Hofmeister Ion-Induced Changes in Water Structure Correlate with

Jan 13, 2016 - Interestingly, this is the opposite trend to the changes in protein solvation. ... Isomers and energy landscapes of micro-hydrated sulf...
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Hofmeister Ion-Induced Changes in Water Structure Correlate with Changes in Solvation of an Aggregated Protein Complex Taylor P. Light, Karen M. Corbett, Michael A. Metrick, II., and Gina MacDonald* Department of Chemistry and Biochemistry, James Madison University, Harrisonburg, Virginia 22807, United States S Supporting Information *

ABSTRACT: RecA is a naturally aggregating Escherichia coli protein that catalyzes the strand exchange reaction utilized in DNA repair. Previous studies have shown that the presence of salts influence RecA activity, aggregation, and stability and that salts stabilize RecA in an inverse-anionic Hofmeister series. Here we utilized attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy and circular dichroism (CD) to investigate how various Hofmeister salts alter the water structure and RecA solvation and aggregation. Spectroscopic studies performed in water and deuterium oxide suggest that salts alter water O−1H and O−2H stretch and bend vibrations as well as protein amide I (or I′) and amide II (or II′) vibrations. Anions have a much larger influence on water vibrations than cations. Water studies also show increased water−water and/or water−ion interactions in the presence of strongly hydrated SO42− salts and evidence for decreased interactions with weakly hydrated Cl− and ClO4− salts. Salt-water difference infrared spectra show that kosmotropic salts are more hydrated than chaotropic salts. Interestingly, this is the opposite trend to the changes in protein solvation. Infrared spectra of RecA show that vibrations associated with protein desolvation were observed in the presence of SO42− salts. Conversely, vibrations associated with protein solvation were observed in the presence of Cl− and ClO4− salts. Difference infrared studies on the dehydration of model proteins aided in identifying changes in RecA−solvent interactions. This study provides evidence that salt-induced changes in water vibrations correlate to changes in protein solvent interactions and thermal stability.

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INTRODUCTION Solution conditions such as pH and temperature have been shown to greatly influence protein stability and aggregation. Solution conditions that are known to stabilize proteins may be of broad interest for materials and medically related research. Previous studies in our laboratory have focused on how solution conditions alter RecA and model protein stability.1−3 RecA is a naturally aggregating Escherichia coli protein involved in the strand exchange process utilized in DNA repair.4 Interestingly, RecA has been used in nanoscience applications such as in the construction of novel molecular devices that can form a functional circuit on a DNA-templated wire network.5,6 These advancements with RecA−DNA nanowires are a promising basis for the production of novel molecular devices.7,8 Investigations into how solution conditions alter protein stability are also widely applicable to a variety of disorders. Protein misfolding and aggregation have been associated with several neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, Huntington’s disease, and prion diseases.9 RecA is an interesting, aggregating protein system as multiple studies have found that the activity, aggregation state, and stability of RecA are influenced by buffer and salt conditions, although the mechanisms of these salt-induced changes remain unclear.1,2,10−12 Multiple studies on Hofmeister salts have worked to obtain a molecular understanding of complex interactions among ions, © XXXX American Chemical Society

water, and proteins. The Hofmeister series ranks cations and anions based on their relative ability to salt-in or salt-out proteins.13−15 The series ranks kosmotropes to chaotropes for anions (CO32− > SO42− > S2O32− > H2PO4− > F− > Cl− > Br− ≈ NO3− > I− > ClO4− > SCN−) and for cations (NH4+ > Cs+ > Rb+ > K+ > Na+ > Li+ > Ca2+ > Mg2+).13−15,20 Ions have effects on proteins, peptides, polymers, amide mimics, protein−water interactions, and the solvation water surrounding macromolecules.1,3,15−24 Studies on proteins in sol−gels have reported that proteins are directly influenced by hydration and solute effects.17,18 Changes in protein solubility are thought to be the result of the weak hydration around chaotropes lending water molecules to the protein and the strong hydration around kosmotropes removing water molecules from the immediate protein hydration shell.15 Additionally, kosmotropes were traditionally thought to strengthen the hydrogen-bonding network of water (“water structure makers”) while chaotropes were thought to weaken the hydrogen-bonding network of water (“water structure breakers”).15 Recent studies suggest that salts can perturb the water structure past the first few hydration layers,25−28 while others suggest that salts affect only the first few hydration layers.15,16,29,30 Previous studies have independently Received: December 8, 2015 Revised: January 11, 2016

