Fast Fluoroalkylation of Proteins Uncovers the Structure and Dynamics of Biological Macromolecules.

Covalent labeling of proteins in combination with mass spectrometry has been established as a complementary technique to classical structural methods, such as X-ray, NMR, or cryogenic electron microscopy (Cryo-EM), used for protein structure determination. Although the current covalent labeling techniques enable the protein solvent accessible areas with sufficient spatial resolution to be monitored, there is still high demand for alternative, less complicated, and inexpensive approaches. Here, we introduce a new covalent labeling method based on fast fluoroalkylation of proteins (FFAP). FFAP uses fluoroalkyl radicals formed by reductive decomposition of Togni reagents with ascorbic acid to label proteins on a time scale of seconds. The feasibility of FFAP to effectively label proteins was demonstrated by monitoring the differential amino acids modification of native horse heart apomyoglobin/holomyoglobin and the human haptoglobin-hemoglobin complex. The obtained data confirmed the Togni reagent-mediated FFAP is an advantageous alternative method for covalent labeling in applications such as protein footprinting and epitope mapping of proteins (and their complexes) in general. Data are accessible via the ProteomeXchange server with the data set identifier PXD027310.

Fenton-Chemistry-Based Oxidative Modification of Proteins Reflects Their Conformation.

In order to understand protein structure to a sufficient extent for, e.g., drug discovery, no single technique can provide satisfactory information on both the lowest-energy conformation and on dynamic changes over time (the 'four-dimensional' protein structure). Instead, a combination of complementary techniques is required. Mass spectrometry methods have shown promise in addressing protein dynamics, but often rely on the use of high-end commercial or custom instruments. Here, we apply well-established chemistry to conformation-sensitive oxidative protein labelling on a timescale of a few seconds, followed by analysis through a routine protein analysis workflow. For a set of model proteins, we show that site selectivity of labelling can indeed be rationalised in terms of known structural information, and that conformational changes induced by ligand binding are reflected in the modification pattern. In addition to conventional bottom-up analysis, further insights are obtained from intact mass measurement and native mass spectrometry. We believe that this method will provide a valuable and robust addition to the 'toolbox' of mass spectrometry researchers studying higher-order protein structure.

The Scavenging Effect of Myoglobin from Meat Extracts toward Peroxynitrite Studied with a Flow Injection System Based on Electrochemical Reduction over a Screen-Printed Carbon Electrode Modified with Cobalt Phthalocyanine: Quantification and Kinetics.

The scavenging activity of myoglobin toward peroxynitrite (PON) was studied in meat extracts, using a new developed electrochemical method (based on cobalt phthalocyanine-modified screen-printed carbon electrode, SPCE/CoPc) and calculating kinetic parameters of PON decay (such as half-time and apparent rate constants). As reactive oxygen/nitrogen species (ROS/RNS) affect the food quality, the consumers can be negatively influenced. The discoloration, rancidity, and flavor of meat are altered in the presence of these species, such as PON. Our new highly thermically stable, cost-effective, rapid, and simple electrocatalytical method was combined with a flow injection analysis system to achieve high sensitivity (10.843 nA µM-1) at a nanomolar level LoD (400 nM), within a linear range of 3-180 µM. The proposed biosensor was fully characterized using SEM, FTIR, Raman spectroscopy, Cyclic Voltammetry (CV), Differential Pulse Voltammetry (DPV), and Linear Sweep Voltammetry (LSV). These achievements were obtained due to the CoPc-mediated reduction of PON at very low potentials (around 0.1 V vs. Ag/AgCl pseudoreference). We also proposed a redox mechanism involving two electrons in the reduction of peroxynitrite to nitrite and studied some important interfering species (nitrite, nitrate, hydrogen peroxide, dopamine, ascorbic acid), which showed that our method is highly selective. These features make our work relevant, as it could be further applied to study the kinetics of important oxidative processes in vivo or in vitro, as PON is usually present in the nanomolar or micromolar range in physiological conditions, and our method is sensitive enough to be applied.

De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins.

