l-Proline and RNA Duplex m-Value Temperature Dependence - The

Article pubs.acs.org/JPCB

L‑Proline

and RNA Duplex m‑Value Temperature Dependence

Jeffrey J. Schwinefus,* Nadia L. Baka,‡ Kalpit Modi,‡ Kaylyn N. Billmeyer,‡ Shutian Lu,‡ Lucas R. Haase,‡ and Ryan J. Menssen‡ Department of Chemistry, St. Olaf College, Northfield, Minnesota 55057, United States S Supporting Information *

ABSTRACT: The temperature dependence of L-proline interactions with the RNA dodecamer duplex surface exposed after unfolding was quantified using thermal and isothermal titration denaturation monitored by uv-absorbance. The mvalue quantifying proline interactions with the RNA duplex surface area exposed after unfolding was measured using RNA duplexes with GC content ranging between 17 and 83%. The m-values from thermal denaturation decreased with increasing GC content signifying increasingly favorable proline interactions with the exposed RNA surface area. However, m-values from isothermal titration denaturation at 25.0 °C were independent of GC content and less negative than those from thermal denaturation. The m-value from isothermal titration denaturation for a 50% GC RNA duplex decreased (became more negative) as the temperature increased and was in nearly exact agreement with the m-value from thermal denaturation. Since RNA duplex transition temperatures increased with GC content, the more favorable proline interactions with the high GC content duplex surface area observed from thermal denaturation resulted from the temperature dependence of proline interactions rather than the RNA surface chemical composition. The enthalpy contribution to the m-value was positive and small (indicating a slight increase in duplex unfolding enthalpy with proline) while the entropic contribution to the m-value was positive and increased with temperature. Our results will facilitate proline’s use as a probe of solvent accessible surface area changes during biochemical reactions at different reaction temperatures.

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INTRODUCTION Biopolymer conformational changes in biochemical processes, such as biopolymer folding and unfolding, biopolymer−ligand complex formation, and aggregation, are sensitive to the presence of solutes. Solutes span a range of species including the Hofmeister salts,1,2 denaturants (urea3 and guanidinium chloride4), and the osmolytes (glycine betaine5 and proline6). Both solutes and water compete for interaction with the biopolymer surface area exposed or buried during a conformational change. If the solvent accessible surface area buried or exposed during a conformational change has thermodynamically favorable interactions with the solute and solvent, burial of that surface area will be discouraged and exposure encouraged. The preferential interaction of solute with the surface area buried or exposed during a conformational change can be quantified by a decrease or increase in the observed equilibrium and kinetic rate constants for the process.1,7 The sensitivity of biopolymer conformational changes to the presence of solutes makes solutes ideal probes of conformational changes in biochemical processes. Solutes, such as urea, glycine betaine, proline, potassium chloride, polyethylene glycol, sodium glutamate, and trimethylamine N-oxide (TMAO), have been used to study conformational changes in E. coli RNA polymerase,8−10 burial of DNA surface area in Lac repressor binding,11 transition states in protein folding,12 the excluded © XXXX American Chemical Society

volume effects on nucleic acid hairpin and secondary structure stability,13,14 and the kinetics of amyloid fibrillogenesis.15−17 The interaction of solutes with the biopolymer solvent accessible surface area exposed or buried during a biochemical reaction is quantified through the m-value18 m‐value =

◦ ∂ΔGobs ∂ln Kobs = −RT ∂m3 ∂m3

(1)

where the observed standard Gibbs energy change for the biopolymer process is ΔG°obs, Kobs is the observed equilibrium constant for the process, and m3 is the molality of the solute. When the m-value is negative, the biochemical process becomes more favorable: ΔGobs ° decreases with increasing solute concentration by either lowering the Gibbs energy of the biopolymer reaction products more than the reactants or by raising the Gibbs energy of the products less than the reactants. The m-value is dependent on the magnitude of the solvent accessible surface area change as well as the chemical functional groups that comprise the surface area exposed or buried in the process.19 Received: April 17, 2017 Revised: June 28, 2017

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DOI: 10.1021/acs.jpcb.7b03608 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