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Figure 1. Buffer infrared spectra in 1 M salt 1H2O and 2H2O buffers, pH 7.5, 25 °C: (A) O−1H and O−2H stretching regions, (B) normalized O−1H stretch, (C) normalized O−2H stretch, and (D) normalized O−1H bend. BioATR cell II with a silicon crystal) equipped with a HeNe laser and an LN-MCT detector by coadding 500 scans (velocity, 20 kHz; apodization, Happ-Genzel; resolution, 4 cm−1; phase resolution, 32; phase correction, Mertz; aperture, 8 mm). Infrared spectra were collected at 25 °C following a 60 min period during which the protein was able to equilibrate on the ATR crystal and every 5−10 °C up to 95 °C with 2 min equilibration periods following heating. Water-buffer absorbance spectra were subtracted from protein spectra by eliminating the wide combination ∼2125 cm−1 water peak as Rahmelow and Hubner34 advise as well as keeping the subtracted spectrum relatively flat in the ∼1750−2000 cm−1 region as suggested by Dong et al.35 Deuterium oxide buffer absorbance spectra were subtracted from protein spectra by eliminating the ∼2480 cm−1 O−2H stretch. All infrared experiments were repeated in triplicate; spectra reported are the result of averaging three trials. ATR-FTIR Studies of Partially Dehydrated Protein Films. RecA was diluted to a final protein concentration of 1.5 mg/mL (∼38−40 μM) into the previously described 1H2O or 2H2O Tris buffer, pH 7.5. Stock protein solutions of 20 mg/mL myoglobin and α-chymotrypsin were diluted to a final protein concentration of 2 mg/mL (∼120−140 μM) into 1H2O or 2H2O Tris buffer, pH 7.5. Proteins were dried under a gentle, constant flow of N2 gas at 25 °C for 40 min, and spectra were measured at 1 min intervals. The average final drying time was approximately 25 ± 1 min for each protein sample. IR spectra were obtained with the same parameters as described above, but 100 scans were coadded instead of 500 scans to keep the data collection time to 1 min. Difference spectra were obtained by subtracting the initial protein solution spectrum from each consecutive spectrum. All infrared dehydration experiments were repeated in triplicate; spectra reported are the result of averaging three trials. Circular Dichroism Studies of RecA. RecA was diluted to a final concentration of 5 μM into the previously described Tris buffer in 1H2O or 2H2O with the addition of one of the salts with a final concentration of 1 M. CD spectra were obtained using a Jasco J-810 spectropolarimeter with a single Peltier cell holder in a 3 mm path length quartz cell from Starna Cells, Inc. CD spectra were obtained at 25 °C from 300 to 180 nm in continuous scanning mode (scanning speed, 100 nm/min; data pitch, 0.1 nm; bandwidth, 1 nm; response time, 4 s). The nitrogen flow rate was 100 mL/min for all CD spectra. CD spectra were obtained at 25 °C and every 5° to 105 °C with 2 min equilibration periods following heating. Three spectra were collected and averaged per experiment. For all experiments, 222 nm voltages were within accepted limits ( Cl− > ClO4−) and cations (NH4+ > Na+ > Ca2+ > Mg2+).13,14 The combined use of CD and ATR-FTIR spectroscopy allows us to correlate ion-induced alterations in water vibrations such as changes in water−water or water−ion interactions and relate these to changes in protein solvation and stability. ATR-FTIR Studies on Water Structure. Figure 1 depicts the infrared spectra of 1H2O or 2H2O buffers in the absence and presence of 1 M salts. These studies expand on previous studies and provide a more complete sudy of how Hofmeister ions influence the water structure (refs 24−30, 37, and 38 and references within.) The water-salt studies were performed in the exact buffers used for the RecA studies such that they allow us to directly compare changes in water−water and/or water−ion interactions to changes in protein solvation. The O−1H and O−2H stretching regions (Figure 1A) provide information on the influence of each salt on water vibrations. In general, each salt showed a primary absorbance peak at ∼3360 cm−1 and a lowfrequency shoulder at ∼3260 cm−1 in the O−1H stretching region. (NH4)2SO4 and MgSO4 show a unique band shape and higher relative intensity of the ∼3260 cm−1 shoulder. The O−2H stretch shows a shape similar to the O−1H stretch, with a primary absorbance at ∼2480 cm−1 and a low-frequency shoulder at ∼2400 cm−1. We observed differences in the stretch vibrations that contain information about salt-induced differences in water−ion interactions and water−water hydrogen bonding. The changes in the frequency of the stretch vibrations are shown in Figure 1. The order of these shifts are as follows from highest to lowest frequency (NaClO4 > NH4Cl > MgCl2 > CaCl2 > NaCl > Tris > Na2SO4 > (NH4)2SO4 > MgSO4) and are consistent in both 1H2O and 2H2O. A decrease in the O−1H stretching frequency (sulfate salts) can reflect increases in water hydrogen bonding interactions while an increase in the O−1H stretching frequency (chloride and perchlorate salts) can reflect decreases in water hydrogen bonding interactions.27,29,39,40 Differences in the frequency of the O−1H bend vibrations in decreasing frequency from 1639 to 1635 cm−1 are as follows: MgSO4 ≈ Na2SO4 ≈ NaCl > (NH4)2SO4 > Tris > CaCl2 ≈ MgCl2 ≈ NH4Cl > NaClO4 (Figure 1B). The sulfate salts increase and the chloride and perchlorate salts decrease the frequency of the O−1H bend, suggesting increased hydrogen bonding between waters in the presence of sulfate salts.27,29,39,40 MgSO4 spectra have more distinct shoulders in the O−1H and O−2H bending vibrations at 1686 and 1242 cm−1. Liu et al. attributed the vibration at 1684 (1686) cm−1 to the first-layer water of Mg2+ and therefore suggest that MgSO4 has a stronger influence on the water structure.29 An infrared study on the water and deuterium oxide substructure fit two Gaussian sub-bands in the O−1H and O−2H stretching vibrations at ∼3460 cm−1 (2545 cm−1) and the O−1H (O−2H) bending overtone at ∼3235 cm−1 (∼2480 cm−1).41 Within the stretching vibrations, several studies have attributed low-frequency vibrations (∼3230 cm−1) to an icelike component that represents water molecules with a fully and linearly hydrogen-bonded structure.29,38,39,41−44 Increases in this low-frequency shoulder suggests an increase in water−water hydrogen bonding.29,38,39,41−44 We normalized the spectra from Figure 1A to the O−1H and O−2H stretching vibrations as shown in Figure 1B,C. Normalization revealed that the shoulders at ∼3260 and ∼2400 cm−1 vary in intensity relative to the salt present and showed that salts have ion-specific influences on the