Enzymes that bear a nonnative or artificially introduced metal center can engender novel reactivity and enable new spectroscopic and structural studies. In the case of metal-organic cofactors, such as metalloporphyrins, no general methods exist to build and incorporate new-to-nature cofactor analogs in vivo. We report here that a common laboratory strain, Escherichia coli BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions. In supplemented minimal media containing CoCl2, the metabolically produced CoPPIX is directly incorporated into multiple hemoproteins in place of native heme b (FePPIX). Five cobalt-substituted proteins were successfully expressed with this new-to-nature cobalt porphyrin cofactor: myoglobin H64V V68A, dye decolorizing peroxidase, aldoxime dehydratase, cytochrome P450 119, and catalase. We show conclusively that these proteins incorporate CoPPIX, with the CoPPIX making up at least 95% of the total porphyrin content. In cases in which the native metal ligand is a sulfur or nitrogen, spectroscopic parameters are consistent with retention of native metal ligands. This method is an improvement on previous approaches with respect to both yield and ease-of-implementation. Significantly, this method overcomes a long-standing challenge to incorporate nonnatural cofactors through de novo biosynthesis. By utilizing a ubiquitous laboratory strain, this process will facilitate spectroscopic studies and the development of enzymes for CoPPIX-mediated biocatalysis.

Structure, functional properties and iron bioavailability of Pneumatophorus japonicus myoglobin and its glycosylation products.

Developing safe and efficient iron supplements is significant for the alleviation of iron-deficient anemia (IDA). Myoglobin (Mb) is a heme-protein rich in bioavailable iron. Pneumatophorus japonicus (P. japonicus), one important economic fish in China, contain a high Mb level in its dark meat normally discarded during processing. The present study aimed to determine the structure, physicochemical properties, and iron bioavailability of Mb extracted from P. japonicus. Meanwhile, the effects of glycosylation, a commonly applied chemical modification of proteins, on these parameters were evaluated. Using Box-Behnken design, the optimal conditions for Mb-chitosan glycosylation were obtained: 45.07 °C, pH 6.10 and Mb/chitosan mass ratio of 6.29. The structure and functional properties of the glycosylated Mb (Mb-gly) were investigated. Compared with the original Mb, Mb-gly obtained a more ordered secondary structure. The surface hydrophobicity of Mb-gly was found to be decreased together with the observations of elevated water solubility. Moreover, glycosylation enhanced the Mb antioxidant capacity, and improved its stability in enzymatic digestion system. Regarding to the iron bioavailability, the cellular uptake of Mb­iron was significantly higher than FeSO4, and further elevated by glycosylation. These results provided a basis for the development of Mb-based iron supplements, promoting the utilization of fish-processing industries wastes.

Redox reactions of heme proteins with flavonoids.

Proteins containing heme groups perform a variety of important functions in living organisms. The heme groups are involved in catalyzing oxidation/reduction reactions, in electron transfer, and in binding small molecules, like oxygen or nitric oxide. Flavonoids, low molecular weight plant polyphenols, are ubiquitous components of human diet. They are also components of many plant extracts used in herbal medicine as well as of food supplements. Due to their relatively low reduction potential, flavonoids are prone to oxidation. This paper provides a review of redox reactions of various heme proteins, including catalase, some peroxidases, cytochrome P450, cytochrome c, myoglobin, and hemoglobin with flavonoids. Potential biological significance of these reactions is discussed, in particular when flavonoids are delivered to the body at pharmacological doses.

Photophysical behavior of heme group: Unfolding of hemoglobin and myoglobin in the presence of Gemini surfactants of different molecular architectures.

Fluorescence studies were performed to determine the photophysical behavior of heme group in the presence of cationic Gemini surfactants of different architectures. Both hemoglobin and myoglobin were used to understand the heme group interactions with Gemini surfactants under the influence of temperature variation and were compared with homologous monomeric surfactants. The results were also supplemented from the size and zeta potential measurements of both proteins. Gemini surfactants showed marked effect on the unfolding behavior of hemoglobin that mainly contributed by the stronger hydrophobic interactions of double hydrocarbon chains as well as methylene spacer in the head group region with the hydrophobic domains of hemoglobin. Myoglobin with single polypeptide chain did not show similar unfolding behavior in the presence of Gemini surfactants rather it was readily solubilized in the surfactant solution and that too in the presence of monomeric surfactants rather than Gemini surfactants. The results highlighted the mechanistic aspects by which water soluble globular proteins interact with amphiphilic molecules of different functionalities and thus, helped to predict the interactions of both hemoglobin and myoglobin with the complex biological molecules possessing similar functionalities.

Interactions of indole alkaloids with myoglobin: A mass spectrometry based spectrometric and computational method.

