Silver nanoparticle interactions with glycated and non-glycated human serum albumin mediate toxicity.

Introduction: Biomolecules bind to and transform nanoparticles, mediating their fate in biological systems. Despite over a decade of research into the protein corona, the role of protein modifications in mediating their interaction with nanomaterials remains poorly understood. In this study, we evaluated how glycation of the most abundant blood protein, human serum albumin (HSA), influences the formation of the protein corona on 40 nm silver nanoparticles (AgNPs) and the toxicity of AgNPs to the HepG2 human liver cell line. Methods: The effects of glycation on AgNP-HSA interactions were quantified using circular dichroism spectroscopy to monitor protein structural changes, dynamic light scattering to assess AgNP colloidal stability, zeta potential measurements to measure AgNP surface charge, and UV-vis spectroscopy and capillary electrophoresis (CE) to evaluate protein binding affinity and kinetics. The effect of the protein corona and HSA glycation on the toxicity of AgNPs to HepG2 cells was measured using the WST cell viability assay and AgNP dissolution was measured using linear sweep stripping voltammetry. Results and Discussion: Results from UV-vis and CE analyses suggest that glycation of HSA had little impact on the formation of the AgNP protein corona with protein-AgNP association constants of ≈2x107 M-1 for both HSA and glycated HSA (gHSA). The formation of the protein corona itself (regardless of whether it was formed from HSA or glycated HSA) caused an approximate 2-fold decrease in cell viability compared to the no protein AgNP control. While the toxicity of AgNPs to cells is often attributed to dissolved Ag(I), dissolution studies showed that the protein coated AgNPs underwent less dissolution than the no protein control, suggesting that the protein corona facilitated a nanoparticle-specific mechanism of toxicity. Overall, this study highlights the importance of protein coronas in mediating AgNP interactions with HepG2 cells and the need for future work to discern how protein coronas and protein modifications (like glycation) may alter AgNP reactivity to cellular organisms.

Silver Nanoparticle Surface Chemistry Determines Interactions with Human Serum Albumin and Cytotoxic Responses in Human Liver Cells.

Engineered nanomaterials (ENMs) are synthesized with a diversity of surface chemistries that mediate biochemical interactions and physiological response to the particles. In this work, silver engineered nanomaterials (AgENMs) are used to evaluate the role of surface charge in protein interactions and cellular cytotoxicity. The most abundant protein in blood, human serum albumin (HSA), was interacted with 40 nm AgENMs with a range of surface-charged coatings: positively charged branched polyethyleneimine (bPEI), negatively charged citrate (CIT), and circumneutral poly(ethylene glycol) (PEG). HSA adsorption to AgENMs was monitored by UV-vis spectroscopy and dynamic light scattering, while changes to the protein structure were evaluated with circular dichroism spectroscopy. Binding affinity for citrate-coated AgENMs and HSA is largest among the three AgENM coatings; yet, HSA lost the most secondary structure upon interaction with bPEI-coated AgENMs compared to the other two coatings. HSA increased AgENM oxidative dissolution across all particle types, with the greatest dissolution for citrate-coated AgENMs. Results indicate that surface coating is an important consideration in transformation of both the particle and protein upon interaction. To connect results to cellular outcomes, we also performed cytotoxicity experiments with HepG2 cells across all three AgENM types with and without HSA. Results show that bPEI-coated AgENMs cause the greatest loss of cell viability, both with and without inclusion of HSA with the AgENMs. Thus, surface coatings on AgENMs alter both biophysical interactions with proteins and particle cytotoxicity. Within this study set, positively charged bPEI-coated AgENMs cause the greatest disruption to HSA structure and cell viability.

Isolation Methods Influence the Protein Corona Composition on Gold-Coated Iron Oxide Nanoparticles.