Figure 2. Intensity of the low-frequency shoulder of the O−1H and O−2H stretching vibrations in the 1 M salt buffer spectra ordered from most intense to least intense in the left column with their respective anionic species in the right column (C, chaotrope; K, kosmotrope; N, neutral). Relative hydration of anions.15,21

relative hydration of the anionic species present.15,21 The order from highest intensity to lowest intensity in 1H2O is as follows: MgSO4 > (NH4)2SO4 > Tris > Na2SO4 ≈ NH4Cl ≈ MgCl2 > NaCl ≈ CaCl2 ≈ NaClO4 (Figures 1B,C and 2). These findings are in agreement with previous studies44 and support the fact that sulfate salts increase water hydrogen bonding and chloride and perchlorate salts decrease water hydrogen bonding. The saltinduced changes in water vibrations can be related to increasing anion hydration and is in agreement with the Hofmesiter series for anions (Figures 1 and 2). Several studies have used difference spectra to isolate distinct changes within the O−1H and O−2H stretching vibrations.24,27,29,37,38 In order to isolate salt-induced changes in water vibrations, we used the data from Figure 1A to generate salt buffer minus Tris buffer difference spectra (Figure 3A,B). The O−1H and O−2H stretch difference spectra show similar features (compare Figure 3A,B). The difference spectrum of NaCl (Figure 3A, purple) has a negative feature at 3145 cm−1 and a positive feature at 3435 cm−1. Previously reported chloride salt minus control difference spectra have similar features at ∼3160 cm−1 (reduction in the hydrogen-bonding network of pure water) and ∼3410 cm−1 (weak hydrogen bonding between the anion and water).27 NaClO4 (Figure 3A, orange) has a large negative feature at 3210 cm−1 similar to that of the Cl− salts and has a novel positive vibration at 3590 cm−1 that has been previously assigned to water molecules weakly hydrogen bonded to ClO4−.24,37,38 The negative feature is the most intense in NaClO4, suggesting that the ClO4− anion has the largest effect on breaking the overall water structure.27,38 The MgCl2 and MgSO4 difference spectra have a unique positive feature at 3095 cm−1 that has been previously assigned to the first-layer water around Mg2+ (Figure 3A, red and light blue).29 The (NH4)2SO4 and NH4Cl difference spectra (Figure 3A, green and yellow) show positive features at 2885 and 3025 cm−1 that have been previously assigned to the first hydration layer of NH4+.38 The Na2SO4 (Figure 3A, blue) difference spectrum has a positive feature at 3475 cm−1 and a slightly negative feature at 3215 cm−1 that is consistent with previous studies.29,38 Our data suggests that MgSO4 and NaClO4 have the largest influence on O−1H stretching and bending vibrations. Changes with MgSO4 and MgCl2 agree with previous studies that show larger water effects in the presence of magnesium.25,26 Our studies show that the change in water structure is dominated by anionic influences. C

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Figure 3. Difference infrared spectra in 1 M salt 1H2O and 2H2O buffers, pH 7.5, 25 °C obtained by subtracting the Tris control buffer spectrum from each salt-buffer spectrum in 1H2O (A, C, E) and 2H2O (B, D, F). (A, B) Salts used with RecA throughout this study, (C, D) Hofmeister anions with the Na+ cation, and (E, F) Hofmeister cations with the Cl− anion. (Dotted line markers are located at (C) 3180 and 2970 cm−1 and (D) 2370 and 2240 cm−1.)