RATIONALE: Interactions of drug molecules and proteins play important roles in physiological and pathological processes in vivo. It is of significance to establish a reliable strategy for studying protein-drug ligand interactions and would be helpful for the design and screening of new drugs in pharmacological research. METHODS: The interactions between four indole alkaloids (IAs) extracted from Ophiorrhiza japonica (O. japonica) and myoglobin (Mb) protein were investigated using a multi-spectrometric and computational method of native electrospray ionization mass spectrometry (native ESI-MS), hydrogen/deuterium exchange mass spectrometry (HDX-MS), circular dichroism (CD) and molecular docking (MD). RESULTS: The IA-bound Mb complexes were analyzed using native ESI-MS, with the obtained protein-to-ligand stoichiometry at 1:1, 1:2 and 1:3. Binding constants were measured according to the interpretation of MS spectra. MD complemented MS measurements, probing the binding sites and modes of the four IAs to Mb. Analyses involving CD and HDX-MS demonstrated that exposure to IAs could affect the conformation of Mb by decreasing the α-helix content and made Mb more susceptible to HDX at the backbone. CONCLUSIONS: A new MS-based integrated analysis method has been developed to successfully study the interactions of Mb and IAs extracted from O. japonica. The experimental and calculation results have good consistency, revealing all of the four IA molecules could bind to Mb to form 1:1, 1:2 and 1:3 Mb-IA complexes. The order of binding ability of these IAs to Mb was ophiorrhine B > compound C > ophiorrhine A > compound D. CD and HDX-MS results indicated that binding with IAs destabilizes Mb. HDX-MS analysis suggests that Mb becomes more susceptible to HDX, indicating that binding with IAs destabilizes the structure of Mb. In addition, the interaction with IAs affected the overall structure of Mb, ascribed to the decrease of α-helix content and less folding of the backbone.

The Intercalation of CORM-2 with Pharmaceutical Clay Montmorillonite (MMT) Aids for Therapeutic Carbon Monoxide Release.

The pharmaceutical clay montmorillonite (MMT) is, for the first time, explored as a carbon monoxide-releasing material (CORMat). MMT consists of silicate double layered structure; its exfoliation feature intercalate the CORM-2 [RuCl(µ-Cl)(CO)3]2 inside the layers to suppress the toxicity of organometallic segment. The infrared spectroscopy (IR) confirmed the existence of ruthenium coordinated carbonyl ligand in MMT layers. The energy-dispersive X-ray spectroscopy (EDX) analysis showed that ruthenium element in this material was about 5%. The scanning electron microscopy (SEM) and transmission electron microscope (TEM) images showed that the layer-structure of MMT has been maintained after loading the ruthenium carbonyl segment. Moreover, the layers have been stretched out, which was confirmed by X-ray diffraction (XRD) analysis. Thermogravimetric (TG) curves with huge weight loss around 100-200 °C were attributed to the CO hot-release of ruthenium carbonyl as well as the loss of the adsorbed solvent molecules and the water molecules between the layers. The CO-liberating properties have been assessed through myoglobin assay. The horse myoglobin test showed that the material could be hydrolyzed to slowly release carbon monoxide in physiological environments. The half-life of CO release was much longer than that of CORM-3, and it has an excellent environmental tolerance and slow release effect.

Defect and Additional Active Sites on the Basal Plane of Manganese-Doped Molybdenum Diselenide for Effective Enzyme Immobilization: In Vitro and in Vivo Real-Time Analyses of Hydrogen Peroxide Sensing.

The defect engineering makes the new concepts and designs to further enhance the electrocatalytic activity of layered structures. In this work, we demonstrated the synthesis of Mn-doped MoSe2 and reported the resultant defective sites. Subsequently, the MnMoSe2 was developed as a new type of electrocatalyst for electrochemical biosensors. The formation of defect/distortion and effective immobilization of myoglobin (Mb) were evidently confirmed by using the transmission electron microscopy and UV-vis spectroscopy analyses, respectively. The result of electrochemical impedance spectroscopy analysis reveals that the Mn doping not only helps  to enzyme immobilization but also enhances the electronic conductivity of layered material.  Owing to the multiple signal amplification strategies, the proposed Mb-immobilized MnMoSe2 (Mb@MnMoSe2) exhibited an ultralow detection limit (0.004 µM) and a higher sensitivity (222.78 µA µM-1 cm-2) of H2O2. In real-sample analysis, the Mb@MnMoSe2 showed a feasible recovery range of H2O2 detection in human serum (95.6-102.1%), urine (101.2-102.3%), and rain water (100.7-102.1%) samples. On the other hand, an in vivo study using HaCaT (7.1 × 105/mL) and RAW 264.7 (1 × 106/mL) living cells showed the feasible current responses of 0.096 and 0.085 µA, respectively. Finally, the Mn doping gives a new opportunity to fabricate a promising electrocatalyst for H2O2 biosensing.

