope. By kinetically characterizing the loop movements of the paratope, we found that, with an increase in affinity, the state populations shift towards the binding competent conformation. The contacts contributing to antigen binding, and those who contribute to the overall stability, show a clear trend towards less variable but more intense contacts. Additionally, these investigated nanobodies clearly follow the conformational selection paradigm, as the binding competent conformation pre-exists within the structural ensembles without the presence of the antigen.

The Ability of Some Polysaccharides to Disaggregate Lysozyme Amyloid Fibrils and Renature the Protein.

The deposition of proteins in the form of insoluble amyloid fibril aggregates is linked to a range of diseases. The supramolecular architecture of such deposits is governed by the propagation of β-strands in the direction of protofilament growth. In the present study, we analyze the structural changes of hen egg-white lysozyme fibrils upon their interactions with a range of polysaccharides, using AFM and FTIR spectroscopy. Linear anionic polysaccharides, such as κ-carrageenan and sodium alginate, are shown to be capable to disaggregate protofilaments with eventual protein renaturation. The results help to understand the mechanism of amyloid disaggregation and create a platform for both the development of new therapeutic agents for amyloidose treatment, and the design of novel functional protein-polysaccharide complex-based nanomaterials.

Cationic liposome of hen egg white lysozyme for enhanced its stability, activity and accessibility in gastro-intestinal tract

Hen egg white lysozyme (HEWL) is a food-derived antimicrobial protein, exhibiting bacteriolytic activity against several bacteria including foodborne enteric pathogens. It has been approved for using in food, pharmacological and therapeutic applications. However, using HEWL through the ingestion is still a challenge due to its poor stability and low bioavailability in the gastro-intestinal tract (GIT). Herein, liposomes incorporating with dimethyl dioctadecyl ammonium bromide (DDAB) were fabricated to encapsulate HEWL (Lip_HEWL_DDAB) for oral delivery. Lip_HEWL_DDAB was spherical with an average diameter of 162.7 ± 1.6 nm and zeta potential of 37.1 ± 4.3 mV. The Lip_HEWL_DDAB had high entrapment efficiency of 80% and 11% loading efficiency of HEWL. The formulated Lip_HEWL_DDAB were highly physicochemical stable in both simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) and capable of enhancing the intestinal absorption via their mucoadhesive property while retaining antimicrobial activity against enteric bacteria. HEWL was released from Lip_HEWL_DDAB via diffusion mechanism at higher extent than those without the DDAB. Additionally, the prepared Lip_HEWL_DDAB was non-toxic at below 6.25 μg/ml to intestinal cells and was able to deliver HEWL across an in vitro intestinal epithelium model, resulting in enhanced anti-microbial/anti-inflammatory activities, compared to their free- and unmodified-liposomal forms. Together, our modified cationic liposome is a new and promising strategy for improving oral delivery of HEWL with enhanced nutraceutical functions.

Effect of Antihypertensive Drug (Chlorothiazide) on Fibrillation of Lysozyme: A Combined Spectroscopy, Microscopy, and Computational Study.

Amyloid fibrils abnormally accumulate together in the human body under certain conditions, which can result in lethal conditions. Thus, blocking this aggregation may prevent or treat this disease. Chlorothiazide (CTZ) is a diuretic and is used to treat hypertension. Several previous studies suggest that diuretics prevent amyloid-related diseases and reduce amyloid aggregation. Thus, in this study we examine the effects of CTZ on hen egg white lysozyme (HEWL) aggregation using spectroscopic, docking, and microscopic approaches. Our results showed that under protein misfolding conditions of 55 °C, pH 2.0, and 600 rpm agitation, HEWL aggregated as evidenced by the increased turbidity and Rayleigh light scattering (RLS). Furthermore, thioflavin-T, as well as trans electron microscope (TEM) analysis confirmed the formation of amyloid structures. An anti-aggregation effect of CTZ is observed on HEWL aggregations. Circular dichroism (CD), TEM, and Thioflavin-T fluorescence show that both CTZ concentrations reduce the formation of amyloid fibrils as compared to fibrillated. The turbidity, RLS, and ANS fluorescence increase with CTZ increasing. This increase is attributed to the formation of a soluble aggregation. As evidenced by CD analysis, there was no significant difference in α-helix content and β-sheet content between at 10 µM CTZ and 100 µM. A TEM analysis of HEWL coincubated with CTZ at different concentrations validated all the above-mentioned results. The TEM results show that CTZ induces morphological changes in the typical structure of amyloid fibrils. The steady-state quenching study demonstrated that CTZ and HEWL bind spontaneously via hydrophobic interactions. HEWL-CTZ also interacts dynamically with changes in the environment surrounding tryptophan. Computational results revealed the binding of CTZ to ILE98, GLN57, ASP52, TRP108, TRP63, TRP63, ILE58, and ALA107 residues in HEWL via hydrophobic interactions and hydrogen bonds with a binding energy of -6.58 kcal mol-1. We suggest that at 10 µM and 100 μM, CTZ binds to the aggregation-prone region (APR) of HEWL and stabilizes it, thus preventing aggregation. Based on these findings, we can conclude that CTZ has antiamyloidogenic activity and can prevent fibril aggregation.