Figure 4. CD spectra of 5 μM RecA in 1 M salt (A) 1H2O and (C) 2H2O buffers, pH 7.5, 25 °C. Buffer-subtracted, infrared spectra of 1.5 mg/mL RecA in 1 M salts (B) 1H2O and (D) 2H2O buffers, pH 7.5, 25 °C. Infrared spectra were normalized for protein content using amide II for RecA−1H2O spectra and amide I′ for RecA−2H2O spectra. (D, inset) Magnified view of the normalized amide I′ shoulder from 1640 to 1600 cm−1. (E) Difference infrared spectra of RecA in the presence of 1 M salt 2 H2O buffers obtained by subtracting the RecA−no salt control spectrum from the RecA−salt spectra. RecA−salt spectra not shown in (A) have been previously published by our group.1

However, as observed in previous studies, our results confirm important, cooperative cationic effects on water.25,26 In order to isolate and further study anionic versus cationic contributions, we performed two separate and complete studies that expand on previous work. Difference spectra were obtained on Hofmeister ions with a neutral cation (Na+) and a neutral anion (Cl−) (Figure 3C−F). A comparison of Figure 3C,D (anions) and Figure 3E,F (cations) reveals that anions have a larger and more variable influence on water and agrees with previous studies comparing cation and anion effects on water.21 The chaotropic anions (warm-colored spectra) have a negative feature attributed to a reduction in the hydrogen-bonding network of water.24,27 The most strongly hydrated, kosmotropic salts (cold-colored spectra) do not have a negative feature but have a positive feature at ∼2970 cm−1 in 1H2O associated with strengthening the hydrogen-bond network of water (Figure 3).24 The cation difference spectra have similar shapes and smaller changes in intensity in the O−1H vibrations as compared to anionic difference spectra (compare Figure 3C/D,E/F), suggesting that anions have a larger influence on water vibrations. CD and ATR-FTIR Studies of RecA in Hofmeister Salts. Circular dichroism studies of RecA in 1 M salt 1H2O and 2H2O buffers are summarized in Figure 4A,C. Our previously published CD data1 did not include studies using NH4Cl, (NH4)2SO4, and NaClO4 salts or any experiments in 2H2O buffers. The CD

spectra of RecA at 25 °C in 1H2O and 2H2O (Figure 4A,C and Cannon et al.1) reveal slight differences in RecA secondary structure in the presence of different salts.1 Sulfate salts show a less-pronounced ∼208 nm feature, suggesting a small decrease in α-helical structure as compared to that for RecA in the presence of the chloride and perchlorate salts (Figure 4A,C).45,46 The structural information obtained using circular dichroism provides general information about the overall secondary structure of RecA and is an important complement to infrared spectroscopic studies that provide information on protein structure and solvation.3,31−33,40,47,48 The ATR-IR spectra of the RecA control and RecA in the presence of various 1 M salt solutions at 25 °C in 1H2O (amide I and amide II) and 2H2O (amide I′ and amide II′) are shown in Figure 4B,D. In order to allow us to directly compare saltinduced changes in RecA, each set (in either 1H2O or 2H2O) of protein spectra were scaled to account for differences in protein content. The spectra obtained in 1H2O were normalized using the amide II (∼1550 cm−1) vibration since the amide I vibration (∼1650 cm−1) contains contributions from protein and solvent O−1H contributions and the amide II vibration contains only protein contributions. The 2H2O spectra were normalized to the amide I′ (∼1650 cm−1) vibration that does not contain overlapping solvent contributions. The amide I or I′ band D

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vibrations associated with deuterium exchange.3,40,47 Our previous studies showed that the strongly hydrated kosmotropic anions (SO42−) hindered the rate of 1H/2H exchange and that the weakly hydrated chaotropic anions (Cl−, ClO4−) enhanced 1 H/2H exchange in myoglobin and lysozyme.3 Similar results were obtained for solution RecA samples (Figure 4D). Figure 4D shows decreased intensities of the amide II′ band at ∼1450 cm−1 in the presence of sulfates (with the exception of MgSO4), suggesting that sulfates inhibit deuterium exchange and/or are more desolvated as compared to RecA samples with the chloride salts. NaClO4 increased the intensities of amide II′ relative to those of all of the other salts, suggesting increased RecA−water interactions. Additional experiments performed in increasing concentrations of sucrose are consistent with previous studies and show a decreased amide II′ intensity similar to that of sulfate salts and an increased amide I (I′) frequency associated with desolvation and support the idea that the intensity of amide II′ can be used to monitor solvation (Figure S3).3,31,50 An initial comparison of RecA−salt spectra in 1H2O and 2H2O suggests that sulfates decrease protein solvation relative to the control and chloride samples. The results obtained in 2H2O (Figure 4D, amide II′) showing that sulfates decrease solvation also agree with the 1H2O spectra (Figure 4B) that suggest a decreased number of waters are associated with sulfate RecA complexes. Many previous studies on polymers and proteins suggest that some of the observed solute-dependent infrared changes may arise from differences in protein solvation.3,31−33 Poly(Nisopropylacrylamide), PNIPA, is considered to be a model system for protein unfolding studies.32 Previous infrared studies on PNIPA identified amide vibrations that are influenced by changes in solvation.32,33 The amide I and amide II vibrations were deconvoluted into three distinct subbands altered by solvation.32,33 The amide I subbands assigned were hydrogenbonded amide−amide at 1630 cm−1, amide−water at 1620 cm−1, and a “free” non-hydrogen-bonded amide at 1643 cm−1.32,33 Amide II subbands assigned were a hydrogen-bonded amide− amide at 1551 cm−1, an amide−water at 1565 cm−1, and a free non-hydrogen-bonded amide band at 1535 cm−1.32,33 Although we may expect small shifts in deuterium oxide, the RecA spectra obtained in 2H2O do not contain O−1H solvent contributions and show some salt-induced differences that correlate with changes observed in the polymer. The increased 1620 cm−1 intensity present in the infrared spectra obtained on RecA in the chloride salts in deuterium oxide would then suggest an increase in amide−water interactions and a greater solvation of RecA in the presence of Cl− and ClO4− salts.20,32 The increased 1634 cm−1 and decreased 1643 cm−1 intensities are consistent with an increase in amide−amide interactions and a decrease in free amide in the RecA−sulfate spectra and suggest greater desolvation and increased RecA−RecA interactions in the presence of SO42− salts.32 Further evidence that these vibrations reflect changes in RecA solvation are observed in Figure S4. The intensity of the previously assigned vibrations (1634 and 1620 cm−1) are dependent on the salt concentration. Figure S4 shows that increasing sulfate concentrations decrease the 1620 cm−1 intensity and increase the 1634 cm−1 intensity, with the exception of the 2 M sodium sulfate sample. Previous experiments in our laboratory have shown that 2 M sodium sulfate more significantly alters the structure and aggregation of RecA such that these changes will influence the shape and intensity of amide I′.1 Increasing perchlorate concentration corresponds to decreases in 1634 cm−1 intensity and increases in 1620 cm−1 intensity (Figure S4). The fact that the changes in the