Upon exposure to a biological environment, nanoparticles (NPs) acquire biomolecular coatings, the most studied of which is the protein corona. This protein corona gives NPs a new biological identity that will determine various biological responses including cellular uptake, biodistribution, and toxicity. The standard method to isolate NPs from a biological matrix in order to study their coronas is centrifugation, but more gentle means of retrieval may enable deeper understanding of both irreversibly bound hard coronas and more loosely bound soft coronas. In this study, magnetic gold-coated iron oxide NPs were incubated with rainbow trout gill cell total protein extracts and mass spectrometric proteomic analysis was conducted to determine the composition of the protein coronas isolated by either centrifugation or magnetic retrieval. The number of washes were varied to strip away the soft coronas and isolate the hard corona. Hundreds of proteins were adsorbed to the NPs. Some proteins were common to all isolation methods and many others were particular to the isolation method. Some qualitative trends in protein character were discerned from quantitative proteomic analyses, but more importantly, a new kind of protein corona was identified, mixed corona, in which the labile or inert nature of the protein-NP interaction is dependent upon sample history.

Environmental dimensions of the protein corona.

The adsorption of biomolecules to the surface of engineered nanomaterials, known as corona formation, defines their biological identity by altering their surface properties and transforming the physical, chemical and biological characteristics of the particles. In the first decade since the term protein corona was coined, studies have focused primarily on biomedical applications and human toxicity. The relevance of the environmental dimensions of the protein corona is still emerging. Often referred to as the eco-corona, a biomolecular coating forms upon nanomaterials as they enter the environment and may include proteins, as well as a diverse array of other biomolecules such as metabolites from cellular activity and/or natural organic matter. Proteins remain central in studies of eco-coronas because of the ease of monitoring and structurally characterizing proteins, as well as their crucial role in receptor engagement and signalling. The proteins within the eco-corona are optimal targets to establish the biophysicochemical principles of corona formation and transformation, as well as downstream impacts on nanomaterial uptake, distribution and impacts on the environment. Moreover, proteins appear to impart a biological identity, leading to cellular or organismal recognition of nanomaterials, a unique characteristic compared with natural organic matter. We contrast insights into protein corona formation from clinical samples with those in environmentally relevant systems. Principles specific to the environment are also explored to gain insights into the dynamics of interaction with or replacement by other biomolecules, including changes during trophic transfer and ecotoxicity. With many challenges remaining, we also highlight key opportunities for method development and impactful systems on which to focus the next phase of eco-corona studies. By interrogating these environmental dimensions of the protein corona, we offer a perspective on how mechanistic insights into protein coronas in the environment can lead to more sustainable, environmentally safe nanomaterials, as well as enhancing the efficacy of nanomaterials used in remediation and in the agri-food sector.

The Primarily Undergraduate Nanomaterials Cooperative: A New Model for Supporting Collaborative Research at Small Institutions on a National Scale.

The Primarily Undergraduate Nanomaterials Cooperative (PUNC) is an organization for research-active faculty studying nanomaterials at Primarily Undergraduate Institutions (PUIs), where undergraduate teaching and research go hand-in-hand. In this perspective, we outline the differences in maintaining an active research group at a PUI compared to an R1 institution. We also discuss the work of PUNC, which focuses on community building, instrument sharing, and facilitating new collaborations. Currently consisting of 37 members from across the United States, PUNC has created an online community consisting of its Web site (nanocooperative.org), a weekly online summer group meeting program for faculty and students, and a Discord server for informal conversations. Additionally, in-person symposia at ACS conferences and PUNC-specific conferences are planned for the future. It is our hope that in the years to come PUNC will be seen as a model organization for community building and research support at primarily undergraduate institutions.

The role of human serum and solution chemistry in fibrinogen peptide-nanoparticle interactions.

In living systems, the biomolecules that coat nanoparticles (NPs) alter the NP biological identity and response. Although some biomolecules are more effective in mediating NP stability or biological fate, it is difficult to monitor an individual biomolecule within the complexity of the biota. To understand the dependence of protein-NP interactions on common variations in blood, we have evaluated binding between silica NPs and a model gamma-fibrinogen (GF) peptide. Fibrinogen is commonly identified within the protein corona fingerprint of human serum, but its abundance on the NP varies. To assess the relative importance of human serum and solution conditions, GF peptide and silica NP interactions were evaluated with and without serum across pH, NaCl concentrations, and glucose concentrations. Initial evaluation of the GF peptide and silica NP complexes using circular dichroism and dynamic light scattering show little change in the secondary structure of the peptide and no significant agglomeration of NPs, suggesting peptide-NP complexes are stable across study conditions. Fluorescence anisotropy was used to monitor GF peptide-NP binding. Both with and without serum, binding constants for the gamma-fibrinogen peptide vary significantly upon addition of diluted HS (1:500) and 29 mM sodium chloride. Yet, results indicated that gamma-fibrinogen binding interactions with silica NPs are comparatively insensitive to physiologically relevant pH changes and dramatic increases in glucose concentrations. Results highlight the importance of blood chemistries, which vary across individuals and disease states, in mediating protein corona formation.