Optimization of hydrogen-deuterium exchange ensemble reweighting (HDXer) to aid the biophysical characterization of proteins.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) experiments provide unique insights into native protein structure and dynamics in solution but have limited spatial resolution. Molecular dynamics (MD) simulations, in contrast, allow for the exploration of protein conformational landscapes with an atomistic level of resolution. However, forcefield inaccuracies, crystallographic biases, and timescale limitations may compromise the validity of the final structural ensemble. Hydrogen Deuterium eXchange Ensemble Reweighting (HDXer) is a maximum entropy approach that was recently developed to (1) quantitatively predict hydrogen-deuterium exchange from a modeled conformational ensemble and (2) refine the candidate ensemble to best fit an experimental HDX-MS dataset. Here, we compare how various computational and experimental parameters impact the performance of HDXer, using extracellular regulated kinase 2 (ERK2) and hen egg white lysozyme (HEWL) as model proteins. Conventional molecular dynamics simulations of ERK2 and HEWL were performed in triplicate using the CHARMM36m, AMBER FF19SB, and Drude forcefields. After predicting the HDX rates of each candidate ensemble, HDXer was used to fit the simulated structures to the corresponding HDX-MS datasets. To assess the influence of simulation length, truncated and strided copies of the full-length simulations were generated. Other factors, including peptide charge state selection, ion mobility mass spectrometry, and independent HDX-MS data acquisitions, were also tested. We find that while some variables (e.g., high vs. low peptide charge states) do not have a significant effect, other parameters (e.g., early vs. late timepoint selection) directly correlate with the disagreement between calculated and experimental deuterium uptake. The results from this benchmarking inform the development of a recommended HDXer workflow and best practices for optimal data acquisition.

Roles of Tryptophan and Charged Residues on the Polymorphisms of Amyloids Formed by K-Peptides of Hen Egg White Lysozyme Investigated through Molecular Dynamics Simulations.

Atomistic molecular dynamics simulations of amyloid models, consisting of the previously reported STDY-K-peptides and K-peptides from the hen egg white lysozyme (HEWL), were performed to address the effects of charged residues and pH observed in an in vitro study. Simulation results showed that amyloid models with antiparallel configurations possessed greater stability and compactness than those with parallel configurations. Then, peptide chain stretching and ordering were measured through the end-to-end distance and the order parameter, for which the amyloid models consisting of K-peptides and the STDY-K-peptides at pH 2 displayed a higher level of chain stretching and ordering. After that, the molecular mechanics energy decomposition and the radial distribution function (RDF) clearly displayed the importance of Trp62 to the K-peptide and the STDY-K-peptide models at pH 2. Moreover, the results also displayed how the negatively charged Asp52 disrupted the interaction networks and prevented the amyloid formation from STDY-K-peptide at pH 7. Finally, this study provided an insight into the interplay between pH conditions and molecular interactions underlying the formation of amyloid fibrils from short peptides contained within the HEWL. This served as a basis of understanding towards the design of other amyloids for biomaterial applications.

Regioselective protein oxidative cleavage enabled by enzyme-like recognition of an inorganic metal oxo cluster ligand

Oxidative modifications of proteins are key to many applications in biotechnology. Metal-catalyzed oxidation reactions efficiently oxidize proteins but with low selectivity, and are highly dependent on the protein surface residues to direct the reaction. Herein, we demonstrate that discrete inorganic ligands such as polyoxometalates enable an efficient and selective protein oxidative cleavage. In the presence of ascorbate (1 mM), the Cu-substituted polyoxometalate K8[Cu2+(H2O)(α2-P2W17O61)], (CuIIWD, 0.05 mM) selectively cleave hen egg white lysozyme under physiological conditions (pH =7.5, 37 °C) producing only four bands in the gel electropherogram (12.7, 11, 10, and 5 kDa). Liquid chromatography/mass spectrometry analysis reveals a regioselective cleavage in the vicinity of crystallographic CuIIWD/lysozyme interaction sites. Mechanistically, polyoxometalate is critical to position the Cu at the protein surface and limit the generation of oxidative species to the proximity of binding sites. Ultimately, this study outlines the potential of discrete, designable metal oxo clusters as catalysts for the selective modification of proteins through radical mechanisms under non-denaturing conditions.