shapes in 1H2O and 2H2O indicate that there are salt-dependent differences in RecA structure and/or solvation (Figure 4B,D). The amide I vibration in the 1H2O spectra shows two distinct positive peaks around 1650 and 1634 cm−1 and decreased intensity around 1643 cm−1 in the presence of sulfate salts. However, these peaks are less resolved in the infrared spectra of RecA obtained in chloride and perchlorate salts (Figure 4B). Relative to the Tris control spectrum, the presence of SO42− salts decreased the 1620 cm−1 intensities while the Cl− and ClO4− salts spectra increased the 1620 cm−1 intensities. In addition, the RecA 1H2O spectra obtained in the SO42− salts also have slightly less 1535 cm−1 intensity than do the Cl− and ClO4− salts. A comparison of the RecA infrared spectra obtained in SO42, Cl−, and ClO4− salts revealed differences in the amide I vibration and provided information about protein−solvent interactions as the CD spectra suggest only minimal changes in protein structure. Infrared spectra of RecA before scaling for protein content are shown in Figure S1. Amide I, II, and I′ intensities suggest that ammonium and sodium sulfate significantly increase protein aggregation on the crystal (Figure S1). Since the 1H2O spectra (Figure 4B) were normalized for protein content using amide II, the amide I intensity contains contributions from the O−1H bend and may reflect how salts influence the amount of water associated with the RecA complexes on the ATR surface. If the protein content is similar for all 1H2O spectra shown in Figure 4B, then the differences in the amide I intensity reflect the different amounts of solvent O−1H contributions to the amide I vibration. In general, RecA−sulfate spectra show decreased intensities and are consistent with the idea that the RecA−sulfate surface complexes have fewer associated waters than do the RecA−chloride and perchlorate complexes, indicating that the salts influence the number of waters solvating the protein. Although the 1H2O infrared spectra may provide useful information on water−protein complexes, the overlapping O−1H bend at ∼1640 cm−1 can complicate assignments.48,49 The results obtained in 2H2O complement those obtained in 1 H2O and suggest that salts differentially alter RecA solvation (Figure 4D). RecA 2H2O samples were further diluted in the appropriate salt 2H2O solution before incubating samples on the ATR crystal and obtaining data. Spectra obtained in deuterium oxide were scaled for protein content using the amide I′ vibration and have small 2−5 cm−1 shifts in the maximum absorbance as compared to amide I.48 Infrared spectra of RecA obtained in the presence of chlorides or perchlorate have increased 1620 cm−1 and decreased 1634 cm−1 intensities as compared to RecA spectra obtained in the presence of the sulfate salts (Figure 4D, inset). Protein−salt minus protein−control difference spectra more clearly isolate salt-induced differences in protein (Figure 4E). Difference spectra obtained in the absence of protein (Figure S2) clearly reveal that the changes observed in Figure 4E arise from protein and not solution differences. The changes in the 1660−1700 cm−1 region may also reflect that some ion interactions with protein side chains such as Arg, Asn, Gln, Asp, Glu or could result from specific or nonspecific ion-protein interactions.40,47 The 1450 cm−1 vibration contains contributions from amide II′ and the 1H−O−2H vibration and reflects the amount of deuterium exchange in each protein sample on the ATR surface. Previous studies in our laboratory followed 1H/2H exchange in partially dehydrated and solution myoglobin and lysozyme samples and allowed us to observe differences in protein solvation or protein−water interactions.3 Increases in the 1450 cm−1 intensity reflect increases in 1H−O−2H and amide II′ E