0.75, and 1.0 m for thermal denaturation and ranged between 0 and 14 m for isothermal titration denaturation (134 mm sodium chloride, 150 mm sodium cation overall). Final RNA duplex concentrations were approximately 2.5 μM for thermal and isothermal titration denaturation. Prior to thermal denaturation, solutions were degassed under vacuum using a ThermoVac pump (MicroCal). RNA duplex thermal transitions were monitored at 260 nm using a Cary 100 spectrophotometer (Varian) equipped with a Peltier temperature controller with a starting temperature between 4 to 20 °C (depending on the RNA duplex) to 100 °C. RNA duplex samples were heated at a rate of 0.3 °C/min and absorbance readings were collected every 0.2 °C. For isothermal titration d e n a t u r a t i o n , a b s o r b a n c e r e a din g s o n t h e 5 ′- r (GAAAGUAUAAAG)-3′, 5′-r(GAUAGUAGAUAG)-3′, 5′-r(GCAAAGUAAACG)-3′, and 5′-r(GCAUAGCAUACG)-3′ duplexes with increasing concentrations of proline were collected at 260 nm and 25.0 °C. Isothermal titration absorbance readings were also collected at 35.0, 45.0, and 55.7 °C for the 5′-r(GCAUAGCAUACG)-3′ duplex. Isothermal titration absorbance values were corrected for the minor proline absorbance at 260 nm. For both thermal and isothermal titration denaturation, RNA duplex and single-stranded plateau regions in the absorbance melting profiles were fit by linear regression.29 The fraction of unfolded single-stranded RNA (unfolded RNA single strands relative to the total single strand concentration), θ, as a function of temperature or proline molality was determined from a ratio of absorbance differences: the difference in the experimentally measured absorbance values and the linear fit to the absorbance of the duplex plateau region relative to the difference in absorbance between the linear fits to the singlestranded and duplex plateau regions.13 The observed unfolding equilibrium constant Kobs for RNA duplex denaturation into single strands S1 and S2 (duplex ⇄ S1 + S2) was determined from

L-Proline is a well-known osmoprotectant, or osmolyte, that accumulates in plants, bacteria, algae and marine invertebrates in response to osmotic stress.20 Proline is also referred to as a compatible solute because it does not have a pronounced effect on enzyme activity nor does it destabilize the secondary or tertiary structure of proteins.21,22 Proline has demonstrated promise as a probe of biopolymer conformational surface area changes.6,23−25 Proline’s preferential interactions with coarsegrained chemical functional groups on protein model compounds have already been quantified at 25.0 °C,6 facilitating the quantitative interpretation of the attenuation or increase in observed equilibrium and kinetic rate constants of biochemical reactions in proline solutions. Proline exhibits similar behavior as glycine betaine,26 preferentially destabilizing GC-rich nucleic acid secondary structure to a greater extent than AT- or AU-rich duplexes.25 Recently, the m-value for glycine betaine interactions with nucleic acid surface area was shown to be temperature dependent.27 For proline to serve as a probe of biopolymer conformational changes, the temperature dependence of proline interactions with biopolymer solvent accessible surface area must be elucidated since biochemical reactions in vitro are often executed at temperatures other than room temperature. This work quantified the temperature dependence of proline’s interactions with the RNA surface area exposed after unfolding using RNA dodecamer duplexes with GC contents ranging between 17 and 83% to facilitate proline’s use as a probe of biopolymer conformational changes.

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EXPERIMENTAL SECTION Materials. Lyophilized single-stranded RNA dodecamers (5′-r(GAAAUUAUAAAG)-3′, 5′-r(GAAAGUAUAAAG)-3′, 5′-r(GAUAGUAGAUAG)-3′, 5′-r(GAAAGUAGAAAC)-3′, 5′-r(GCAAAGUAAACG)-3′, 5′-r(GCAUAGCAUACG)-3′, 5′-r(GCAAAGCAAACG)-3′, 5′-r(GCGAAGCCAACG)-3′, 5′-r(GCUCCGCCAACG)-3′, 5′-r(GCGCAGCCAGCG)-3′, and ten additional complementary strands) ranging in GC content from 17 to 83% were purchased from Integrated DNA Technologies (IDT). L-Proline (BioUltra, ≥ 99.5%) was purchased from Sigma. Sodium phosphate monobasic monohydrate, sodium phosphate dibasic, and sodium chloride for sodium phosphate buffer were purchased from Fisher Scientific. All reagents were used without further purification. RNA Duplex Thermal and Isothermal Titration Denaturation. Lyophilized RNA single strands were dissolved in enough phosphate buffer (133 mM sodium chloride, 10 mM sodium phosphate pH 6.9) to yield approximately 50 μM solutions based on the reported number of moles of each single strand from IDT. Exact single strand concentrations were determined by uv-absorbance at 260 nm with extinction coefficients determined from the nearest-neighbor method.28 RNA duplexes were annealed by mixing complementary single strands at a 1:1 mol ratio, heating to 65 °C, and slowly cooling to room temperature. All stock RNA duplex solutions were stored at 4 °C. RNA duplex-proline solutions for thermal and isothermal titration denaturation14 were prepared gravimetrically by massing stock RNA solution, stock proline in phosphate buffer solution, and phosphate buffer solution to maintain constant salt molality (m) with varying proline molality. Density measurements were made with an Anton Paar DMA 5000 densitometer to verify the micromolar concentration of each RNA duplex. Final proline concentrations were 0, 0.25, 0.50,

Kobs =

[S1][S2] θ 2C T = [duplex] 2(1 − θ)

