An Experimental and Molecular Dynamics Study of ... - ACS Publications

Apr 20, 2017 - Department of Biomedical and Translational Sciences, Rowan University, 201. Mullica Hill Road, Glassboro, New Jersey 08028, United Stat...
0 downloads 0 Views 4MB Size
Download PDF
Article pubs.acs.org/JPCB

An Experimental and Molecular Dynamics Study of Red Fluorescent Protein mCherry in Novel Aqueous Amino Acid Ionic Liquids Kelsey L. Borrell,† Christine Cancglin,† Brittany L. Stinger,† Kelsey G. DeFrates,† Gregory A. Caputo,†,‡ Chun Wu,†,‡ and Timothy D. Vaden*,† †

Department of Chemistry and Biochemistry amd ‡Department of Biomedical and Translational Sciences, Rowan University, 201 Mullica Hill Road, Glassboro, New Jersey 08028, United States S Supporting Information *

ABSTRACT: The search for biocompatible ionic liquids (ILs) with novel biochemical and biomedical applications has recently gained greater attention. In this report, we characterize the effects of two novel amino acid−based aqueous ILs composed of tetramethylguanidinium (TMG) and amino acids on the structure and stability of a widely used red fluorescent protein (mCherry). Our experimental data shows that while the aspartic acid-based IL (TMGAsp) has effects similar to previously studied conventional ILs (BMIBF4, EMIAc, and TMGAc), the alanine-based IL (TMGAla) has a much stronger destabilization effect on the protein structure. Addition of 0.30 M TMGAla to mCherry decreases the unfolding temperature from 83 to 60 °C. Even at 25 °C, TMGAla results in a blue shift of the mCherry absorbance and fluorescence peaks and an increased Stokes shift. Molecular dynamics simulations show that the chromophore conformation and its interaction with mCherry with TMGAla are changed relative to those with TMGAsp or in the absence of ILs. Protein-ILs contact analysis indicates that the mCherry-Asp interactions are hydrophilic but the (fewer) mCherry-Ala interactions are more hydrophobic and may modulate the TMG interaction with the protein. Hence, the anion hydrophobicity may explain the special TMGAla destabilization of mCherry. with other literature reports on α-helical proteins (such as bovine serum albumin, lysozyme, and chymotrypsin) that show butyl-methyl-imidazolium tetrafluoroborate (BMIBF4) and other hydrophobic ILs at moderate concentrations do not directly denature proteins but destabilize their structures.15,18,21,31−33 Studies on β-sheet proteins have also yielded interesting fundamental and applied results.28,34 BMIBF4 in aqueous solution affects β-sheet sections differently relative to α-helical sections of luciferase.35 ILs affect β-sheet aggregation in β−amyoid and silk proteins, which presents interesting applications in modulation or inhibition of amyloid fiber formation.36 The β-barrel green fluorescent protein has been utilized in ILs in solutions for reporting biomass solubilization, which suggests the β-barrel structure is stable in the presence of ILs.37 In this work we study the structure and stability of the βbarrel red fluorescent protein (RFP) mCherry 38,39 in moderately dilute aqueous solutions of ILs. RFPs have many applications throughout biomedical and biochemical research fields. The RFP class of proteins, including mCherrry, have been widely utilized as fluorescent reporters in a variety of in

1. INTRODUCTION Ionic liquids (ILs) have recently found a variety of novel biomedical and biochemical applications. ILs have been used to solubilize biomaterials such as cellulose,1−3 inhibit or enhance enzyme activities,4−6 enhance antibiotic effectiveness, or act as novel antimicrobial compounds,7−10 and act as drug delivery systems.11−13 A natural question arises when studying ILs and biological systems regarding the nature of interactions between ILs and biomolecules. Hence, numerous recent investigations have reported protein−IL interactions and protein structures and stabilities in the presence of ILs in aqueous solution. Constantinescu et al. have reported protein unfolding thermodynamics of different systems,14 and Venkatesu and co-workers have reported detailed investigations of myoglobin and other proteins utilizing thermal unfolding methods as well as molecular dynamics (MD) simulations.15−20 More recent reports have shown that MD simulations can help characterize IL effects on proteins and elucidate stabilization and/or destabilization mechanisms.21−25 Such studies have identified direct IL−protein binding interactions as well as indirect effects.5,26−28 We have recently reported studies of myoglobin in dilute aqueous IL solutions,29,30 including unfolding ΔG measurements,29 unfolding kinetics measurements, and hydrogen− deuterium exchange experiments.30 The studies are consistent © 2017 American Chemical Society

Received: April 16, 2017 Revised: April 19, 2017 Published: April 20, 2017 4823

DOI: 10.1021/acs.jpcb.7b03582 J. Phys. Chem. B 2017, 121, 4823−4832

Article

The Journal of Physical Chemistry B vitro and in vivo assays40−45 with over 1000 references in PubMed. The mCherry protein, similar to other RFPs and FPs in general, has been used extensively in flow-cytometry and fluorescence microscopy, with applications ranging from superresolution microscopy to high-throughput screening.46,47 The β-barrel structure of RFP with the strongly bound red chromophore also presents an interesting target for fundamental studies of IL effects in aqueous solution environments. The mCherry variant of RFP is especially interesting because it, unlike the DsRed parent it was evolved from, is a monomer in solution and the chromophore exhibits known sensitivity to pH and the local amino acid environment within the core of the barrel.48,49 Concurrent with the development of biomedical applications of ILs has been the search for biocompatible ILs. BMIBF4 and other conventional ILs are often not amenable to biological systems (for example, BF4− can generate HF in aqueous solution). ILs containing amino acids as molecular anions have been developed for potential applications50,51 and used with proteins.52 ILs utilizing the tetramethylguanidinium (TMG) molecular cation have been prepared via very simple acid−base synthesis techniques and used as biocompatible additives for various biomolecular applications.28 We can combine the simplicity of TMG-based ILs synthesis with the potential biomedical applicability of amino acid−based ILs by preparing TMG-amino acid ILs. Such ILs may have interesting applications as well as unexpected effects on protein structures and stabilities. We can characterize the effects of these ILs using conventional protein thermal unfolding studies combined with UV circular dichroism (CD) spectroscopy of the mCherry chromophore.53 We can also use MD simulations to understand the protein−IL interactions at a molecular level and compare the simulation predictions to the experimental results. In this work, we present two novel ILs, TMG-Alanine (TMGAla) and TMG-Aspartic acid (TMGAsp), and characterize the effects of these ILs in dilute aqueous solutions on the structure and stability of the mCherry RFP. The TMGAsp appears to have effects similar to previously studied ILs but the TMGAla has a much stronger destabilization effect on the protein structure. The CD spectra and MD simulations provide a consistent picture of the special effects of the TMGAla in the protein environment and conclusively demonstrate that the IL destabilizes the local protein environment around the red chromophore, which results in a conformational structural destabilization of the entire protein tertiary structure.