Low-Dose Silver Nanoparticle Surface Chemistry and Temporal Effects on Gene Expression in Human Liver Cells.

Silver nanoparticles (AgNPs) are widely incorporated into consumer and biomedical products for their antimicrobial and plasmonic properties with limited risk assessment of low-dose cumulative exposure in humans. To evaluate cellular responses to low-dose AgNP exposures across time, human liver cells (HepG2) are exposed to AgNPs with three different surface charges (1.2 µg mL-1 ) and complete gene expression is monitored across a 24 h period. Time and AgNP surface chemistry mediate gene expression. In addition, since cells are fed, time has marked effects on gene expression that should be considered. Surface chemistry of AgNPs alters gene transcription in a time-dependent manner, with the most dramatic effects in cationic AgNPs. Universal to all surface coatings, AgNP-treated cells responded by inactivating proliferation and enabling cell cycle checkpoints. Further analysis of these universal features of AgNP cellular response, as well as more detailed analysis of specific AgNP treatments, time points, or specific genes, is facilitated with an accompanying application. Taken together, these results provide a foundation for understanding hepatic response to low-dose AgNPs for future risk assessment.

Emerging investigator series: characterization of silver and silver nanoparticle interactions with zinc finger peptides.

In biological systems, chemical and physical transformations of engineered silver nanomaterials (AgENMs) are mediated, in part, by proteins and other biomolecules. Metalloprotein interactions with AgENMs are also central in understanding toxicity and antimicrobial and resistance mechanisms. Despite their readily available thiolate and amine ligands, zinc finger (ZF) peptides have thus far escaped study in reaction with AgENMs and their Ag(I) oxidative dissolution product. We report spectroscopic studies that characterize AgENM and Ag(I) interactions with two ZF peptides that differ in sequence, but not in metal binding ligands: the ZF consensus peptide CP-CCHC and the C-terminal zinc finger domain of HIV-1 nucleocapsid protein p7 (NCp7_C). Both ZF peptides catalyze AgENM (10 and 40 nm, citrate coated) dissolution and agglomeration, two important AgENM transformations that impact bioreactivity. AgENMs and their oxidative dissolution product, Ag(I)(aq), mediate changes to ZF peptide structure and metalation as well. Spectroscopic titrations of Ag(I) into apo-ZF peptides show an Ag(I)-thiolate charge transfer band, indicative of Ag(I)-ZF binding. Fluorescence studies of the Zn(II)-NCp_7 complex indicate that the Ag(I) also effectively competes with the Zn(II) to drive Zn(II) displacement from the ZFs. Upon interaction with AgENMs, Zn(II) bound ZF peptides show a secondary structural change in circular dichroism spectroscopy toward an apo-like structure. The results suggest that Ag(I) and AgENMs may alter ZF protein function within the cell.

Publisher Correction: On the issue of transparency and reproducibility in nanomedicine.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Publisher Correction: On the issue of transparency and reproducibility in nanomedicine.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Best practice in reporting corona studies: Minimum information about Nanomaterial Biocorona Experiments (MINBE).

Characterisation of the adsorption of biomolecules, or a biocorona, on nanomaterials has proliferated in the past 10 years, as protein corona studies provide molecular level insight into mechanisms of cellular recognition, uptake, and toxicity of nanomaterials. At the crossroads of two rapidly evolving orthogonal fields, nanoscience and proteomics, the interdisciplinarity of protein corona studies creates challenges for experimental design and reporting. Here we propose a flexible checklist for experimental design and reporting guidelines to outline Minimum Information about Nanomaterial Biocorona Experiments (MINBE). The checklist for experimental design, compiled after review of reporting within the protein corona literature, provides researchers with prompts to ensure best practice experimental approaches for each stage of the workflow, collated from the nanoscience, proteomics, and bioinformatics fields. Reporting guidelines are also assembled from established sources, integrated to span the entire workflow and extended and modified to aid interdisciplinary researchers in the most challenging stages of the workflow. Where appropriate, de novo guidelines to address areas specific to protein corona studies, including exposure conditions and isolation of adsorbed proteins, were written. The MINBE guidelines provide protein corona researchers with a conduit between materials science techniques and proteomics. Implementation of these guidelines is anticipated to catalyse enhanced quality, impact, and extent of data mining and computational modelling of protein corona composition and its role in nanosafety and nanomedicine. Furthermore, high quality experimental design and reporting in the bio-nanosciences will enhance the next phase of targeted nanomedicines and sustainable nanotechnologies.