(2)

where CT represents the total concentration of single strands.29 Values of Kobs were determined for θ values between 0.2 and 0.8.13 The m-values for the thermal denaturation experiments were calculated at multiple temperatures. First, the transition temperatures for RNA duplexes in 0.50 m proline were identified at θ = 0.5. The Kobs values for the 0, 0.25, 0.50, 0.75, and 1.0 m proline solutions were then determined at the transition temperature of the duplex in 0.50 m proline and respective θ values. Additionally, Kobs values were determined at five evenly spaced temperatures in the transition region starting with θ = 0.2 (the lowest temperature used in the transition region) with no added proline and θ = 0.8 (the highest temperature used in the transition region) in 1 m proline. These temperature ranges spanned approximately 4 °C for low GC-content duplexes and approximately 2 °C for high GCcontent duplexes. For clarification, Figure 1 contains the fraction of unfolded 5′-r(GCGAAGCCAACG)-3′ total strand as a function of temperature indicating the transition temperature in 0.50 m proline, the temperature at θ = 0.2 in 0 m proline, and the temperature at θ = 0.8 in 1 m proline. Values of lnKobs were averaged from duplicate or triplicate trials and standard errors propagated. Linear regression of B


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probe radius of 1.4 Å and the set of van der Waals radii from Richards.32 In the stacked model, A-form single strands had stacked nucleobases. Starting at the 5′ end of the single strands, the torsion angles about the O3′−P bonds were rotated 120 degrees in UCSF Chimera33 to disrupt base stacking. In the unstacked model, single strands had unstacked nucleobases. In the half-stacked model, the ASA for single strands was calculated by averaging the ASA for stacked and unstacked single strands. The change in solvent accessible surface area for denaturing each RNA duplex into single strands, ΔASA, was calculated for the stacked, half-stacked, and unstacked models by summing the ASA of each single strand and subtracting the ASA of the duplex.

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RESULTS AND DISCUSSION m-Values from Thermal Denaturation. Figure 1 contains a representative plot of the fraction of unfolded single-stranded RNA for the 5′-r(GCGAAGCCAACG)-3′ RNA duplex as a function of temperature obtained from thermal denaturation monitored by absorbance at 260 nm. Proline was a very strong destabilizer of RNA duplexes, in agreement with previous studies.20,25,34,35 The transition temperature at an RNA single strand unfolded fraction of 0.5 in 0 m proline decreased by approximately 5.5 °C to the same unfolded fraction in 1 m proline. Solutes, such as urea and glycine betaine, exhibited transition temperature attenuations of 2.6 °C29 and 2.7 °C,27 respectively, with a 12 base pair RNA duplex with similar GC content. Proline’s strong destabilization capability made proline a candidate as a chemical denaturant to fully unfold RNA duplexes, subject to proline’s solubility limit (next section). The natural logarithm of the unfolding equilibrium constant Kobs at the transition temperature in 0.5 m proline is plotted in Figure 1 as a function of proline molality for three representative RNA duplexes (plots for all RNA duplexes can be found in Figure S1 in Supporting Information). There was no indication of curvature in any of the lnKobs versus proline molality plots for the RNA duplexes. The increase in lnKobs with proline molality for all duplexes indicated net favorable interaction of proline with the RNA surface area exposed upon unfolding. As GC content increased, the slope of lnKobs versus proline molality increased, indicating stronger or more proline interactions with the surface area exposed upon duplex unfolding. To quantify the strength of proline interactions with the RNA surface area exposed after unfolding, m-values from thermal denaturation for each RNA duplex were calculated using eq 1 and the slopes from linear regression of lnKobsversus proline molality (Figure 1). The m-values from thermal denaturation are plotted in Figure 2 as a function of the GC content for each RNA duplex. The RNA-proline m-values from thermal denaturation were all negative indicating favorable proline interactions with the RNA surface area exposed upon unfolding. In comparison to popular solutes such as urea29 and glycine betaine27 with DNA and RNA dodecamer duplexes, proline m-values were more negative and signified a greater dependence of the observed unfolding equilibrium constant Kobs on proline molality than urea or glycine betaine concentration. The m-values from thermal denaturation had a linear dependence on GC content, with larger GC content duplexes having more negative m-values (Figure 2). The higher GC content duplexes had stronger or more interactions between proline and the RNA surface area exposed upon unfolding. The

Figure 1. Top: Representative plot of the fraction 5′-r(GCGAAGCCAACG)-3′ RNA duplex total strand unfolded during thermal denaturation as a function of temperature and L-proline molality. The dashed vertical line intersects the x-axis at the 0.50 m proline transition temperature (where the fraction total strand unfolded was 0.5). Solid vertical lines intersect the x-axis at temperatures corresponding to total strand unfolded fractions of 0.2 in the absence of proline and 0.8 in 1.0 m proline. The solid vertical lines define the temperature range used to measure the duplex unfolding equilibrium constant Kobs. Bottom: Determination of the 5′r(GAAAGUAUAAAG)-3′ (crosses), 5′-r(GCAUAGCAUACG)-3′ (filled circle), and 5′-r(GCGAAGCCAACG)-3′ (open diamond) duplex m-values from the slope of ln Kobs versus proline molality at the duplex transition temperatures in 0.50 m proline.