breaks. The sonicated sample was centrifuged again at the same speeds above and decanted retaining the supernatant and discarding the pellet. Ammonium sulfate was slowly added to a final concentration of 50% while stirring at ambient temperature. The sample stirred for 1 h and was then centrifuged (Sorvall RC 5B+ centrifuge; Rotor SS-34; 17000 rpms; 20 min). The 50% ammonium sulfate pellet was discarded and the supernatant was transferred into a beaker for further addition of ammonium sulfate to achieve a final concentration of 55%. The sample was again allowed to stir for 1 h at ambient temperature and subsequently pelleted via centrifuge (centrifuge same as above). The supernatant was decanted brought to 60% ammonium sulfate, stirred, and pelleted as above. The process was subsequently repeated for 65% and 70% final concentrations of ammonium sulfate. The resultant 5 pellets were individually resuspended in sodium phosphate buffer (20 mM, pH 7) and put into 7000 MWCO dialysis tubing (SnakeSkin, ThermoFisher). The sample was dialyzed overnight against 4 L of 10 mM phosphate buffer supplemented with 1% Tween-20 (Amresco), pH 7.4. The dialyzed samples were individually frozen and lyophilized. Lyophilized samples were each resuspended in 3−5 mL phosphate buffer (20 mM, pH 7) and vortexed until no precipitates were visible. Samples were loaded onto a 30 mL Q-Sepharose column (GE Healthcare) pre-equilibrated with three bed volumes of low salt buffer (10 mM phosphate, 10 mM NaCl). A salt gradient was creating using a dual-chamber gradient maker with the low salt buffer and a high salt buffer (10 mM phosphate 2 M NaCl, pH 7.0). Fractions were collected and those with the most visible pink color were again dialyzed and lyophilized. Gel electrophoresis was performed using 4−20% gradient gels (Jule Inc.) and subsequently visualized by coomassie staining to confirm sample purity. A representative gel is shown in Figure S1 in the Supporting Information. 2.2. Preparation of TMG-Amino Acid ILs. TMG (99%) and L-alanine (98%) were purchased from Alfa Aesar and used without further purification. L-aspartic (99%) was purchased from Acros Organic and used without further purification. To prepare the TMG-amino acid ILs, TMG and the amino acid were mixed together in a 1:1 molar ratio in distilled water. The amino acid was dissolved in water first and TMG was added volumetrically to the solution under stirring. The mixture was stirred overnight and then the water was removed first by evaporation under vacuum at 45 °C and then under high vacuum at 25 °C. IR, Raman, and NMR spectra were measured to verify the synthesized IL chemical identities and purities. IR and Raman spectra are shown in Figure S2 in the Supporting Information section. The chief impurity (∼2−3%) is water, which is clearly not a problem in our experiments. 2.3. Preparation of Samples. BMIBF4 (97%) and ethylmethyl-imidazolium acetate (EMIAc, 97%) were purchased from Aldrich and used without purification (main contaminants are water and NaCl). TMG-acetate (TMGAc) and TMGlactate (TMGLa) was synthesized and purified as previously reported.28,30 All ILs used in the experiments were prepared in a stock solution of 3.0 M in 20 mM pH 7 phosphate buffer. Control experiments were performed with 500 mM pH 7 phosphate buffer. Stock solutions were prepared immediately prior to use to prevent degradation in water. Lyophilized mCherry (solid) was used to prepare stock mCherry solutions of 5 mg/mL in pH 7 phosphate buffer. Samples containing mCherry and ILs were prepared with protein concentrations of 0.5 mg/mL in all cases. All protein samples were studied within

2. EXPERIMENTAL SECTION 2.1. Protein Expression and Purification. Escherichia coli containing the pet28b plasmid carrying the gene for mCherry was streaked onto an LB-agar plate and grown overnight at 37 °C. A single colony was inoculated in LB broth (Difco) supplemented with kanamycin (10 mg/mL) and grown overnight (25 mL). This overnight culture was transferred into 1 L of fresh LB-kanamycin broth and grown at 37 °C in a shaking incubator. When culture density reached OD600 ∼ 1.0, isopropyl β-D-1-thiogalactopyranoside (IPTG, Alfa Aesar) was added to a final concentration of 0.1 mM to induce expression. Upon optical density plateau (∼5 h), the culture was centrifuged (Sorvall RC 5B+ centrifuge; 4700 rpm; Rotor SS34; 20 min) and the supernatant was decanted. The pellet was resuspended in PBS lysis buffer (10 mM sodium phosphate, 100 mM NaCl, 5 mM EDTA, pH 7.0) and sonicated on ice using a Sonic Vibra Cell 10 times in 30 s intervals with 2 min 4824

DOI: 10.1021/acs.jpcb.7b03582 J. Phys. Chem. B 2017, 121, 4823−4832

Article

The Journal of Physical Chemistry B

solute heavy atoms for 12 ps, and finally simulation under NPT with a high temperature of 400 K, pressure of 1 bar and no restraints for 1.5 ns to randomize ion position. After the equilibration phase, a 500 ns production run at 300 K was conducted for each of the ten systems under the NPT ensemble using the default protocol.56 The temperature was controlled using the Nosé−Hoover chain coupling scheme with a coupling constant of 1.0 ps. Pressure was controlled using the Martyna− Tuckerman−Klein chain coupling scheme56 with a coupling constant of 2.0 ps. M-SHAKE57 was applied to constrain all bonds connecting hydrogen atoms, enabling a 2.0 fs time step in the simulations. The k-space Gaussian split Ewald method58 was used to treat long-range electrostatic interactions under periodic boundary conditions (charge grid spacing of ∼1.0 Å, and direct sum tolerance of 10−9). The cutoff distance for shortrange nonbonded interactions was 9 Å, with the long-range van der Waals interactions based on a uniform density approximation. To reduce the computation, nonbonded forces were calculated using an r-RESPA integrator59 were updated every three steps. The trajectories were saved at 100.0 ps intervals for analysis. The protein backbone RMSD matrix was used in a hierarchical cluster average linkage method60 to group the structures in the simulation trajectory. The merging distance cutoff was set to be 2.5 Å. The centroid structure (the structure having the largest number of neighbors in the structural family) was used to represent the structural family of RFP. Detailed protein-chromophore interactions were analyzed using Simulation Interaction Diagram (SIM) module of Schrödinger suite. The 2D protein−ligand interaction diagrams were used to explore four types of specific and nonspecific protein−ligand interactions: hydrogen bonds, hydrophobic, ionic, and waterbridge.