Machine learning provides predictive analysis into silver nanoparticle protein corona formation from physicochemical properties.

Proteins encountered in biological and environmental systems bind to engineered nanomaterials (ENMs) to form a protein corona (PC) that alters the surface chemistry, reactivity, and fate of the ENMs. Complexities such as the diversity of the PC and variation with ENM properties and reaction conditions make the PC population difficult to predict. Here, we support the development of predictive models for PC populations by relating biophysicochemical characteristics of proteins, ENMs, and solution conditions to PC formation using random forest classification. The resulting model offers a predictive analysis into the population of PC proteins in Ag ENM systems of various ENM size and surface coatings. With an area under the receiver operating characteristic curve of 0.83 and F1-score of 0.81, a model with strong performance has been constructed based upon experimental data. The weighted contribution of each variable provides recommendations for mechanistic models based upon protein enrichment classification results. Protein biophysical properties such as pI and weight are weighted heavily. Yet, ENM size, surface charge, and solution ionic strength also proved essential to an accurate model. The model can be readily modified and applied to other ENM PC populations. The model presented here represents the first step toward robust predictions of PC fingerprints.

Beyond the passive interactions at the nano-bio interface: evidence of Cu metalloprotein-driven oxidative dissolution of silver nanoparticles.

BACKGROUND: In a biological system, an engineered nanomaterial (ENM) surface is altered by adsorbed proteins that modify ENM fate and toxicity. Thus far, protein corona characterizations have focused on protein adsorption, interaction strength, and downstream impacts on cell interactions. Given previous reports of Ag ENM disruption of Cu trafficking, this study focuses on Ag ENM interactions with a model Cu metalloprotein, Cu(II) azurin. The study provides evidence of otherwise overlooked ENM-protein chemical reactivity within the corona: redox activity. RESULTS: Citrate-coated Ag ENMs of various sizes (10-40 nm) reacted with Cu(II) azurin resulted in an order of magnitude more dissolved ionic silver (Ag(I)(aq)) than samples of Ag ENMs only, ENMs mixed Cu(II) ions, or control proteins such as cytochrome c and horse radish peroxidase. This dramatic increase in ENM oxidative dissolution was observed even when Cu(II) azurin was combined with a diverse mixture of Escherchia coli proteins to mimic the complexity of the cellular conona. SDS PAGE results confirm that the multiprotein ENM corona includes azurin. A Cu(I)(aq) colorimetric indicator confirms Cu(II) azurin reduction upon interaction with Ag ENMs, but not with the addition of ionic silver, Ag(I)(aq). CONCLUSIONS: Cu(II) azurin and 10-40 nm Ag ENMs react to catalyze Ag ENM oxidative dissolution and reduction of the model Cu metalloprotein. Results push the current evaluation of protein-ENM characterization beyond passive binding interactions and enable the proposal of a mechanism for reactivity between a model Cu metalloprotein and Ag ENMs.

Spectroscopic characterization of copper(I) binding to apo and metal-reconstituted zinc finger peptides.