lnKobs with proline molality was used to calculate RNA duplex m-values (eq 1 with errors from linear regression) at the temperatures of interest. For isothermal titration denaturation, lnKobsvalues for a given duplex were plotted at each proline molality. Linear regression of lnKobs with proline molality was used to determine m-values at the experimental temperature. ASA Calculations. The xleap module in AMBER 1030 was used to construct RNA duplexes in the A-form conformation. The A-form solvent accessible surface area (ASA) of the duplex and two single strands were calculated using naccess31 with a C


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carboxylate oxygen, phosphate oxygen, and cationic nitrogen.6 To use eq 3 in this work, we made the following assumptions: (1) the αi-values for phosphorus, O4′, and O5′ were assigned values of zero because of the small change in the surface areas of these atoms upon unfolding the duplex and there are no corresponding values in Diehl et al.,6 (2) nitrogen atoms in the nucleobase rings were treated as aromatic carbon surface areas except for N1 on guanine and N3 on uracil and thymine which were treated as amide nitrogens (which also have a similar αvalue to aromatic carbon surface area), (3) primary amines on nucleobases were treated as amide nitrogen (not cationic nitrogens) because of their ability to donate and accept hydrogen bonds (regardless, amide and cationic nitrogens have similar α-values), 4) O6 on guanine and O2 and O4 on uracil were treated as amide oxygens, 5) O2 on cytosine was treated as amide oxygen only because it does not qualify as a carboxylate oxygen. For each of the three single strand conformational models considered (stacked, half-stacked, unstacked nucleobases), ΔASA increased by ∼200 Å2 from 17% to 100% GC content (tables for stacked and half-stacked ΔASAmodels are reported elsewhere,27 Table S1 for unstacked ΔASA can be found in the Supporting Information). Much of this increase in ΔASA came from an increase in the primary amine ΔASA with GC content that was not balanced by a reduction in aromatic ΔASA. Figure 2 plots predicted m-values at 25.0 °C as a function of GC content for the RNA duplexes. Predicted m-values were negative, indicating net favorable interactions between proline and the surface area exposed after unfolding the RNA duplexes. The largest contributions to the predicted m-values included very favorable proline interactions with the aromatic nucleobase ring and amide-like surface area, presumably due to cation-π and hydrogen bonding interactions, respectively. Unfavorable contributions to the predicted m-values included proline interactions with aliphatic carbon and carbonyl oxygen surface area. The most negative predicted m-values manifested for the unstacked model because the unstacked single strand conformational model had the largest surface area change upon unfolding. Thus, the ranking of predicted m-values according to unstacked < half stacked < stacked models did not represent stronger interactions of proline with the surface area exposed upon unfolding the duplex, but more proline interactions with the newly exposed surface area. The predicted m-values for all of the three models were nearly independent of GC content, unlike m-values from thermal denaturation. Additionally, if the predicted m-values were accurate, unfolded single strands during thermal denaturation existed somewhere between half-stacked and unstacked single strand conformations. Guinn et al. instead found that single strands of RNA and DNA duplexes existed in conformations between the stacked and half-stacked conformations after isothermal unfolding in urea solutions at room temperature.29 Additionally, the m-value determined from extrapolation of the thermal denaturation mvalues to zero GC content did not fall between the stacked and half-stacked models (Figure 2). It is important to note that the m-values from thermal denaturation were all obtained at temperatures above 25.0 °C while the predicted m-values were all determined at 25.0 °C.6 High GC content duplexes were also destabilized to a greater extent in glycine betaine solutions when destabilization was monitored by thermal denaturation.26,27,36 The greater destabilization of GC-rich relative to AT- or AU-rich nucleic

Figure 2. RNA duplex m-values as a function of GC content from thermal denaturation determined at the duplex transition temperature in 0.5 m proline: 5′-r(GAAAUUAUAAAG)-3′ (plus), 5′-r(GAAAGUAUAAAG)-3′ (cross), 5′-r(GAUAGUAGAUAG)-3′ (open square), 5′-r(GAAAGUAGAAAC)-3′ (filled square), 5′r(GCAAAGUAAACG)-3′ (open triangle), 5′-r(GCAAAGCAAACG)-3′ (hatched circle), 5′-r(GCAUAGCAUACG)-3′ (black-filled circle), 5′-r(GCGAAGCCAACG)-3′ (open diamond), 5′-r(GCUCCGCCAACG)-3′ (filled dash), 5′-r(GCGCAGCCAGCG)-3′ (open dash). Linear regression of m-values from thermal denaturation yielded a 0% GC y-intercept of −0.899 ± 0.039 kcal mol−1 m−1. Open circles represent m-values from isothermal titration at 25.0 °C for the 5′-r(GAAAGUAUAAAG)-3′, 5′-r(GAUAGUAGAUAG)-3′, 5′-r(GCAAAGUAAACG)-3′, 5′-r(GCAUAGCAUACG)-3′ duplexes. The horizontal solid black line represents the average of the m-values from isothermal titration at 25.0 °C (−0.995 ± 0.067 kcal mol−1 m−1). Light, medium, and dark gray circles represent m-values from isothermal titration at 35.0, 45.0, and 55.7 °C (the transition temperature in 0.5 m proline), respectively, for the 5′-r(GCAUAGCAUACG)-3′ duplex. Predicted RNA duplex m-values for stacked (red), half-stacked (green), and unstacked (blue) RNA nucleobases after unfolding calculated using proline interaction parameters from Diehl et al.6