a few hours of preparation, and measurements of samples either immediately or 3 h after preparation yielded identical results. 2.4. Absorbance and Fluorescence Measurements. Electronic absorbance spectra of mCherry samples in the presence of ILs in aqueous solution were recorded with a PerkinElmer Lambda 35 UV/vis spectrometer. All samples were prepared in 20 mM pH 7 phosphate buffer. The same phosphate buffer was used for background correction. Fluorescence spectra were recorded with a Horiba Fluoromax-4 spectrofluorometer. The excitation wavelength was chosen as the λmax value from the corresponding absorbance spectrum. The excitation and emission slit widths were 3 nm for all measurements. 2.5. Circular Dichroism Experiments for Measuring Protein Stabilities. CD spectra were recorded with a Jasco J810 spectropolarimeter equipped with a Peltier-type temperature controller. Spectra were measured at 25 °C and the spectrum of 20 mM pH 7 phosphate buffer at 25 °C was subtracted as background. All reported spectra, including background, were generated as averages of three separate scans. For thermal unfolding experiments, the sample was placed in a cuvette capped and sealed with parafilm to prevent water evaporation. The CD spectrum was recorded from 200 to 600 nm at different temperatures from 25 to 90 °C. For each data point, the sample was held at the constant temperature for 10 min prior to data acquisition to allow for thermal equilibration. 2.6. Molecular Dynamics Simulations. MD simulations were used to probe protein-chromophore conformation change and that occurs with addition of different TMG-amino acid− based ILs. Three systems were constructed: mCherry only, mCherry with TMGAla and mCherry with TMGAsp (Figure S3). Each simulation system was built using the high-resolution structure of mCherry (PDB ID: 2H5Q). Please note that the residue numbering in this PDB structure differs from that of mCherry we purified due to the N-terminal missing residues in the PDB structure (e.g Lys 70 in the PDB structure is actually 75 of our expressed mCherry). The protein was then prepared using the Protein Preparation Wizard of Maestro program.54 Three steps were performed: preprocessing, optimization of protonation state at pH = 7, and geometry optimization using the default parameters for restrained minimization. Ions representative of the ionic liquid were prepared using Maestro software and manually added to the protein system. Enough ions were added to represent 0.1 M concentration of ionic liquid in the system. The ions were placed surrounding the protein at random (Figure S3), and the ion placement was further randomized during the relaxation protocol (described later). A system was then built using SPC as water solvent with an orthorhombic solvent box with 76 Å × 76 Å × 76 Å dimensions. An OPLS3 force field55 was used to represent the ternary complex. The total number of atoms for these systems was around 40 000 atoms. The Desmond simulation module of Maestro was used to run each simulation. The prepared systems were first equilibrated using the default equilibration protocol: Brownian dynamics simulation under the NVT ensemble with temperature 10 K for 100 ps, simulation under the 10 NVT ensemble with temperature of 10 K, small time steps and restraints on solute heavy atoms for 12 ps, simulation under the NVT ensemble with temperature of 10 K and restraints on solute heavy atoms for 12 ps, simulation under the NPT ensemble with temperature of 10 K, pressure of 1 bar and restraints on

3. RESULTS AND DISCUSSION 3.1. Thermal Stability of mCherry in the Presence of ILs in Aqueous Solution. We performed CD-based temperature unfolding experiments to characterize the thermal stability of mCherry in the presence of ILs in pH 7 buffered (aqueous) solutions. Thermal unfolding experiments have been used as a relatively quick measure of protein stabilities in the presence of ILs.15,17,18,61 The CD spectra recorded at different temperatures are shown for mCherry in aqueous solution in Figure 1. The 25 °C spectrum is also shown as Figure S4 in the Supporting Information section and, importantly, is identical to the CD spectra reported in the literature.53,62 This spectrum, seen as

Figure 1. CD spectra of 0.5 mg/mL mCherry in 20 mM pH = 7 phosphate buffer with increasing temperatures. The arrows indicate that band intensities decrease with increasing temperature. 4825

DOI: 10.1021/acs.jpcb.7b03582 J. Phys. Chem. B 2017, 121, 4823−4832

Article

The Journal of Physical Chemistry B

alanine (in free amino acid form) is included; alanine destabilizes mCherry about the same as TMGAsp. In fact, TMGAsp, BMIBF4, and alanine all have roughly the same minor destabilizing effect on mCherry. This demonstrates that it is not simply the alanine that strongly destabilizes the protein (via protein-alanine interactions or other effects). Rather, the IL has a special strongly destabilizing effect on the protein conformational structure. We can also confirm that this destabilization effect is not simply due to ionic strength, as demonstrated by NaCl control experiments shown in Figure S5 of the Supporting Information (in which NaCl does not destabilize mCherry). While we cannot qualify the effects of the IL on the β-sheet secondary structure, due to the inability of measuring the 230 nm CD bands in the IL-containing solutions, it is clear that the TMGAla destabilizes the tertiary protein structure. It is notable from the results in Figure 2B that the TMGAsp IL appears to have no significant destabilizing effect. This illustrates the sensitivity of the destabilizing IL effect(s). The main difference between the Ala and Asp molecular anion is that the carboxylic acid side chain in Asp can donate a proton whereas Ala has no ionizable side chain. This subtle but significant difference in the context of the IL in aqueous solution apparently leads to differences in how the ILs affects the mCherry structure. In principle, the ILs can change the aqueous solution pH. To ensure that the IL effects on mCherry are not simply due to pH changes, we performed experiments with 500 mM phosphate buffer (pH 7) concentration and ensured that the pH values are not significantly affected by the 300 mM ILs. The results confirm that the specific strong destabilization of mCherry by TMGAla is still observed with well-controlled pH conditions and are shown in Figure S6) 3.2. Effect of TMGAla on the mCherry Chromophore Signals. Figure 2 essentially shows that TMGAla strongly destabilizes the protein tertiary structure. To investigate the nature of the mCherry-IL interaction, we first look at the CD spectra in more detail. Figure 3 presents the CD spectra of

the low-temperature trace in Figure 1, exhibits a negative band at 580 nm. This corresponds to the red chromophore signal.53 The spectrum also exhibits two positive bands at 280 and 340 nm that correspond to the tertiary and quaternary protein structure.62 The strong negative bands below 230 nm correspond to the β-sheet secondary structure signals but we do not further analyze these bands because they are not observable in the presence of the TMG-based ILs (TMG-based ILs, like most ILs, absorb light below 250 nm). As temperature increases in Figure 1, the band intensities at 280, 340, and 580 nm decrease. This indicates thermal unfolding and loss of tertiary/quaternary structure, and we can assume that the percentage of native protein structure (100% folded) is proportional to the integrated peak areas. Figure 2 shows thermal unfolding curves generated by graphing

Figure 2. Thermal melting curves for mCherry in different ILs in aqueous solution. All ILs are present at 0.3 M. Integrated peak areas normalized to the 25 °C buffer-only data are shown versus temperature. (A) TMGAc and EMIAc have no effect on mCherry thermal unfolding but BMIBF4 slightly destabilizes the protein; (B) TMGAla strongly destabilizes the protein (note the lower TMGAla concentration; higher IL concentrations completely denature the protein), while Ala and TMGLa have no effects.

the integrated peak areas as a function of temperature. All data points are normalized to the integrated peak areas for the 25 °C “buffer-only” spectrum. We quantify the onset of unfolding temperature of the protein as the temperature at which significant denaturation (at least 20% unfolded) begins to happen. In the absence of ILs (black squares) mCherry this onset is at ∼83 °C. mCherry is a very stable protein62 and may not be completely unfolded above 90 °C but partial denaturation clearly occurs. The thermal unfolding curves provide a way to assess the effects of ILs on the protein stability in a straightforward manner. Figure 2A shows unfolding curves for mCherry in the presence of conventional ILs (TMGAc, BMIBF4, and EMIAc) at 0.30 M concentration. With TMGAc and EMIAc, the protein unfolding temperature is essentially unchanged, indicating that these ILs have no detectable effect on the stability. With BMIBF4, the unfolding temperature decreases to ∼78 °C, which indicates that the protein is slightly destabilized by the IL. In general the ILs in Figure 2A have no or (with BMIBF4) minor effects on the protein structure. Figure 2B shows results for mCherry in the presence of 0.30 M TMGAla and TMGAsp ILs. TMGAsp has a minor destabilizing effect (onset of unfolding temperature decreases to ∼78 °C) but in the presence of TMGAla the onset of unfolding temperature decreases to 60 °C, which represents a significant protein destabilization. As a control experiment, mCherry thermal unfolding data in the presence of 0.30 M