Cu(I) exhibits high affinity for thiolate ligands, suggesting that thiol-rich zinc or iron binding sites may be subject to disruption during copper stress conditions. Zinc fingers constitute a large class of metalloproteins that use a combination of cysteine and histidine residues that bind Zn(II) as a structural element. Despite the shared preference of both copper and zinc for thiolate and amine coordination, the susceptibility of zinc finger domains toward copper substitution is not well studied. We report spectroscopic studies that characterize the Cu(I) binding properties of the zinc finger consensus peptides CP-CCHH, CP-CCHC, and CP-CCCC and the C-terminal zinc finger domain of HIV-1 nucleocapsid protein p7 (NCp7_C). Cu(I) binds to both the apopeptides and the Co(II)-substituted peptides, and the stoichiometry of Cu(I) binding is dependent on the number of cysteine thiols at the metal binding site. Fluorescence studies of the Zn(II)-NCp7_C complex indicate that Cu(I) also effectively competes with Zn(II) at the metal binding site, despite the high affinity of Zn(II) for the CCHC binding motif. Circular dichroism studies on both CP-CCHC and NCp7_C show that the conformations of the Cu(I)-bound complexes differ substantially from those of the Zn(II) species, implying that Cu(I) substitution is likely to impact zinc finger function. These results show that for the peptides studied here, Cu(I) is the thermodynamically favored metal despite the known high Zn(II) affinity of zinc finger domains, suggesting that Cu(I)-substituted zinc finger domains might be relevant in the context of both copper toxicity mechanisms and copper-responsive transcription factors.

Structural and functional effects of Cu metalloprotein-driven silver nanoparticle dissolution.

Interactions of a model Cu-metalloprotein, azurin, with 10-100 nm silver nanoparticles (NPs) were examined to elucidate the role of oxidative dissolution and protein interaction on the biological reactivity of NPs. Although minimal protein and NP structural changes were observed upon interaction, displacement of Cu(II) and formation of Ag(I) azurin species under aerobic conditions implicates Cu(II) azurin as a catalyst of NP oxidative dissolution. Consistent with NP oxidation potentials, largest concentrations of Ag(I) azurin species were recorded in reaction with 10 nm NPs (>50%). Apo-protein was also observed under anaerobic reaction with NPs of all sizes and upon aerobic reaction with larger NPs (>20 nm), where NP oxidation is slowed. Cu(II) azurin displacement upon reaction with NPs was significantly greater than when reacted with Ag(I)(aq) alone. Regardless of NP size, dialysis experiments show minimal reactivity between azurin and the Ag(I)(aq) species formed as a result of NP oxidative dissolution, indicating Cu displacement from azurin occurs at the NP surface. Mechanisms of azurin-silver NP interaction are proposed. Results demonstrate that NP interactions not only impact protein structure and function, but also NP reactivity, with implications for targeting, uptake, and cytotoxicity.

Metal affinity enrichment increases the range and depth of proteome identification for extracellular microbial proteins.

Many key proteins, such as those involved in cellular signaling or transcription, are difficult to measure in microbial proteomic experiments due to the interfering presence of more abundant, dominant proteins. In an effort to enhance the identification of previously undetected proteins, as well as provide a methodology for selective enrichment, we evaluated and optimized immobilized metal affinity chromatography (IMAC) coupled with mass spectrometric characterization of extracellular proteins from an extremophilic microbial community. Seven different metals were tested for IMAC enrichment. The combined results added ∼20% greater proteomic depth to the extracellular proteome. Although this IMAC enrichment could not be conducted at the physiological pH of the environmental system, this approach did yield a reproducible and specific enrichment of groups of proteins with functions potentially vital to the community, thereby providing a more extensive biochemical characterization. Notably, 40 unknown proteins previously annotated as "hypothetical" were enriched and identified for the first time. Examples of identified proteins includes a predicted TonB signal sensing protein homologous to other known TonB proteins and a protein with a COXG domain previously identified in many chemolithoautotrophic microbes as having a function in the oxidation of CO.

Characterization of extracellular polymeric substances from acidophilic microbial biofilms.

We examined the chemical composition of extracellular polymeric substances (EPS) extracted from two natural microbial pellicle biofilms growing on acid mine drainage (AMD) solutions. The EPS obtained from a mid-developmental-stage biofilm (DS1) and a mature biofilm (DS2) were qualitatively and quantitatively compared. More than twice as much EPS was derived from DS2 as from DS1 (approximately 340 and 150 mg of EPS per g [dry weight] for DS2 and DS1, respectively). Composition analyses indicated the presence of carbohydrates, metals, proteins, and minor quantities of DNA and lipids, although the relative concentrations of these components were different for the two EPS samples. EPS from DS2 contained higher concentrations of metals and carbohydrates than EPS from DS1. Fe was the most abundant metal in both samples, accounting for about 73% of the total metal content, followed by Al, Mg, and Zn. The relative concentration profile for these metals resembled that for the AMD solution in which the biofilms grew, except for Si, Mn, and Co. Glycosyl composition analysis indicated that both EPS samples were composed primarily of galactose, glucose, heptose, rhamnose, and mannose, while the relative amounts of individual sugars were substantially different in DS1 and DS2. Additionally, carbohydrate linkage analysis revealed multiply linked heptose, galactose, glucose, mannose, and rhamnose, with some of the glucose in a 4-linked form. These results indicate that the biochemical composition of the EPS from these acidic biofilms is dependent on maturity and is controlled by the microbial communities, as well as the local geochemical environment.