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Table 1. Values Obtained by Dividing the Infrared Intensity of the Amide−Water (1620 cm−1) or Amide−Amide (1634 cm−1) Peaks of the Normalized, 2H2O, and 1 M RecA−Salt Spectra by the Tris Spectruma and Values Obtained by Dividing the Intensities of the Amide−Water (1620 cm−1) or Amide−Amide (1634 cm−1) Peaks by the Intensity of the 1650 cm−1 Peak Attributed to Overall Protein Content Using the Prenormalized 1 M RecA−Salt Spectrab amide−water interactions salt

salt 1620 Tris

Na2SO4 MgSO4 (NH4)2SO4 Tris NaCl CaCl2 MgCl2 NH4Cl NaClO4 a

cm−1 intensity

amide−amide interactions

1620 cm−1 intensity 1650 cm−1 intensity

0.88 1.06 0.99 1.00 1.07 1.07 1.07 1.11 1.20

0.48 0.57 0.53 0.54 0.58 0.58 0.58 0.60 0.65

salt 1634 Tris

cm−1 intensity 1.02 1.01 1.00 1.00 0.98 0.96 0.94 0.94 0.95

1634 cm−1 intensity 1650 cm−1 intensity

1.01 0.99 0.98 0.99 0.97 0.96 0.94 0.93 0.94

Columns 1 and 3. bColumns 2 and 4.

influence structure and aggregation.20,32,33 In either case, it is clear that there are salt-dependent trends that correlate with the influence of salts on water vibrations. In order to confirm our assignments, additional experiments were performed to better identify protein vibrations that are altered by desolvation and dehydration. Protein dehydration was directly monitored using FTIR. Various protein solution samples were dehydrated in either 1 H2O or 2H2O Tris buffer solutions with gentle nitrogen flow over time, and infrared spectra were obtained every minute during the dehydration in order to directly follow changes in protein hydration (Figure 5). Difference spectra were obtained

intensity of vibrations associated with amide−amide (1634 cm−1) or amide−water (1620 cm−1) interactions are concentration-dependent provides additional evidence that these vibrations reflect salt-dependent changes in RecA solvation (Figure S4). RecA amide I′ intensities contain a large number of overlapping contributions from the protein backbone including those associated with differences in protein structure, solvation, and aggregation. Thus, we are not able to unambiguously assign these vibrations solely to differences in RecA−water interactions, especially given that we do not have specific information about salt-induced changes in RecA aggregation. Most likely, differences in RecA solvation would also influence aggregation. However, it is very interesting that the RecA−salt spectra show salt-dependent and concentration-dependent differences that may be explained by PNIPA solvation studies.32,33,47,48 Further analysis of RecA spectra confirmed our initial observations. RecA−salt spectra were divided by the RecA− Tris spectrum (Figure 4D), and the intensities of 1634 cm−1 (amide−amide) and 1620 cm−1 (amide−water) vibrations are reported in Table 1. RecA spectra obtained in the presence of Cl− and ClO4− salts have increased 1620 cm−1 intensities, as compared to those of control or SO42− salt samples (Table 1, column 1). RecA spectra obtained in the presence of SO42− salts have increased 1634 cm−1 intensities (Table 1, column 3). Additional calculations were performed in order to confirm that the differences observed in Table 1 (columns 1 and 3) were not artifacts of the normalization of amide I′. Using unscaled RecA−2H2O spectra (Figure S1), the intensities of the 1634 or 1620 cm−1 peaks were divided by the intensity of the 1650 cm−1 peak attributed to the overall protein content (Table 1, columns 2 and 4). The new ratios reveal the same order of intensity and confirm that SO42− salts have decreased 1620 cm−1 vibrations and increased 1634 cm−1 intensities as compared to those of RecA in the presence of Cl− and ClO4− salts.20,32,33 However, previous infrared protein studies have assigned vibrations at 1650 cm−1 to buried, desolvated helices and a 1633 cm−1 vibration to solvated helices.51−54 The 1620 cm−1 vibration is often assigned to aggregated protein in addition to solvated polymer amide.20,32,33,48 These salt-dependent differences in RecA vibrational intensities may be directly explained by differences in RecA solvation such as those observed in the PNIPA studies.32,33 However, these differences may also be due to small differences in RecA structure and/or aggregation that are altered by the salts or the salt-induced differences in solvation that

Figure 5. Difference infrared spectra obtained for partially dehydrated proteins. Myoglobin (α-helical),35 α-chymotrypsin (β-sheet),35 and RecA (α and β).4 Solutions contained 2 mg/mL myoglobin or αchymotrypsin or 1.5 mg/mL RecA in (A) 1H2O and (B) 2H2O buffers, pH 7.5, 25 °C. Difference spectra represent the final (dried) minus the initial (solution) spectrum.