(R)-α-Lipoic acid inhibits fructose-induced myoglobin fructation and the formation of advanced glycation end products (AGEs) in vitro.

BACKGROUND: Fructose-mediated protein glycation (fructation) has been linked to an increase in diabetic and cardiovascular complications due to over consumption of high-fructose containing diets in recent times. The objective of the present study is to evaluate the protective effect of (R)-α-lipoic acid (ALA) against fructose-induced myoglobin fructation and the formation of advanced glycation end products (AGEs) in vitro. METHODS: The anti-glycation activity of ALA was determined using the formation of AGEs fluorescence intensity, iron released from the heme moiety of myoglobin and the level of fructosamine. The fructation-induced myoglobin oxidation was examined using the level of protein carbonyl content and thiol group estimation. RESULTS: The results showed that co-incubation of myoglobin (1 mg/mL), fructose (1 M) and ALA (1, 2 and 4 mM) significantly inhibited the formation of AGEs during the 30 day study period. ALA markedly decreased the levels of fructosamine, which is directly associated with the reduction of AGEs formation. Furthermore, ALA significantly reduced free iron release from myoglobin which is attributed to the protection of myoglobin from fructose-induced glycation. The results also demonstrated a significant protective effect of ALA on myoglobin oxidative damages, as seen from decreased protein carbonyl content and increased protein thiols. CONCLUSION: These findings provide new insights into the anti-glycation properties of ALA and emphasize that ALA supplementation is beneficial in the prevention of AGEs-mediated diabetic and cardiovascular complications.

Effects of dietary coated cysteamine hydrochloride on pork color in finishing pigs.

BACKGROUND: Coated cysteamine hydrochloride (CC) was applied as a feed additive in animal production. The influence and the mechanisms of CC used as a feed additive in promoting meat quality in finishing pigs were investigated. RESULTS: Dietary CC supplementation increased (P < 0.05) the a* and H* values and reduced (P < 0.05) the L* value in the longissimus dorsi muscles at 48 h postmortem (P < 0.05). The deoxymyoglobin content was enhanced (P < 0.05) and the metmyoglobin and malondialdehyde contents were reduced (P < 0.05) in pigs fed the dietary CC. Pigs fed a dietary CC of 0.035 g kg-1 had a lower cooking loss (P < 0.05) and a higher a* (24 h) value in the longissimus dorsi muscles than pigs on control treatment. The messenger RNA expression of superoxide dismutase 1 was upregulated (P < 0.05) in the longissimus dorsi. CONCLUSION: Dietary supplementation with CC could improve antioxidant status and delay meat discoloration by improving glutathione levels and antioxidase activity after longer chill storage (for 48 h after slaughter). Dietary supplementation with CC at 0.035 g kg-1 may promote the stability of pork color by reducing oxidation. © 2017 Society of Chemical Industry.

Comparative study of the influence of hawthorn (Crataegus monogyna) berry ethanolic extract and butylated hydroxylanisole (BHA) on lipid peroxidation, myoglobin oxidation, consistency and firmness of minced pork during refrigeration.

BACKGROUND: Following public concern on the use of synthetic food antioxidants, there is an increasing demand for the application of mixed or purified natural antioxidants to maintain quality of meat products quality during storage. The aim of this research was to investigate the effect of ethanolic extract of hawthorn berry, compared to butylated hydroxylanisole (BHA), on lipid peroxidation, myoglobin oxidation, protein electrophoresis pattern, consistency and firmness of minced pork during refrigeration at 4 °C, and to identify the relationship between chemical modifications and consistency variation. RESULTS: After 6 days of refrigeration it was found that the thiobarbituric acid reactive substances value of minced pork containing 200 mg GAE kg-1 total phenolics in minced meat (200 HP) was significantly lower (0.1543 ± 0.006 mg) compared to BHA-treated meat. The ratio of oxymyoglobin to metmyoglobin in treated minced pork was respectively 0.845 for 200 HP and 0.473 for BHA-treated minced meat. Concentrations of 100 HP or 300 HP will generate statistically higher firmness than BHA in minced pork. CONCLUSION: Hawthorn berry ethanolic extract was more effective than BHA in reducing lipid oxidation and protein degradation, for maintaining firmness and consistency of minced pork during 6 days of refrigeration at 4 °C. © 2017 Society of Chemical Industry.