m-values had little dependence on RNA sequence. The two 33% GC and two 50% GC duplexes had very similar m-values given standard errors. Extrapolation of the thermal denaturation data to 0% GC content yielded an m-value of −0.899 ± 0.039 kcal mol−1 m−1. For comparison to the m-values obtained from thermal denaturation, we calculated predicted m-values based on proline interaction potentials (α-values) at 25.0 °C with individual chemical function groups on RNA duplexes and single strands.6 The α-values are normalized by the surface area of a particular functional group i. Therefore, m-values can be predicted by summing the αi-values weighted by the change in solvent accessible surface area ΔASAi of surface type i6,12,29 m‐value = RT ∑ αiΔASA i i

(3)

In eq 3, the contributions of sodium and chloride anions were neglected since the m-values determined in this work represent proline’s interaction with the surface area exposed upon unfolding the RNA duplexes.12,19 The proline αi-values from Diehl et al. are specific to aliphatic carbon, aromatic carbon, hydroxyl oxygen, amide oxygen, amide nitrogen, D


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The Journal of Physical Chemistry B acids was attributed to glycine betaine’s “isostabilizing” ability.26 Glycine betaine’s isostabilizing ability originated from the temperature dependence of glycine betaine interactions with nucleic acid surfaces.27 Since proline m-values obtained from thermal denaturation were obtained at temperatures well above room temperature, any temperature dependence of proline interactions with the unfolded RNA surface area would make comparison of m-values from thermal denaturation and predicted m-values in Figure 2 tenuous at best. The temperature dependence of RNA duplex denaturation is examined in the next two sections. m-Values from Isothermal Titration Denaturation. Since proline was such a strong destabilizer of RNA duplexes as demonstrated with thermal denaturation, isothermal titration denaturation of four RNA duplexes (5′-r(GAAAGUAUAAAG)-3′, 5′-r(GAUAGUAGAUAG)-3′, 5′-r(GCAAAGUAAACG)-3′, and 5′-r(GCAUAGCAUACG)-3′) was accomplished by preparing a series of solutions of constant RNA concentration with increasing molality of proline and monitoring the absorbance at 260 nm and 25.0 °C. Analysis of the isothermal titration denaturation absorbance curves generated the fraction of RNA single strands as a function of proline molality (Figure 3). This analysis was limited to RNA duplexes with GC content less than 50%. RNA duplexes with GC content greater than 50% proved too robust and conversion to single strands could not be accomplished prior to the solubility limit of proline at 25.0 °C. In general, the RNA duplex unfolding transition region shifted to greater proline concentration with increasing GC content. Figure 3 also contains plots of lnKobs as a function of proline molality calculated with the fraction of unfolded single strands between 0.2 and 0.8.13 Slopes of lnKobs versus proline molality from linear regression were used to calculate m-values at 25.0 °C for the four RNA duplexes (eq 1). Figure 2 plots the m-values from isothermal titration at 25.0 °C as a function of GC content. Given standard errors, the mvalues from isothermal titration were independent of GC content. Since the m-values were independent of GC content, in agreement with predicted m-values, and the m-values for each duplex were determined over different ranges of proline molality (Figure 3), isothermal titration m-values were also independent of proline concentration. The m-values from isothermal titration agreed with predicted m-values assuming half-stacked nucleobases in the unfolded single strands, although Guinn et al. demonstrated the unfolding of DNA and RNA duplexes in urea solutions comprised unfolded single strand conformations between stacked and half-stacked conformations.29 The discrepancy may have arisen from the assumptions made in extending proline interaction potentials assessed with amino acid model compounds to nucleic acids.6 Additionally, the high concentrations of proline used in isothermal titration may have promoted less stacking in the single strands and increased duplex ΔASA. However, the isothermal titration proline m-values from this work were nearly independent of GC-content, in agreement with the unfolding of DNA and RNA duplexes in urea solutions29 and predicted proline m-values (Figure 2). Proline m-values from isothermal denaturation at 25.0 °C and thermal denaturation were in agreement only for RNA duplexes with 25% GC or less. The m-values from thermal denaturation became more negative than those from isothermal denaturation at 25.0 °C as the GC content increased (Figure 2).