by subtracting the initial solution spectrum from the final dehydrated spectrum such that positive features result from vibrations associated with dehydrated protein and negative features result from vibrations associated with solvated protein. Myoglobin (α-helical)35 shows positive features at around 1655 cm−1 and a negative feature at around 1620 cm−1 as dehydration occurs (Figure 5). However, α-chymotrypsin (β-sheet)35 shows positive features at around 1637 and 1686 cm−1 and a negative feature at around 1620 cm−1 during dehydration (Figure 5). The dehydration of RecA, (α helix and β sheet)4 shows positive features at 1657 and 1635 cm−1 and negative features at 1643 and 1618 cm−1 vibrations during dehydration (Figure 5).32 The negative feature at ∼1620 cm−1 is present in each protein spectrum and suggests that this vibration is associated with F

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Langmuir solvated protein, supporting previous assignments of the ∼1620 cm−1 vibration to amide−water.20,32 Studies in 2H2O show similar difference spectral features in amide I (I′) for each protein (compare Figure 5A,B). These experiments agree with the assignments from the polymer and clearly show that desolvated and solvated amide vibrations are dependent on the secondary structure of the protein. In summary, small anion-dependent changes in RecA secondary structure are observed in 1H2O and 2H2O. RecA in the presence of sulfate salts shows vibrational changes consistent with increased amide−amide and decreased amide−water interactions. These results suggest that sulfates decrease RecA−water and increase RecA−RecA interactions. In the presence of chloride and perchlorate salts, RecA spectra show vibrational changes consistent with decreased amide−amide and increased amide−water interactions. Additional experiments were performed in attempts to follow salt-dependent changes in protein solvation during unfolding and to further study aniondependent changes in protein solvation. CD and ATR-FTIR Studies of RecA Unfolding in Hofmeister Salts. In efforts to study the influence of Hofmeister salts on solvation changes during RecA unfolding, infrared spectra of RecA were obtained from 25 to 105 °C every 5−10 °C. CD unfolding profiles on similar samples are presented in Figure 6A,B. Previously published CD spectra and melting

The Tris control and sodium sulfate unfolding have more gradual transitions in 2H2O than in 1H2O (compare Figure 6A,C). There are some additional differences between 1H2O and 2 H2O solutions (Figure 6A,C). RecA in NH4Cl is stabilized up to 105 °C in 1H2O but unfolds at ∼93 °C in 2H2O (Figure 6A,C). The melting temperatures of RecA in sulfate salts increase in 2 H2O, suggesting that 2H2O has a stabilizing effect on RecA compared to RecA in 1H2O (Figure 6A,C and Cannon et al.1). Figure 6B (1H2O) and Figure 6D (2H2O) are the turbidity (aggregation) plots generated by plotting the HT voltage at 285 nm versus temperature.36 The turbidity plots presented here and in Cannon et al.1 reveal that the Tris control and the sulfate salt RecA samples aggregate upon unfolding. RecA in chlorides and perchlorate salts does not aggregate during unfolding (Figure 6B,D). In RecA−NH4Cl samples and only slightly in RecA− NaCl samples, RecA aggregation increases at high temperatures. Interestingly, a comparison of turbidities at 25 °C shows that all salts increase the RecA aggregation in 1H2O as compared to the Tris control while all salts decrease RecA aggregation in 2H2O as compared to the Tris control (Figure 6B,D and Cannon et al.1). Infrared spectroscopy was also used to study RecA unfolding. The results from these studies are summarized in Figure 7. Figure 7A shows RecA spectra in the absence and presence of 1 M salts in 2H2O at 95 °C, and panels B−J show the thermal unfolding profiles for each salt from 25 to 95 °C (Figure 7). The amide I′ band shows large differences in intensity and shape compared to the spectra at 25 °C (compare Figures 4D and 7A). At 95 °C, the amide I′ of all salts has a double-maximum shape with features at 1650 cm−1 and ∼1620 cm−1. The band at 1620 cm−1 has been assigned to nonspecific protein aggregation, and the increasing prominence of a 1620 cm−1 feature suggests an increase in RecA aggregation at higher temperatures.48 This band associated with aggregation has the same assignment as the amide−water interaction.32 The sulfate salts have the most intense maximum at 1620 cm−1 that correlates to the unfolded, aggregated protein observed in the turbidity plots (Figures 6D and 7A). RecA unfolding results in increases in the amide I′ intensity that suggest increased RecA on the ATR crystal (Figure 7). Similar to CD and turbidity measurements during unfolding, the Tris control and the chloride and perchlorate salts gradually increase the intensity of amide I′, with the exception of NH4Cl, while the sulfate salts and NH4Cl have sharper unfolding transitions (Figures 6 and 7). RecA in the presence of the Tris control, chloride, and perchlorate salts does not show large increases in amide II′ relative to amide I during unfolding. RecA in sulfate salts results in larger increases in amide I′ and amide II′ during RecA unfolding. Interestingly, this result suggests that unfolding-induced changes in solvation are greater for RecA in the presence of sulfate salts as compared to chlorides or perchlorate. The unfolding data supports the infrared and dehydration studies performed at room temperature and are summarized in Figure 8. Sulfates decrease protein−water interactions, and chlorides and perchlorates increase protein− water interactions.