Figure 3. CD spectra of 0.5 mg/mL mCherry in the presence of increasing TMGAla concentrations (0.01 to 0.25 M) in pH = 7 phosphate buffer solutions. The chromophore peak at 580 nm clearly shifts to shorter wavelength.

mCherry in the presence of increasing TMGAla concentrations from 0.01 to 0.25 M. The bands at 280 and 340 nm are not significantly affected by the IL, which is consistent with the conclusion that the protein retains its tertiary structure in the presence of ILs at all concentrations considered here. However, the chromophore band at 580 nm clearly shifts to shorter wavelength (and decreases in intensity) with increasing 4826

DOI: 10.1021/acs.jpcb.7b03582 J. Phys. Chem. B 2017, 121, 4823−4832

Article

The Journal of Physical Chemistry B

observed with alanine alone as shown in Figure 5B. Finally, the TMGAla-specific chromophore shift is also observed in the high-buffer concentration control experiments (Figure S6). 3.3. MD Simulations of mCherry in the presence of TMGAla and TMGAsp. Figures 2, S4, and 5 provide the key experimental evidence to suggest that TMGAla has a specific destabilizing interaction with mCherry that results in the viscosity and polarity increase of the red chromophore, thus leading to a blue-shifted absorbance and fluorescence, an increased Stokes shift, and a weakening of the protein tertiary structure. In contrast, the TMGAsp IL appears to not exhibit this destabilizing interaction. To further investigate these effects and elucidate the IL-protein interactions at the molecular level, we performed MD simulations as described in Section 2.6. The RMSD plots over the entire 500 ns simulation are shown for both the protein backbone (mCherry peptide chain) and the chromophore in Figure 6. The protein RMSD

TMGAla. Hence, the CD absorption of the chromophore is changed with the IL. This is shown more completely in Figure 4, which presents absorbance (Figure 4A) and fluorescence (Figure 4B) spectra of mCherry with increasing TMGAla concentration.

Figure 4. (A) Absorbance spectra of mCherry shown in the presence of increasing TMGAla in solution, with concentrations labeled to the right of the traces. (B) Fluorescence spectra of the solutions from (A).

Figure 4A clearly demonstrates that when TMGAla is introduced to the mCherry in solution, the chromophore absorbance (λmax value) shifts by ∼20 nm from 580 to 560 nm. Figure 4B demonstrates TMGAla also induces a shift in the fluorescence spectra (λmax) of ∼15 nm, from 610 to 595 nm. The Stokes shift computed from Figure 4 is included in Figure S7 of Supporting Information, which shows an increase of ∼15 nm. It is notable that the fluorescence intensities are not significantly affected by the TMGAla molecular ions; the IL does not strongly quench mCherry fluorescence under these conditions. Interestingly, the TMGAsp does not exhibit these effects on the mCherry absorbance spectra (shown in Figure S8 in Supporting Information). It therefore appears that the TMGAla in aqueous solution has a specific effect on the mCherry chromophore. From Sections 3.1 and 3.2, together we can draw the preliminary conclusion that TMGAla interacts with mCherry in a way that changes the chromophore and destabilizes the protein tertiary conformational structure. The effect is specific to TMGAla and is not observed with TMGAsp or any of the conventional ILs (or NaCl, data not shown), as shown in Figure 5A. It is also specific to the IL as it is not

Figure 6. Protein (black) and ligand (red) RMSD plots from the MD simulations. (A) With no IL; (B) with TMGAsp; (C) with TMGAla.

fluctuations and evolution are about the same without IL (Figure 6A), with TMGAsp (Figure 6B), and with TMGAla (Figure 6C) indicating that the protein secondary/tertiary structure is not significant changed by the ILs. This is consistent with experiments because the simulations were performed at 25 °C. However, the RMSD plots for the chromophore are different with and without the ILs. Without ILs the chromophore changes conformation from 250−350 ns and with TMGAla the chromophore conformational change lasts from 100−350 ns. The chromophore conformational changes with TMGAsp present are unclear.

Figure 5. (A) CD spectra of mCherry in the presence of different ILs showing that the chromophore band is only affected by TMGAla. (B) CD spectra of mCherry in the presence of TMGAla and alanine showing that the alanine alone is not responsible for the chromophore band shift. 4827

DOI: 10.1021/acs.jpcb.7b03582 J. Phys. Chem. B 2017, 121, 4823−4832

Article

The Journal of Physical Chemistry B

calculated from the MD simulations in Figure 8. Figure 8A−C shows the nature and frequency of the contacts and Figure

It appears the TMGAla IL has some effects on the chromophore conformation and minor (if any) effects on the overall protein structure. This is supported by our clustering and secondary structure analyses. Figure 7 shows the most

Figure 8. Protein−ligand interactions (interactions per residue versus residue) from the MD simulations. Green = H-bonds; black = water bridges; gray = hydrophobic; red = ionic. (A) With no IL; (B) with TMGAsp; (C) with TMGAla. Representative structures from the clustering analysis showing protein−ligand 3D interactions: Yellow Dash Line = H-bonds (D) with no IL, (E) with TMGAsp, (F) with TMGAla.

8D−F shows the structure of the protein-chromophore interactions. The chromophore is covalently bound to the protein at Phe65 and Ser69 and interacts via hydrogen bonds with Lys70, Arg95, Trp 93, and Gln109. In the presence of TMGAla (Figure 8C), the interactions with Lys70 and Arg95 gain more ionic character and also the simulations predict a significant increase in hydrogen bonding with Gln163 (indicated with an asterisk in Figure 8C and circled in Figure 8F) and a slight decrease in hydrogen bonding with Gln109. This new hydrogen bond likely is responsible for altering the chromophore conformation. In the presence of TMGAsp, the Gln163 hydrogen bond is present but the interactions with Lys70 and Arg95 are not ionic (Figure 8E). Figures 6−8 demonstrate that TMGAla somehow changes the chromophore conformation. This effect could be due to direct interactions between mCherry and the IL molecular ions, and/or it could be an indirect effect such as the ion-induced water hydrogen bond networking change. We can investigate the direct protein-IL interactions by characterizing the atom contacts predicted during the MD simulations. Figure 9 shows the protein interacting with IL molecular ions. Clearly, both TMG and amino acids (Asp and Ala) do directly interact with proteins. Compared with the anion (Asp/Ala), the TMG cation in both systems has fewer contacts with the protein than the amino acids, as the red traces in Figure 9D,E essentially stay

Figure 7. Protein and chromophore most-common conformations from MD Simulations. (A) Front view; (B) back view; (C) hairpin of β-strand 7 and 8; (D) chromophore. Red = No IL; blue = TMGAsp; green = TMGAla. N- and C-termini are indicated by red and blue balls, respectively.