Figure 3. Top: Fraction 5′-r(GAAAGUAUAAAG)-3′ (×), 5′r(GAUAGUAGAUAG)-3′ (open squares), 5′-r(GCAAAGUAAACG)-3′ (triangles), and 5′-r(GCAUAGCAUACG)3′ (filled circles) RNA single strands unfolded during isothermal titration denaturation monitored by uv-absorbance as a function of Lproline molality at 25.0 °C. Bottom: Determination of the duplex mvalues from the slope of ln Kobs versus proline molality at 25.0 °C.

To investigate whether isothermal titration and thermal denaturation m-values might agree at higher temperatures, isothermal titration denaturation with proline was performed on the 50% GC duplex 5′-r(GCAUAGCAUACG)-3′ at 35.0, 45.0, and 55.7 °C (the transition temperature in 0.5 m proline). The fraction of unfolded 5′-r(GCAUAGCAUACG)-3′ duplex single strands is shown in Figure 4 as a function of temperature and proline molality. As the temperature was increased, the unfolding transition region shifted to lower proline molality. Figure 4 also contains isothermal plots of lnKobs as a function of proline molality calculated with the fraction of unfolded single strands between 0.2 and 0.8. The m-values for the 5′r(GCAUAGCAUACG)-3′ duplex calculated from linear regression of lnKobs with proline molality (eq 1) are plotted E


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discrepancy in m-values between the two denaturation techniques. We did not study a 100% AU duplex due to potential end fraying at room temperature.14,37 However, the predicted m-value for a 100% AU duplex from extrapolation of the thermal denaturation data in Figure 2 (−0.899 ± 0.039 kcal mol−1 m−1) was in excellent agreement with the average mvalue from isothermal titration denaturation (−0.995 ± 0.067 kcal mol−1 m−1) at 25.0 °C. Additionally, as the isothermal titration denaturation working temperature was increased, agreement between isothermal titration and thermal denaturation m-values was achieved for the 5′-r(GCAUAGCAUACG)3′ duplex (Figure 2). Figure 5 plots m-values from thermal denaturation as a function of temperature for the RNA duplexes. For each duplex,

Figure 5. L-Proline m-values for the ten RNA duplexes as a function of temperature. For each duplex, m-values were determined over a temperature range corresponding to an unfolded strand fraction of 0.2 in the absence of proline (smallest temperature) and 0.8 in 1.0 m proline (highest temperature). According to eq 4, linear regression ° /dm3 = 2.83 ± 0.13 kcal mol−1 m−1 and dΔSobs ° /dm3 = yielded dΔHobs 0.0129 ± 0.0004 kcal mol−1 K−1 m−1.

Figure 4. Top: Fraction 5′-r(GCAUAGCAUACG)-3′ RNA duplex total strand unfolded during isothermal titration denaturation monitored by uv-absorbance as a function of L-proline molality at 25.0 °C (open circles), 35.0 °C (light gray circles), 45.0 °C (medium gray circles), and 55.7 °C (dark gray circles). Bottom: Determination of the 5′-r(GCAUAGCAUACG)-3′ m-values from the slope of ln Kobs versus proline molality at 25.0, 35.0, 45.0, and 55.7 °C.

m-values were determined over a temperature range corresponding to an unfolded strand fraction of 0.2 in the absence of proline (smallest temperature) and 0.8 in 1.0 m proline (highest temperature) as demonstrated in Figure 1. Figure S2 in Supporting Information contains plots of lnKobs versus proline molality for each of the duplexes at the temperatures in Figure 5. Linear regression of lnKobs with proline molality was used to determine the m-values in Figure 5. Except for the 17% GC duplex, the m-values for all RNA duplexes decreased with temperature and indicated the change in m-value was due to temperature and not GC content. The degree of nucleobase stacking in the RNA single strands was acknowledged as a potential contributor to the magnitude of the m-values obtained from thermal denaturation since mvalues are proportional to ΔASA. To investigate the dependence of stacking on proline concentration during thermal denaturation, hyperchromicities for each of the RNA duplexes in Figure 5 were calculated from the change in absorbance in the thermal denaturation transition region, normalized by the RNA duplex concentration, and plotted as a function of GC content in Figure S3.27 Similar hyperchromicity magnitudes

in Figure 2. As the temperature increased, the m-value of the 5′r(GCAUAGCAUACG)-3′ duplex from isothermal titration denaturation decreased (became more negative). There was near exact agreement with the m-values obtained from thermal and isothermal titration denaturation evaluated at 55.7 °C. Destabilization of RNA Duplexes by Proline Is Entropy Driven. The m-values for glycine betaine interaction with RNA have been shown to be temperature dependent, indicating glycine betaine interactions with biopolymer surface areas are temperature dependent.27 We argue a similar mechanism accounts for the discrepancy between m-values obtained from isothermal titration denaturation at 25.0 °C and thermal denaturation. All duplexes studied in this work had transition temperatures greater than 25.0 °C. Those duplexes with the lowest transition temperatures (AU-rich) had the best agreement between m-values from thermal denaturation and isothermal titration at 25.0 °C while those duplexes with the largest transition temperatures (GC-rich) had the largest F