Figure 6. (A, C) 222 nm ellipticity and (B, D) 285 nm turbidity (HT voltage) versus temperature (25−105 °C in 5 °C increments) during RecA unfolding. All solutions contained 5 μM RecA in Tris buffer, pH 7.5 and the addition of 1 M salts in 1H2O (A, B) and 2H2O (C, D). Additional salts not shown in (A, B) have been previously published.1

profiles in 1H2O are complimented by the addition of RecA melting and turbidity profiles obtained in three additional salts: NH4Cl, (NH4)2SO4, and NaClO4 (Figure 6A,B).1 Figure 6A,C plots CD intensity at 222 nm against temperature in 1H2O and 2 H2O, respectively. The stability of RecA in the presence of these additional salts is consistent with the previously published data that shows RecA follows an inverse anionic Hofmeister series (Figure 6A,C).1 The sulfate salts destabilize RecA while the chloride and perchlorate salts stabilize RecA up to 105 °C in 1 H2O (Figure 6A).

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CONCLUSIONS Attenuated total reflectance infrared (ATR-IR) spectroscopy and circular dichroism (CD) were used to investigate how Hofmeister salts influence water structure and RecA solvation, aggregation, and stability. Our study shows that observed trends associated with changes in water vibrations can be associated with changes in protein solvation and stability. We have observed G

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Figure 7. (A) Overlay of RecA infrared spectra in the absence and presence of 1 M salts at 95 °C. (B−J) Buffer-subtracted, infrared spectra of 1.5 mg/mL RecA in 1 M salts in 2H2O buffers, pH 7.5 at temperatures ranging from 25 to 95 °C in 5−10 °C increments. Light-gray spectra represent lower temperatures, and dark-gray/black spectra represent higher temperatures.

O−2H stretching and the O−1H bending modes suggest increased water hydrogen bonding in kosmotropic anionic salts and decreased water hydrogen bonding in chaotropic anionic salts. We confirm and expand on previous studies that show that infrared spectra are useful for studying the extent to which ions influence water structure and confirm that Hofmeister anions have a larger influence on water vibrations as compared to the Hofmeister cations. The anion-induced changes in water vibrations should in turn influence RecA aggregation and RecA’s interaction with water. Our results are also consistent with the fact that sulfate salts increase RecA−RecA interactions and decrease RecA−water interactions while chlorides and perchlorate anions show relative decreases in RecA−RecA interactions and increases in RecA−water interactions. While we are unable to unambiguously assign vibrations to those associated with RecA solvation, protein dehydration studies clearly show that the infrared signature of the protein changes upon dehydration. The results presented within clearly show saltdependent trends that correlate with the salts’ influence on water vibrations. Figure 8 illustrates the influence of Hofmeister anions on water hydrogen bonding and RecA solvation. Here we provide evidence that the kosmotropic anions increase water hydrogen bonding and promote RecA desolvation while chaotropic anions decrease water hydrogen bonding and promote RecA−solvent interactions (Figure 8).

Figure 8. Cartoon depiction showing anionic species and their influence on water hydrogen bonding and protein solvation. Infrared studies on water show that kosmotropic salts increase water−water hydrogen bonding while chaotropic salts decrease hydrogen bonding between waters. A variety of RecA infrared studies show that salts alter the RecA solvation. Infrared absorption spectra of RecA obtained in water or deuterium oxide and infrared dehydration and RecA unfolding studies all suggest that kosmotropic anions decrease RecA−water interactions (solvation) while chaotropic anions increase RecA−water interactions. Protein surface (black circles), anions (blue and orange spheres), and water molecules (red/white).

ion-induced changes in the O−1H and O−2H stretching vibrations in water that correlate with ion-induced differences in protein vibrations associated with protein−water interactions. We have also shown that vibrational changes associated with altered protein solvation can be dependent on the protein’s secondary structure. Changes in the frequency of the O−1H and H

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.5b04489. IR spectra of RecA in 1 M salts before normalization, salt buffer difference spectra in the protein IR amide I′ region, RecA−sucrose CD and IR spectra, and RecA IR spectra in 0−2 M Na2SO4 and NaClO4 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: 540-568-6852. Fax: 540-568-7938. E-mail: macdongx@ jmu.edu. Author Contributions

The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Funding

This research was supported by National Science Foundation grants NSF-RUI 0814716, NSF-REU CHE-1062629, and NSFREU CHE-1461175. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Dr. Yanjie Zhang for her invaluable discussions and contributions.

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ABBREVIATIONS ATR-FTIR, attenuated total reflectance Fourier transform infrared spectroscopy; CD, circular dichroism; Tris, tris(hydroxymethyl)aminomethane; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid

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REFERENCES

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