abundant conformation from the clustering analysis of the simulation trajectory and Figure S9 shows the secondary structure abundance. Although the protein secondary/tertiary structure is very similar without IL or with TMGAsp or TMGAla, a short fragment 142−144 of β-strand 7 was converted into a coiled conformation due to loss of interstrand hydrogen bonds in the presence of TMGAsp and TMGAla. Similarly, the chromophore conformation (Figure 7d) with TMGAla is different from that with TMGAsp or without IL. While the conformational differences in Figure 7 are subtle, they are significant and may provide clues to the specific protein destabilization by TMGAla. To further elucidate the TMGAla effect on the mCherry chromophore, we present the protein-chromophore contacts 4828

DOI: 10.1021/acs.jpcb.7b03582 J. Phys. Chem. B 2017, 121, 4823−4832

Article

The Journal of Physical Chemistry B

However, there are significant nonpolar−nonpolar and nonpolar−polar interactions. Therefore, we can conclude that there are fewer mCherry−Ala interactions than mCherry Asp interactions, but the protein−IL interactions with Ala are more hydrophobic than those with Asp. 3.4. Discussion. We have characterized the effects of TMGbased amino acid ILs on the stability and structure of mCherry using CD spectroscopy, thermal unfolding experiments, and MD simulations. The CD spectra (Figures 3 and 5) indicate that the chromophore is affected by TMGAla but not TMGAsp. Further, other conventional ILs (BMIBF4, EMIAc, and TMGAc) do not affect the chromophore. This blueshift is possibly explained by the MD simulation results. Simulations clearly show that in the presence of TMGAla, the chromophore conformation is slightly changed relative to the conformation exhibited in the absence of ILs. While we cannot infer how the IL induces this change, it appears to result from a new hydrogen bond between the chromophore and the protein (Gln163). This can explain the chromophore absorbance (and CD) shift from 580 to 560 nm. Previous work has shown that the mCherry chromophore absorbance is very sensitive to its local environment47−49,62 and subtle changes can shift the absorbance by 20 nm or more.39,64 We therefore can suggest that the minor effect of the IL on the mCherry-chromophore interaction is identified experimentally by the chromophore shift. Extending beyond the amino acid-IL systems, local amino acid environment effects on spectroscopic properties of fluorophores are widely reported. Many amino acids, such as Met and Gln as well as the backbone peptide bond, are known to quench the fluorescence of Trp and other fluorophores.65−67 More specifically, a Gln-indole interaction has been reported to quench fluorescence by an excited state electron transfer mechanism.65,68 In addition to quenching, specific amino acidfluorophore interactions that affect fluorescence emission have also been reported. One example is the Asp-Trp interaction that results in a significantly red-shifted Trp emission spectrum.69,70 The Gln-chromophore interaction is also noted in the structure of green fluorescent protein (GFP) with an hydrogen bond between Gln94 and the chromophore carbonyl as well as a number of hydrogen bonds between polar residues and the phenolic OH.71 Gln-chromophore interactions were also observed in both rhodopsin and bacteriorhodopsin resulting in shifts in absorbance bands in both the dark adapted and light adapted states.72,73 Taken together, these findings strongly support the role of the Gln163-chromophore interaction in modulating the absorbance and fluorescence emission properties of the mCherry. The thermal unfolding results (Figure 2) clearly show that the TMGAla has a significant destabilizing effect on mCherry. While hydrophobic ILs such as BMIBF4 have been shown to strongly destabilize α-helices and other proteins,15,18,20,26,74 in this work the BMIBF4 does not have a very dramatic destabilization effect on the β-sheet/β-barrel structure exhibited by mCherry. TMGAla appears to have a significantly stronger effect than BMIBF4, and also destabilizes mCherry more than TMGAsp. It would make sense to attribute this to a mCherryAla interaction, but Ala by itself has minimal effect on mCherry (at the same concentration). Also the MD simulations do not predict any specific mCherry-Ala interactions at 25 °C. The mCherry-amino acid interactions predicted by the simulations may modulate the TMG-protein interaction and together

Figure 9. mCherry-IL interactions. (A) With no IL; (B) with TMGAsp; (C) with TMGAla; (D) atom-contacts with mCherry in TMGAsp; (E) atom-contacts mCherry in TMGAla.

between 0 and 10. The anion (black traces) has somewhat more direct contact with the protein. Interestingly, the Asp anion has more direct atom contact with the protein than the Ala anion outside the protein β-barrel, probably due to its large size. But the nature of Ala-RFP contacts is more hydrophobic than Asp-RFP contacts, given the neutral side chain of Ala but the ionizable side chain of Asp. It appears that the increase of anion hydrophobicity of IL contributes to the destabilization of the mCherry protein structure. This is consistent with a recent computational study63 of unfolding of miniproteins in IL solutions, in which the more hydrophobic anions lead to faster protein unfolding. Of course, the cation TMG in TMG-Ala should also play some roles in the RFP destabilization, because Ala alone does not lead to the destabilization as shown in our Ala-only experiment. We can further elucidate the nature of the anion hydrophobicity effect on the protein and protein-IL interactions by separating the atom contacts in Figure 9D,E into atom types and classifying these in terms of the interaction types. We present this analysis in Figure 10, which shows the polar−polar,

Figure 10. Breakdown of Figure 9D,E in terms of interaction types between mCherry and amino acids. (A) With TMGAsp; (B) with TMGAla.

polar−nonpolar, and nonpolar−nonpolar interactions between the IL and the protein. With TMGAsp (Figure 10A) there are no nonpolar−nonpolar interactions, which makes sense as the Asp anion is polar. Most of the interactions are polar−polar. With TMGAla (Figure 10B), the total number of protein−IL contacts is less than with TMGAsp (as shown also in Figure 9). 4829

DOI: 10.1021/acs.jpcb.7b03582 J. Phys. Chem. B 2017, 121, 4823−4832

Article

The Journal of Physical Chemistry B

on mCherry at the same concentration. The MD simulations predict that the mCherry-Asp interactions are hydrophilic and the mCherry-Ala interactions are more hydrophobic. Therefore, we speculate that the hydrophilicity/hydrophobicity of the amino acid anion modulates the destabilizing TMG-mCherry interaction, and with TMGAla causes a slight conformational change in the β-strand structure and destabilizes the protein structure.