Community genomic and proteomic analyses of chemoautotrophic iron-oxidizing "Leptospirillum rubarum" (Group II) and "Leptospirillum ferrodiazotrophum" (Group III) bacteria in acid mine drainage biofilms.

We analyzed near-complete population (composite) genomic sequences for coexisting acidophilic iron-oxidizing Leptospirillum group II and III bacteria (phylum Nitrospirae) and an extrachromosomal plasmid from a Richmond Mine, Iron Mountain, CA, acid mine drainage biofilm. Community proteomic analysis of the genomically characterized sample and two other biofilms identified 64.6% and 44.9% of the predicted proteins of Leptospirillum groups II and III, respectively, and 20% of the predicted plasmid proteins. The bacteria share 92% 16S rRNA gene sequence identity and >60% of their genes, including integrated plasmid-like regions. The extrachromosomal plasmid carries conjugation genes with detectable sequence similarity to genes in the integrated conjugative plasmid, but only those on the extrachromosomal element were identified by proteomics. Both bacterial groups have genes for community-essential functions, including carbon fixation and biosynthesis of vitamins, fatty acids, and biopolymers (including cellulose); proteomic analyses reveal these activities. Both Leptospirillum types have multiple pathways for osmotic protection. Although both are motile, signal transduction and methyl-accepting chemotaxis proteins are more abundant in Leptospirillum group III, consistent with its distribution in gradients within biofilms. Interestingly, Leptospirillum group II uses a methyl-dependent and Leptospirillum group III a methyl-independent response pathway. Although only Leptospirillum group III can fix nitrogen, these proteins were not identified by proteomics. The abundances of core proteins are similar in all communities, but the abundance levels of unique and shared proteins of unknown function vary. Some proteins unique to one organism were highly expressed and may be key to the functional and ecological differentiation of Leptospirillum groups II and III.

Dynamic docking of cytochrome b5 with myoglobin and alpha-hemoglobin: heme-neutralization "squares" and the binding of electron-transfer-reactive configurations.

Intracomplex electron transfer (ET) occurs most often in intrinsically transient, low affinity complexes. As a result, the means by which adequate specificity and reactivity are obtained to support effective ET is still poorly understood. We report here on two such ET complexes: cytochrome b5 (cyt b5) in reaction with its physiological partners, myoglobin (Mb) and hemoglobin (Hb). These complexes obey the Dynamic Docking (DD) paradigm: a large ensemble of weakly bound protein-protein configurations contribute to binding in the rapid-exchange limit, but only a few are ET-active. We report the ionic-strength dependence of the second-order rate constant, k2, for photoinitiated ET from within all four combinations of heme-neutralized Zn deuteroporphyrin-substituted Mb/alphaHb undergoing ET with cyt b5, the four "corners" of a "heme-neutralization square". These experiments provide insights into the relative importance of both global and local electrostatic contributions to the binding of reactive configurations, which are too few to be observed directly. To interpret the variations of k2 arising from heme neutralization, we have developed a procedure by which comparisons of the ET rate constants for a heme-neutralization square permit us to decompose the free energy of reactive binding into individual local electrostatic contributions associated with interactions between (i) the propionates of the two hemes and (ii) the heme of each protein with the polypeptide of its partner. Most notably, we find the contribution from the repulsion between propionates of partner hemes to the reactive binding free energy to be surprisingly small, DeltaG(Hb) approximately +1 kcal/mol at ambient temperature, 18 mM ionic strength, and we speculate about possible causes of this observation. To confirm the fundamental assumption of these studies, that the structure of a heme-neutralized protein is unaltered either by substitution of Zn or by heme neutralization, we have obtained the X-ray structure of ZnMb prepared with the porphyrin dimethyl ester and find it to be nearly isostructural with the native protein.