Differential effects of DTT on HEWL amyloid fibrillation and fibril morphology at different pH

Proteins can transform from their native state to a state having fibrillar aggregates characterized by cross β sheet structure. The fibrillar aggregates are known as amyloid and have been linked to several disorders. Disulfide bonds in proteins are one of the important factors that determine the propensity of aggregation. Hen Egg White Lysozyme (HEWL) was used by us as a model protein to decipher the role disulfide bonds play in the amyloid fibril formation and fibril morphology by using Dithiothreitol (DTT) as reducing agent at pH 2.7 and pH 7.4. We found that DTT can have different effects on HEWL amyloid depending on pH and the buffer used for preparing the amyloid fibrils. Our studies highlight the critical role of non-native disulfide bonds in amyloidogenesis and how disruption of these bonds can greatly affect the fibrillation process. Overall, these studies throw light on the fibrillation mechanism and can be explored further in designing effective inhibitors against amyloidosis.

Elucidating the inhibitory effects of rationally designed novel hexapeptide against hen egg white lysozyme fibrillation at acidic and physiological pH

Inhibition of highly ordered cross-β-sheet-rich aggregates of misfolded amyloid proteins using rationally designed sequence-based short peptides is a promising therapeutic strategy for the treatment of neurodegenerative diseases. Here, we have explored the anti-amyloidogenic potency of a rationally designed hexapeptide (Tyr-Pro-Gln-Ile-Pro-Asn) on in vitro hen egg white lysozyme (HEWL) amyloid fibril formation at acidic pH and physiological pH using computational docking as well as various biophysical techniques such as fluorescence spectroscopy, UV–vis spectroscopy, FTIR spectroscopy, confocal microscopy and TEM. The peptide was designed based on the aggregation-prone region (APR) of HEWL and thus referred to as SqP1 (Sequence-based Peptide 1). SqP1 showed over 70% inhibition of HEWL amyloid formation at pH 2.2 and approximately 50% inhibition at pH 7.5. We propose that SqP1 binds to the APR of HEWL and interacts strongly with the Trp62/Trp63, ultimately stabilizing monomeric HEWL at both the pH conditions and preventing conformation changes in the structure of HEWL, leading to the formation of amyloidogenic fibrillar structures. A sequence-based peptide inhibitor of HEWL amyloid formation was not reported previously, making this a critical study that will further emphasize the importance of short synthetic peptides as amyloid inhibitors.

Replica-exchange optimization of antibody fragments

In the framework of the rational design of macromolecules capable of binding to a specific target for biosensing applications, we here further develop an evolutionary protocol designed to optimize the binding affinity of protein binders. In particular we focus on the optimization of the binding portion of small antibody fragments known as nanobodies (or VHH) and choose the hen egg white lysozyme (HEWL) as our target. By implementing a replica exchange scheme for this optimization, we show that an initial hit is not needed and similar solutions can be found by either optimizing an already known anti-HEWL VHH or a randomly selected binder (here a VHH selective towards another macromolecule). While we believe that exhaustive searches of the mutation space are most appropriate when only few key residues have to be optimized, in case a lead binder is not available the proposed evolutionary algorithm should be instead the method of choice.

The Effect of Arginine on the Phase Stability of Aqueous Hen Egg-White Lysozyme Solutions.

The effect of arginine on the phase stability of the hen egg-white lysozyme (HEWL) has been studied via molecular dynamics computer simulations, as well as experimentally via cloud-point temperature determination. The experiments show that the addition of arginine increases the stability of the HEWL solutions. The computer simulation results indicate that arginine molecules tend to self-associate. If arginine residues are located on the protein surface, the free arginine molecules stay in their vicinity and prevent the way protein molecules "connect" through them to form clusters. The results are not sensitive to a particular force field and suggest a possible microscopic mechanism of the stabilizing role of arginine as an excipient.

Effect of Equatorial Ligand Substitution on the Reactivity with Proteins of Paddlewheel Diruthenium Complexes: Structural Studies

The paddlewheel [Ru2Cl(O2CCH3)4] complex was previously reported to react with the model protein hen egg white lysozyme (HEWL), forming adducts with two diruthenium moieties bound to Asp101 and Asp119 side chains upon the release of one acetate. To study the effect of the equatorial ligands on the reactivity with proteins of diruthenium compounds, X-ray structures of the adducts formed when HEWL reacts with [Ru2Cl(D-p-FPhF)(O2CCH3)3] [D-p-FPhF = N,N'-bis(4-fluorophenyl)formamidinate] under different conditions were solved. [Ru2Cl(D-p-FPhF)(O2CCH3)3] is bonded through their equatorial positions to the Asp side chains. Protein binding occurs cis or trans to D-p-FPhF. Lys or Arg side chains or even main-chain carbonyl groups can coordinate to the diruthenium core at the axial site. Data help to understand the reactivity of paddlewheel diruthenium complexes with proteins, providing useful information for the design of new artificial diruthenium-containing metalloenzymes with potential applications in the fields of catalysis, biomedicine, and biotechnology.