illustrate that the hydrophobicity of the anion plays a role in protein destabilization. The simulation results summarized in Figures 9 and 10 help elucidate the conclusions of this work. The TMG interacting with the protein likely imparts some destabilization, as TMG is similar to the well-known guanidinium denaturant. The Asp anion interacts with the protein via hydrophilic (polar−polar) interactions and may offset or modulate the destabilizing TMG interactions. However, the Ala is more hydrophobic and interacts with the protein to a lesser extent, and in a more hydrophobic (nonpolar−nonpolar) nature. Either this hydrophobic interaction destabilizes the mCherry structure or the does not help offset the TMG destabilization. The simulations ultimately are consistent with the hypothesis that the anion hydrophobicity can help explain the special TMGAla destabilizing effect on mCherry. Regarding the two experimental results (chromophore shift and protein destabilization) the MD results regarding the chromophore conformational change may provide further clues regarding the protein destabilization. Previous work on myoglobin has demonstrated that ILs can interact with the heme moiety, and the altered heme conformation correlates with a destabilization of the protein tertiary structure.15,17,19 We can hypothesize that the same effect(s) that induce a hydrogen bond between Gln163 and the chromophore result in a less stable protein structure. Experimentally, the protein unfolding temperature decreases from 85 to 60 °C. A subtle conformational change could have minimal effect on the overall structure at 25 °C but still have magnified effects at higher temperatures that ultimately destabilize the protein.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b03582. The SDS gel and CD spectrum of purified mCherry, supplemental absorbance and fluorescence spectra, Stokes shift analysis, and high-buffer-concentration control experiments (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +1-856-256-5457. E-mail: [email protected]. ORCID

Timothy D. Vaden: 0000-0002-2648-0300 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by NSF Grant CHE-1362493. We also acknowledge the High Performance Computing Facility at Rowan funded by the National Science Foundation under Grant MRI-1429467 and computational time funded through XSEDE MCB160004/160164/160173. We would like to thank Mariel Enriquez, Russell Sapio, and Meagan Costello for help with the protein purification. We would like to thank Dr. Bryan Berger at Lehigh University for the gift of the mCherry plasmid.

5. CONCLUSION Because of the novel applications of ILs in biochemistry and biomedical technology, the search for biocompatible ILs has recently gained increasing attention. In this study, we present two novel biocompatible ILs, TMGAla and TMGAsp, and characterize their effects in dilute aqueous solutions on the structure and stability of the widely used RFP mCherry. Our temperature-dependent spectroscopy data show that while TMGAsp have effects similar to previously studied conventional ILs (BMIBF4, EMIAc, and TMGAc), TMGAla has a much stronger destabilization effect and lower the mCherry unfolding temperature from 80 to 60 °C. At 25 °C, the chromophore is modulated by TMGAla but not TMGAsp or the other conventional ILs, leading to a blueshift in absorbance and fluorescence peaks and an increased Stokes shift. Whereas the blueshift in absorbance and fluorescence indicates an increase of the viscosity of the chromophore local environment, the redshift to the Stokes shift implies an increase of polarity of the chromophore environment. Our MD data at 25 °C show that the chromophore conformation and its interaction with the protein are slightly changed relative to the conformation exhibited in the absence of ILs or TMGAsp. While the key interactions with Lys70, Arg95, and Trp93 are maintained, a hydrogen bond between the chromophore and Gln163 present more pronouncedly in mCherry with TMGAla than TMGAsp or no ILs. These changes might explain the increase of the local polarity and viscosity of the chromophore that cause the spectra shifts. In addition, the conversion of a short fragment 142−144 of the β-strand 7 into coiled conformation was observed in mCherry with TMGAla and TMGAsp. This loss of an interstrand hydrogen bond might partially explain the destabilization effects of ILs. Ala by itself has minimal effect

■

REFERENCES

(1) Pinkert, A.; Marsh, K. N.; Pang, S.; Staiger, M. P. Ionic liquids and their interaction with cellulose. Chem. Rev. 2009, 109, 6712−6728. (2) Moulthrop, J. S.; Swatloski, R. P.; Moyna, G.; Rogers, R. D. Highresolution 13c nmr studies of cellulose and cellulose oligomers in ionic liquid solutions. Chem. Commun. 2005, 41, 1557−1559. (3) Muzzarelli, R. A. Biomedical exploitation of chitin and chitosan via mechano-chemical disassembly, electrospinning, dissolution in imidazolium ionic liquids, and supercritical drying. Mar. Drugs 2011, 9, 1510−1533. (4) Du, P.; Liu, S.; Wu, P.; Cai, C. Preparation and characterization of room temperature ionic liquid/single-walled carbon nanotube nanocomposites and their application to the direct electrochemistry of heme-containing proteins/enzymes. Electrochim. Acta 2007, 52, 6534− 6547. (5) Nordwald, E. M.; Kaar, J. L. Mediating electrostatic binding of 1butyl-3-methylimidazolium chloride to enzyme surfaces improves conformational stability. J. Phys. Chem. B 2013, 117, 8977−8986. (6) Naushad, M.; Alothman, Z. A.; Khan, A. B.; Ali, M. Effect of ionic liquid on activity, stability, and structure of enzymes: A review. Int. J. Biol. Macromol. 2012, 51, 555−560. (7) Pendleton, J. N.; Gilmore, B. F. The antimicrobial potential of ionic liquids: A source of chemical diversity for infection and biofilm control. Int. J. Antimicrob. Agents 2015, 46, 131−139. (8) Rengstl, D.; Kraus, B.; Van Vorst, M.; Elliott, G. D.; Kunz, W. Effect of choline carboxylate ionic liquids on biological membranes. Colloids Surf., B 2014, 123, 575−581.