Valorization of functional properties of extract and powder of olive leaves in raw and cooked minced beef meat.

BACKGROUND: Olive leaves (OL), available in huge amounts from pruning, are known to be a useful source of biologically active compounds. This study investigated the potential application of OL as a supplement to minced beef meat in order to develop a functional product. The effect of OL extract or powder (100 and 150 µg phenols g-1 meat) on the quality and stability of raw and cooked meat during refrigerated storage was examined. RESULTS: Microwave drying at 600 W gave OL with the highest antioxidant quality (evaluated by TEAC/[phenols] (mg mg-1 ) and DPPH/[phenols] (mg mg-1 )) compared with other methods. OL showed an ability to inhibit (P < 0.05) lipid oxidation (TBARS values (mg MDA kg-1 ) were reduced by 25-65%) and myoglobin oxidation (metmyoglobin production was 43-65% in control samples and 14-35% in treated samples). OL also improved the technological quality of the meat, decreasing (P < 0.05) storage loss (%) and defrosting loss (%) without affecting cooking loss (%) and Napole yield (%). Sensory properties were not modified by the added ingredient at the tested levels (P < 0.05). CONCLUSION: OL (extract or powder) may have applications in the development of functional meat products of good technological quality that remain stable during storage. © 2016 Society of Chemical Industry.

Proof of concept of a "greener" protein purification/enrichment method based on carboxylate-terminated carbosilane dendrimer-protein interactions.

Protein sample preparation is a critical and an unsustainable step since it involves the use of tedious methods that usually require high amount of solvents. The development of new materials offers additional opportunities in protein sample preparation. This work explores, for the first time, the potential application of carboxylate-terminated carbosilane dendrimers to the purification/enrichment of proteins. Studies on dendrimer binding to proteins, based on protein fluorescence intensity and emission wavelengths measurements, demonstrated the interaction between carboxylate-terminated carbosilane dendrimers and proteins at all tested pH levels. Interactions were greatly affected by the protein itself, pH, and dendrimer concentration and generation. Especially interesting was the interaction at acidic pH since it resulted in a significant protein precipitation. Dendrimer-protein interactions were modeled observing stable complexes for all proteins. Carboxylate-terminated carbosilane dendrimers at acidic pH were successfully used in the purification/enrichment of proteins extracted from a complex sample. Graphical Abstract Images showing the growing turbidity of solutions containing a mixture of proteins (lysozyme, myoglobin, and BSA) at different protein:dendrimer ratios (1:0, 1:1, 1:8, and 1:20) at acidic pH and SDS-PAGE profiles of the corresponsing supernatants. Comparison of SDS-PAGE profiles for the pellets obtained during the purification of proteins present in a complex sample using a conventional "no-clean" method based on acetone precipitation and the proposed "greener" method using carboxylate-terminated carbosilane dendrimer at a 1:20 protein:dendrimer ratio.

Proteomic studies with a novel nano-magnetic chelating system to capture metalloproteins and its application in the preliminary study of monocyte and macrophage sub-secretome.

A new chelating chromatography method was developed based in the use of magnetic iron oxide nanoparticles functionalized with EDTA-TMS ((N-(trimethoxysilylpropyl)ethylenediaminetriacetate trisodium salt). These particles combine a high surface area, biocompatibility and magnetic removal from solution, with the chelating affinity towards metal ions. The particles were used to selectively capture metallo-dependant proteins in secretome obtained from human monocytes and mouse macrophages. Secreted metallo-dependant proteins are highly important sources of information since they are involved in several pathological processes. The identification of secreted proteins involved in these processes is highly important for diagnosis or monitoring the progression of a disease. In this multiple-approach study it was possible to not only selectively capture several secreted metallo-dependant proteins, but also to significantly avoid masking proteins such as the highly abundant albumin form the fetal bovine serum used to supplement the cell culture medium. Overall, the magnetic nanoparticle-based chelating chromatography method developed here has proved to be a sensitive, low cost, and a quick tool for sample treatment in order to selectively enrich metalloproteins while overcoming the contamination of highly abundant proteins.

Muscle antioxidant (vitamin E) and major fatty acid groups, lipid oxidation and retail colour of meat from lambs fed a roughage based diet with flaxseed or algae.