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Figure 6 plots dΔH°obs/dm3 and TdΔS°obs/dm3 as a function of temperature. Our analysis assumed no temperature dependence

and GC dependence for RNA duplexes have been reported elsewhere.27 The hyperchromicity for each RNA duplex had a weak dependence on proline concentration in the range of 0 to 1 m and we therefore anticipated proline had little effect on nucleobase stacking during thermal denaturation. We also recognized the degree of nucleobase stacking could have depended on transition temperature (which was a function of GC content). Previous work demonstrated that a 66% DNA single strand was approximately 75% stacked at 41 °C, but the stacking dropped to 52% at 80 °C.38 However, RNA duplex mvalues with urea were best predicted assuming 60−70% stacking after thermal denaturation except for a 100% GC duplex that was predicted to have almost 100% stacking after thermal denaturation.29 Lambert and Draper found that urea mvalues were best predicted when the stacked nucleobase conformation was used for RNA single strands with a variety of GC contents.39 These studies suggested RNA single strands of different GC content did not possess markedly different degrees of nucleobase stacking at their different transitions temperatures. If the dependence of m-values with temperature was solely due to less stacking in the higher GC content duplexes, the ΔASA of the 83% GC duplex would have to be nearly twice as big as the 17% GC duplex based on the near doubling of the m-values from thermal denaturation. Previous work does not support this degree of unstacking in higher GC content duplexes. While we cannot ascertain the exact contribution of temperature dependent nucleobase unstacking to thermal denaturation m-values, previous work suggested it was not a major contributor. Using the definition of the m-value from eq 1, the m-value can also be written as m‐value =

◦ ◦ dΔHobs dΔSobs −T dm3 dm3

Figure 6. dΔHobs ° /dm3 (blue), TdΔSobs ° /dm3 (red), and errors (dotted lines) as functions of temperature.

of dΔH°obs/dm3 and dΔS°obs/dm3. While the average dΔH°obs/ dm3 value was constant, TdΔS°obs/dm3 increased with temperature. The mechanism of the greater destabilization of GC-rich RNA duplexes with proline becomes clear. Higher GC content RNA duplexes unfolded at higher transition temperatures. As the difference between dΔH°obs/dm3 and TdΔS°obs/dm3 values increased with transition temperature, the m-value for proline interaction with the RNA surface area exposed upon unfolding decreased (became more negative). The dependence of the mvalue on temperature in Figure 5 was a result of proline interactions that were entropically driven. Since isothermal mvalues at 25.0 °C were GC independent (Figure 2), dΔHobs ° / dm3 and dΔS°obs/dm3 components can be used to temperature correct proline m-values for biopolymer conformational changes. Glycine betaine interactions with biopolymer surface areas have also been shown to be temperature dependent. Felitsky et al. demonstrated that glycine betaine stabilization of the lacI HTH binding domain was also entropically driven.40 Since lacI HTH was stabilized in glycine betaine solutions, glycine betaine had a net unfavorable interaction with the lacI HTH surface area exposed after unfolding. dΔS°obs/dC3(where C3 is the molarity of glycine betaine) was negative for glycine betaine interaction with the unfolded lacI HTH surface area with dΔH°obs/dC3 ≈ 0. The glycine betaine m-values for lacI HTH unfolding therefore increased with temperature. In contrast, glycine betaine destabilized RNA duplexes, but not to the extent as proline.27,41 Glycine betaine m-values were approximately 20−50% of proline m-values with RNA dodecamer duplexes.27 dΔH°obs/dm3 values for unfolding RNA duplexes in glycine betaine solutions were positive with corresponding positive dΔSobs ° /dm3 values such that the m-value decreased with temperature.27 The numerical signs of dΔH°obs/dm3 and dΔS°obs/dm3 for both proline and glycine betaine were the same, indicative of a general mechanism of zwitterion amino acid interaction with nucleic acid solvent accessible surface areas. It is the interactions of solutes with specific functional groups on the biopolymer surface area exposed upon unfolding that determined the net dΔHobs ° /dm3 and dΔSobs ° /dm3 values. The stabilization of lacI HTH with glycine betaine can be attributed to unfavorable glycine betaine interactions with amide oxygens within the amide backbone exposed upon unfolding.5,6 RNA duplex destabilization was driven by very favorable glycine betaine interactions with the aromatic nucleobases through