4830

DOI: 10.1021/acs.jpcb.7b03582 J. Phys. Chem. B 2017, 121, 4823−4832

Article

The Journal of Physical Chemistry B

guanidinium hydrochloride and ionic liquids in solution. J. Phys. Chem. B 2014, 118, 406−412. (30) Miller, M. C.; Hanna, S. L.; DeFrates, K. G.; Fiebig, O. C.; Vaden, T. D. Kinetics and mass spectrometric measurements of myoglobinunfolding in aqueous ionic liquid solutions. Int. J. Biol. Macromol. 2016, 85, 200−207. (31) Yang, Z. Hofmeister effects: An explanation for the impact of ionic liquids on biocatalysis. J. Biotechnol. 2009, 144, 12−22. (32) Bharmoria, P.; Rao, K. S.; Trivedi, T. J.; Kumar, A. Biamphiphilic ionic liquid induced folding alterations in the structure of bovine serum albumin in aqueous medium. J. Phys. Chem. B 2014, 118, 115−124. (33) Paul, B. K.; Ganguly, A.; Guchhait, N. Deciphering the interaction of a model transport protein with a prototypical imidazolium room temperature ionic liquid: Effect on the conformation and activity of the protein. J. Photochem. Photobiol., B 2014, 133, 99−107. (34) Sankaranarayanan, K.; Sreedhar, B.; Nair, B. U.; Dhathathreyan, A. Microviscosity-induced conformational transition in β-lactoglobulin in the presence of an ionic liquid. J. Phys. Chem. B 2013, 117, 1234− 1240. (35) Ghaedizadeh, S.; Emamzadeh, R.; Nazari, M.; Rasa, S. M. M.; Zarkesh-Esfahani, S. H.; Yousefi, M. Understanding the molecular behaviour of renilla luciferase in imidazolium-based ionic liquids, a new model for the a/b fold collapse. Biochem. Eng. J. 2016, 105, 505−513. (36) Debeljuh, N.; Barrow, C. J.; Byrne, N. The impact of ionic liquids on amyloid fibrillization of abeta16−22: Tuning the rate of fibrillization using a reverse hofmeister strategy. Phys. Chem. Chem. Phys. 2011, 13, 16534−16536. (37) Wolski, P. W.; Clark, D. S.; Blanch, H. W. Green fluorescent protein as a screen for enzymatic activity in ionic liquid−aqueous systems for in situ hydrolysis of lignocellulose. Green Chem. 2011, 13, 3107−3110. (38) Campbell, R. E.; Tour, O.; Palmer, A. E.; Steinbach, P. A.; Baird, G. S.; Zacharias, D. A.; Tsien, R. Y. A monomeric red fluorescent protein. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 7877−7882. (39) Shu, X.; Shaner, N. C.; Yarbrough, C. A.; Tsien, R. Y.; Remington, S. J. Novel chromophores and buried charges control color in mfruits. Biochemistry 2006, 45, 9639−9647. (40) Yamamoto, N.; Tsuchiya, H.; Hoffman, R. M. Tumor imaging with multicolor fluorescent protein expression. Int. J. Clin. Oncol. 2011, 16, 84−91. (41) Snaith, H. A.; Anders, A.; Samejima, I.; Sawin, K. E. New and old reagents for fluorescent protein tagging of microtubules in fission yeast; experimental and critical evaluation. Methods Cell Biol. 2010, 97, 147−172. (42) Polyak, D.; Krivitsky, A.; Scomparin, A.; Eliyahu, S.; Kalinski, H.; Avkin-Nachum, S.; Satchi-Fainaro, R. Systemic delivery of sirna by aminated poly(alpha)glutamate for the treatment of solid tumors. J. Controlled Release 2016, DOI: 10.1016/j.jconrel.2016.06.034. (43) Hung, S. S.; Chrysostomou, V.; Li, F.; Lim, J. K.; Wang, J. H.; Powell, J. E.; Tu, L.; Daniszewski, M.; Lo, C.; Wong, R. C. Aavmediated crispr/cas gene editing of retinal cells in vivo. Invest. Ophthalmol. Visual Sci. 2016, 57, 3470−3476. (44) Gambhir, H. S.; Raharjo, E.; Forden, J.; Kumar, R.; Mishra, C.; Guo, G. F.; Grochmal, J.; Shapira, Y.; Midha, R. Improved method to track and precisely count schwann cells post-transplantation in a peripheral nerve injury model. J. Neurosci. Methods 2016, 273, 86−95. (45) Bhat, S. A.; Iqbal, I. K.; Kumar, A. Imaging the nadh:Nad+ homeostasis for understanding the metabolic response of mycobacterium to physiologically relevant stresses. Front. Cell. Infect. Microbiol. 2016, 6, 145. (46) Dempsey, G. T. A user’s guide to localization-based superresolution fluorescence imaging. Methods Cell Biol. 2013, 114, 561− 592. (47) Piatkevich, K. D.; Verkhusha, V. V. Guide to red fluorescent proteins and biosensors for flow cytometry. Methods Cell Biol. 2011, 102, 431−461.

(9) Singh, U. K.; Dohare, N.; Mishra, P.; Singh, P.; Bohidar, H. B.; Patel, R. Effect of pyrrolidinium based ionic liquid on the channel form of gramicidin in lipid vesicles. J. Photochem. Photobiol., B 2015, 149, 1− 8. (10) Lee, H.; Kim, S. M.; Jeon, T.-J. Effects of imidazolium-based ionic liquids on the stability and dynamics of gramicidin a and lipid bilayers at different salt concentrations. J. Mol. Graphics Modell. 2015, 61, 53−60. (11) Singh, G.; Kang, T. S. Ionic liquid surfactant mediated structural transitions and self-assembly of bovine serum albumin in aqueous media: Effect of functionalization of ionic liquid surfactants. J. Phys. Chem. B 2015, 119, 10573−10585. (12) Riduan, S. N.; Zhang, Y. Imidazolium salts and their polymeric materials for biological applications. Chem. Soc. Rev. 2013, 42, 9055− 9070. (13) Liu, G. Applications of ionic liquids in biomedicine. Biophys. Rev. Lett. 2012, 7, 121−134. (14) Constatinescu, D.; Herrmann, C.; Weingartner, H. Patterns of protein unfolding and protein aggregation in ionic liquids. Phys. Chem. Chem. Phys. 2010, 12, 1756−1763. (15) Kumar, A.; Venkatesu, P. A comparative study of myoglobin stability in the presence ofhofmeister anions of ionic liquids and ionic salts. Process Biochem. 2014, 49, 2158−2169. (16) Vasantha, T.; Attri, P.; Venkatesu, P.; Devi, R. S. R. Structural basis for the enhanced stability of protein model compounds and peptide backbone unit in ammonium ionic liquids. J. Phys. Chem. B 2012, 116, 11968−11978. (17) Kumar, A.; Venkatesu, P. Does the stability of proteins in ionic liquids obey the hofmeister series? Int. J. Biol. Macromol. 2014, 63, 244−253. (18) Kumar, A.; Venkatesu, P. Overview of the stability of αchymotrypsin in different solvent media. Chem. Rev. 2012, 112, 4283− 4307. (19) Jha, I.; Attri, P.; Venkatesu, P. Unexpected effects of the alteration of structure and stability of myoglobin and hemoglobin in ammonium-based ionic liquids. Phys. Chem. Chem. Phys. 2014, 16, 5514−5526. (20) Attri, P.; Venkatesu, P. Thermodynamic characterization of the biocompatible ionic liquid effects on protein model compounds and their functional groups. Phys. Chem. Chem. Phys. 2011, 13, 6566−6575. (21) Jaeger, V. W.; Pfaendtner, J. Destabilization of human serum albumin by ionic liquids studied using enhanced molecular dynamics simulations. J. Phys. Chem. B 2016, 120, 12079−12087. (22) Shao, Q. On the influence of hydrated imidazolium-based ionic liquid on protein structure stability: A molecular dynamics simulation study. J. Chem. Phys. 2013, 139, 115102. (23) Jaeger, V. W.; Pfaendtner, J. Structure, dynamics, and activity of xylanase solvated in binary mixtures of ionic liquid and water. ACS Chem. Biol. 2013, 8, 1179−1186. (24) Tomé, L. I. N.; Jorge, M.; Gomes, J. R. B.; Coutinho, J. o. A. P. Molecular dynamics simulation studies of the interactions between ionic liquids and amino acids in aqueous solution. J. Phys. Chem. B 2012, 116, 1831−1842. (25) Sprenger, K. G.; Choudhury, A.; Kaar, J. L.; Pfaendtner, J. Lytic polysaccharide monooxygenases sclpmo10b and sclpmo10c are stable in ionic liquids as determined by molecular simulations. J. Phys. Chem. B 2016, 120, 3863−3872. (26) Nordwald, E. M.; Armstrong, G. S.; Kaar, J. L. Nmr-guided rational engineering of an ionic-liquid-tolerant lipase. ACS Catal. 2014, 4, 4057−4064. (27) Haberler, M.; Schroder, C.; Steinhauser, O. Solvation studies of a zinc finger protein in hydrated ionic liquids. Phys. Chem. Chem. Phys. 2011, 13, 6955−6969. (28) Ding, X.; Wang, Y.; Zeng, Q.; Chen, J.; Huang, Y.; Xu, K. Design of functional guanidinium ionic liquid aqueous two-phase systems for the efficient purification of protein. Anal. Chim. Acta 2014, 815, 22−32. (29) Fiebig, O. C.; Mancini, E.; Caputo, G.; Vaden, T. D. Quantitative evaluation of myoglobin unfolding in the presence of 4831