The effect of feeding flaxseed or algae supplements to lambs on muscle antioxidant potential (vitamin E), major fatty acid groups, lipid oxidation and retail colour was investigated. Lambs (n=120) were randomly allocated to one of 4 dietary treatments according to liveweight and fed the following diets for eight weeks: Annual ryegrass hay [60%]+subterranean clover hay [40%] pellets=Basal diet; Basal diet with flaxseed (10.7%)=Flax; Basal diet with algae (1.8%)=Algae; Basal diet with flaxseed (10.7%) and algae (1.8%)=FlaxAlgae. Flaxseed or algae supplementation significantly affected major fatty acid groups in muscle. The addition of algae (average of Algae and FlaxAlgae) resulted in lower vitamin E concentration in muscle (P<0.003; 1.0 vs 1.3mg/kg of muscle) compared with lambs fed a diet without algae (average of Basal and Flax). Increasing muscle EPA+DHA by algae supplementation significantly increased lipid oxidation, but retail display colour of fresh meat was not affected.

Competitive reduction of perferrylmyoglobin radicals by protein thiols and plant phenols.

Radical transfer from perferrylmyoglobin to other target species (myofibrillar proteins, MPI) and bovine serum albumin (BSA), extracts from green tea (GTE), maté (ME), and rosemary (RE), and three phenolic compounds, catechin, caffeic acid, and carnosic acid) was investigated by electron paramagnetic resonance (EPR) spectroscopy to determine the concentrations of plant extracts required to protect against protein oxidation. Blocking of MPI thiol groups by N-ethylmaleimide was found to reduce the rate of reaction of MPI with perferrylmyoglobin radicals, signifying the importance of protein thiols as radical scavengers. GTE had the highest phenolic content of the three extracts and was most effective as a radical scavenger. IC50 values indicated that the molar ratio between phenols in plant extract and MPI thiols needs to be >15 in order to obtain efficient protection against protein-to-protein radical transfer in meat. Caffeic acid was found most effective among the plant phenols.

Antioxidant diet supplementation and lamb quality throughout preservation time.

Diet supplementation (DS) (100, 200, 300ppm vitamin E -VE; 150ppm product rich in flavonoids-PRF; 100+100ppm VE-PRF; no supplementation) effect was evaluated on lamb quality throughout 10days after sampling (preservation time: PT). pH, colour, myoglobin forms and lipid oxidation were analyzed on Longissimus muscle. Trained panellists evaluated colour intensity of chops packaged in modified atmosphere under display up to 12days. PT had a larger effect on quality than DS. DS showed a clear antioxidant effect on lipids, especially at long PT and at high doses of VE. Visual test showed statistical differences among DS from day 4 of display where 200 and 300ppm VE improved visual colour score. In general, supplementation with antioxidants showed better meat quality and diets higher than 100ppm VE showed higher antioxidant capacity than the rest. The PRF diet was similar for a short PT but lower at a long PT. More research on flavonoids is necessary.

Quantitative evaluation of myoglobin unfolding in the presence of guanidinium hydrochloride and ionic liquids in solution.

The use of ionic liquids in biochemical and biophysical applications has increased dramatically in recent years due to their interesting properties. We report results of a thermodynamic characterization of the chaotrope-induced denaturation of equine myoglobin in two different ionic liquid aqueous environments using a combined absorption/fluorescence spectroscopic approach. Denaturation by guanidinium hydrochloride was monitored by loss of heme absorptivity and limited unfolding structural information was obtained from Förster resonance energy transfer experiments. Results show that myoglobin unfolding is generally unchanged in the presence of ethylmethylimidazolium acetate (EMIAc) in aqueous solution up to 150 mM concentration but is facilitated by butylmethylimidazolium boron tetrafluoride (BMIBF4) in solution. The presence of 150 mM BMIBF4 alone does not induce unfolding but destabilizes the structure as observed by a decrease in threshold denaturant concentration for unfolding and an 80% decrease in the magnitude of ΔGunfolding from 44 kJ/mol in the absence of BMIBF4 to 8 kJ/mol in the presence of 150 mM BMIBF4. Thus, the BMIBF4 significantly destabilizes the myoglobin structure while the EMIAc does not, likely due to differences in anion interaction capabilities. This is confirmed with control studies using NaAc and LiBF4 solutions. EMIAc may be chosen as cosolvent additive with minimal effects on protein structure while BMIBF4 may be used as a supplement in protein folding experiments, potentially allowing access to proteins which have been traditionally difficult to denature as well as designing ionic liquids to match protein characteristics.