(4)

where dΔHobs ° /dm3 represents the dependence of the observed unfolding enthalpy change on proline molality and dΔSobs ° /dm3 represents the dependence of the observed unfolding entropy change on proline molality. Linear regression of the data in Figure 5 (with temperature units in Kelvin) yielded dΔH°obs/ dm3 = 2.83 ± 0.13 kcal mol−1 m−1 and dΔSobs ° /dm3= 0.0129 ± 0.0004 kcal mol−1 K−1 m−1. Both of these thermodynamic quantities should be considered average values since they were obtained on the set of the ten RNA duplexes. A positive dΔHobs ° /dm3 value indicated an increase in the average unfolding enthalpy change with proline molality and agreed with dΔHobs ° /dm3 obtained from analysis of the d2 lnKobs/d(1/ T)dm3 derivatives for each duplex (data not shown).27 Glycine betaine also exhibited a positive correlation in the unfolding enthalpy change with glycine betaine molality.27 The increase in unfolding enthalpy with proline molality was not large; the average unfolding enthalpy change for the ten duplexes was approximately 90 kcal mol−1.27 At 1 molal proline, the expected increase in RNA duplex unfolding enthalpy change was approximately 3%. The temperature dependence of the m-value was contained within TdΔSobs ° /dm3 (eq 4). The dΔSobs ° /dm3 value was positive (Figure 5), so the average unfolding entropy change increased with proline molality. Since −dΔS°obs/dm3 was the slope of the m-value versus temperature (eq 4), the m-value for proline interactions with the RNA duplex surface area exposed upon unfolding decreased (became more negative) with temperature. G



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cation-π interactions.27 Proline exhibited unfavorable interactions with oxygen surface area, but not to such a large extent as glycine betaine.6 The very favorable interaction of proline with aromatic, amide-like nitrogen, and amine surface area on the nucleobases exposed upon unfolding drove RNA destabilization in proline solutions. The fact that dΔHobs ° /dm3 was positive did not imply the unfolded state in proline solutions possessed weaker or less bonding interactions relative to the unfolded state in the absence of proline. Since the m-values measured in this work were specific to proline interactions with the RNA surface area exposed upon unfolding, we had no information regarding the enthalpy of the folded state in proline solutions relative to proline-free solutions. A positive dΔH°obs/dm3 was simply interpreted as a greater reduction in bonding interactions between the unfolded and folded states in proline solutions relative to proline-free solutions. Greater amounts of water, ion, or proline released from the duplex surface area upon unfolding or water released with hydrated proline-unfolded RNA surface area interactions could have accounted for a positive dΔH°obs/ dm3 value. In addition, proline interactions had a minor disruption on nucleobase stacking in the unfolded RNA single strands in a proline concentration dependent manner. Similarly, a positive dΔSobs ° /dm3 value was interpreted as a greater gain in entropy between the unfolded and folded states in proline solutions relative to that for proline-free solutions. Proline interactions with the RNA duplex and unfolded surface areas could have resulted in water, ion release, or disrupted nucleobase stacking to ensure dΔS°obs/dm3 > 0.

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Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b03608. Plots of the natural logarithm of the observed equilibrium constant from thermal denaturation versus proline molality at the duplex transition temperatures for all RNA duplexes; table of unstacked RNA accessible surface area values for different surface area types; Plot of hyperchromicity from thermal denaturation as a function of GC content and proline molality (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]; Telephone: (507) 786-3105. ORCID

Jeffrey J. Schwinefus: 0000-0002-8212-9902 Author Contributions ‡

N.L.B., K.M., K.N.B., S.L., L.R.H., and R.J.M. contributed equally to this work. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by National Institutes of Health grant R15-GM093331 to J.J.S. and a Howard Hughes Medical Institute grant to St. Olaf College. K. Modi was supported by a grant from the Dreyfus Foundation.

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ABBREVIATIONS ASA, solvent accessible surface area; AT, adenosine−thymine base pair; AU, adenosine−uracil base pair; GC, guanine− cytosine base pair

CONCLUSIONS

Proline destabilization of RNA duplexes was temperature dependent. Proline interaction values6 with coarse-grained surface area types predicted m-values in reasonable agreement with m-values from isothermal titration denaturation at 25.0 °C, but failed at higher temperatures as proline interactions became more favorable. A minor contribution to m-values from potential nucleobase unstacking in RNA single strands at higher temperatures was not ruled out. Similar to the solute glycine betaine, the unfolding enthalpy and entropy change for RNA duplexes in proline solutions increased with proline concentration. The m-value decreased (became more negative) with temperature, indicating that the temperature dependence of proline interactions with the RNA solvent accessible surface exposed upon unfolding was entropically driven. Since the m-value is proportional to the change in solvent accessible surface area of a biopolymer during a conformational change, m-values provide a method to gauge the magnitude of the surface area change and the chemical types of surface area exposed. However, not all m-values are experimentally accessible at ambient temperature. The temperature dependence of the proline m-value from this work can be used to temperature correct m-values of other biopolymer systems (along with a ratio of the change in solvent accessible surface area of the biopolymer under investigation to that for the RNA duplexes used in this study).40,41 At the very least, this work will serve to caution comparison of surface areas determined from kinetic rate or observed equilibrium constants in proline or other solute solutions at different temperatures.

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l-Proline and RNA Duplex m-Value Temperature Dependence - The

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