DOI: 10.1021/acs.jpcb.7b03582 J. Phys. Chem. B 2017, 121, 4823−4832

Article

The Journal of Physical Chemistry B

as a probe for studying protein-protein interactions. Chem. Commun. 2013, 49, 490−492. (68) Chen, Y.; Liu, B.; Yu, H.-T.; Barkley, M. D. The peptide bond quenches indole fluorescence. J. Am. Chem. Soc. 1996, 118, 9271− 9278. (69) Jones, J. D.; Gierasch, L. M. Effect of charged residue substitutions on the membrane-interactive properties of signal sequences of the escherichia coli lamb protein. Biophys. J. 1994, 67, 1534−1545. (70) Caputo, G. A.; London, E. Position and ionization state of asp in the core of membrane-inserted alpha helices control both the equilibrium between transmembrane and nontransmembrane helix topography and transmembrane helix positioning. Biochemistry 2004, 43, 8794−8806. (71) Orm, M.; Cubitt, A. B.; Kallio, K.; Gross, L. A.; Tsien, R. Y.; Remington, S. J. Crystal structure of the aequorea victoria green fluorescent protein. Science 1996, 273, 1392−1395. (72) Sakmar, T. P.; Franke, R. R.; Khorana, H. G. Glutamic acid-113 serves as the retinylidene schiff base counterion in bovine rhodopsin. Proc. Natl. Acad. Sci. U. S. A. 1989, 86, 8309−8313. (73) Stern, L. J.; Khorana, H. G. Structure-function studies on bacteriorhodopsin. X. Individual substitutions of arginine residues by glutamine affect chromophore formation, photocycle, and proton translocation. J. Biol. Chem. 1989, 264, 14202−14208. (74) Shu, Y.; Liu, M.; Chen, S.; Chen, X.; Wang, J. New insight into molecular interactions of imidazolium ionic liquids with bovine serum albumin. J. Phys. Chem. B 2011, 115, 12306−12314.

(48) Hendrix, J.; Flors, C.; Dedecker, P.; Hofkens, J.; Engelborghs, Y. Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy. Biophys. J. 2008, 94, 4103−4113. (49) Shaner, N. C.; Steinbach, P. A.; Tsien, R. Y. A guide to choosing fluorescent proteins. Nat. Methods 2005, 2, 905−909. (50) Fukumoto, K.; Yoshizawa, M.; Ohno, H. Room temperature ionic liquids from 20 natural amino acids. J. Am. Chem. Soc. 2005, 127, 2398−2399. (51) Ohno, H.; Fukumoto, K. Amino acid ionic liquids. Acc. Chem. Res. 2007, 40, 1122−1129. (52) Sankaranarayanan, K.; Sathyaraj, G.; Nair, B. U.; Dhathathreyan, A. Reversible and irreversible conformational transitions in myoglobin: Role of hydrated amino acid ionic liquid. J. Phys. Chem. B 2012, 116, 4175−4180. (53) Visser, N. V.; Hink, M. A.; Borst, J. W.; van der Krogt, G. N. M.; Visser, A. J. W. G. Circular dichroism spectroscopy of fluorescent proteins. FEBS Lett. 2002, 521, 31−35. (54) Sastry, G. M.; Adzhigirey, M.; Day, T.; Annabhimoju, R.; Sherman, W. Protein and ligand preparation: Parameters, protocols, and influence on virtual screening enrichments. J. Comput.-Aided Mol. Des. 2013, 27, 221−234. (55) Harder, E.; Damm, W.; Maple, J.; Wu, C.; Reboul, M.; Xiang, J. Y.; Wang, L.; Lupyan, D.; Dahlgren, M. K.; Knight, J. L. Opls3: A force field providing broad coverage of drug-like small molecules and proteins. J. Chem. Theory Comput. 2016, 12, 281−296. (56) Ikeguchi, M. Partial rigid-body dynamics in npt, npat and npγt ensembles for proteins and membranes. J. Comput. Chem. 2004, 25, 529−541. (57) Bailey, A. G.; Lowe, C. P. Milch shake: An efficient method for constraint dynamics applied to alkanes. J. Comput. Chem. 2009, 30, 2485−2493. (58) Shan, Y.; Klepeis, J. L.; Eastwood, M. P.; Dror, R. O.; Shaw, D. E. Gaussian split ewald: A fast ewald mesh method for molecular simulation. J. Chem. Phys. 2005, 122, 054101. (59) Stuart, S. J.; Zhou, R.; Berne, B. J. Molecular dynamics with multiple time scales: The selection of efficient reference system propagators. J. Chem. Phys. 1996, 105, 1426−1436. (60) Bowers, K. J.; Chow, E.; Xu, H.; Dror, R. O.; Eastwood, M. P.; Gregersen, B. A.; Klepeis, J. L.; Kolossvary, I.; Moraes, M. A.; Sacerdoti, F. D. Scalable algorithms for molecular dynamics: Simulations on commodity clusters; Proc. 2006 ACM/IEEE Conf. Supercomputing; Tampa, FL, 2006. (61) Das, N. K.; Ghosh, N.; Kale, A. P.; Mondal, R.; Anand, U.; Ghosh, S.; Tiwari, V. K.; Kapur, M.; Mukherjee, S. Temperature induced morphological transitions from native to unfolded aggregated states of human serum albumin. J. Phys. Chem. B 2014, 118, 7267− 7276. (62) Stepanenko, O. V.; Verkhusha, V.; Kasakov, V. I.; Shavlovsky, M. M.; Kuznetsova, I. M.; Uversky, V. N.; Turoverov, K. K. Comparative studies on the structure and stability of fluorescent proteins egfp, zfp506, mrfp1, “dimer2”, and dsred1. Biochemistry 2004, 43, 14913−14923. (63) Tung, H.-J.; Pfaendtner, J. Kinetics and mechanism of ionicliquid induced protein unfolding: Application to the model protein hp35. Mol. Syst. Des. Eng. 2016, 1, 382−390. (64) Bravaya, K. B.; Subach, O. M.; Korovina, N.; Verkhusha, V.; Krylov, A. I. Insight into the common mechanism of the chromophor formation in the red fluorescent proteins: The elusive blue intermediate revealed. J. Am. Chem. Soc. 2012, 134, 2807−2814. (65) Chen, Y.; Barkley, M. D. Toward understanding tryptophan fluorescence in proteins. Biochemistry 1998, 37, 9976−9982. (66) Weljie, A. M.; Vogel, H. J. Tryptophan fluorescence of calmodulin binding domain peptides interacting with calmodulin containing unnatural methionine analogues. Protein Eng., Des. Sel. 2000, 13, 59−66. (67) Moroz, Y. S.; Binder, W.; Nygren, P.; Caputo, G. A.; Korendovych, I. V. Painting proteins blue: Beta-(1-azulenyl)-l-alanine 4832

DOI: 10.1021/acs.jpcb.7b03582 J. Phys. Chem. B 2017, 121, 4823−4832