Functional Equilibrium of the KcsA Structure Revealed by NMR*

Background: The selectivity filter of KcsA undergoes an equilibrium between permeable and impermeable conformations under acidic conditions. Results: Truncation of the intracellular region or addition of 2,2,2-trifluoroethanol modulates the equilibrium. Conclusion: Membrane environments affect dynamics of KcsA. Significance: This is the first evidence that a structural equilibrium in the membrane is related to the inactivation of a potassium channel. KcsA is a tetrameric K+ channel that is activated by acidic pH. Under open conditions of the helix bundle crossing, the selectivity filter undergoes an equilibrium between permeable and impermeable conformations. Here we report that the population of the permeable conformation (pperm) positively correlates with the tetrameric stability and that the population in reconstituted high density lipoprotein, where KcsA is surrounded by the lipid bilayer, is lower than that in detergent micelles, indicating that dynamic properties of KcsA are different in these two media. Perturbation of the membrane environment by the addition of 1–3% 2,2,2-trifluoroethanol increases pperm and the open probability, revealed by NMR and single-channel recording analyses. These results demonstrate that KcsA inactivation is determined not only by the protein itself but also by the surrounding membrane environments.


KcsA is a tetrameric K ؉ channel that is activated by acidic pH.
Under open conditions of the helix bundle crossing, the selectivity filter undergoes an equilibrium between permeable and impermeable conformations. Here we report that the population of the permeable conformation (p perm ) positively correlates with the tetrameric stability and that the population in reconstituted high density lipoprotein, where KcsA is surrounded by the lipid bilayer, is lower than that in detergent micelles, indicating that dynamic properties of KcsA are different in these two media. Perturbation of the membrane environment by the addition of 1-3% 2,2,2-trifluoroethanol increases p perm and the open probability, revealed by NMR and single-channel recording analyses. These results demonstrate that KcsA inactivation is determined not only by the protein itself but also by the surrounding membrane environments.
KcsA is a prokaryotic K ϩ channel from Streptomyces lividans and functions as a tetramer. The crystal structures of the wild type and mutants of KcsA (1)(2)(3)(4)(5), together with their electrophysiological properties (3,6), revealed that two "gates" are located along the K ϩ permeation pathway on the 4-fold symmetry axis of the tetramer (Fig. 1A): the selectivity filter on the extracellular side and the helix bundle crossing on the intracellular side. The former is responsible for the selective permeation of K ϩ against other ions, and the latter interferes with K ϩ permeation. These fundamental architectures are widely conserved among the pore domains of the eukaryotic voltage-dependent (K v ) and inwardly rectifying (K ir ) K ϩ channels (7).
KcsA is a pH-dependent K ϩ channel whose helix bundle crossing is closed at neutral pH and open at acidic pH (6, 8 -11). In macroscopic current analyses, in which many KcsA channels are reconstituted in an asolectin bilayer (9,12), no current is observed at neutral pH. When the intracellular pH is dropped to around 3, a peak K ϩ current and the following exponential decay on a time constant of seconds are observed. Eventually, the current reaches a plateau where the current is only 10 -15% of the peak current. This process, called activation-coupled inactivation, is also observed in C-type inactivation for many K v channels upon depolarization of the membrane (13). On the basis of the structural similarity of the pore domains, the inactivation of KcsA is assumed to be caused by a similar mechanism to that of the C-type inactivation of K v channels.
The activation-coupled inactivation cannot be explained by the opening and closing of the helix bundle crossing because it is kept open under the acidic condition where the exponential decay of the current is observed. The single-channel current analyses revealed that KcsA undergoes an equilibrium between the permeable and impermeable states at acidic pH (6,14). The equilibrium is affected by mutations near the selectivity filter (3), suggesting that the selectivity filter is responsible for the equilibrium. In a previous study, we demonstrated that the selectivity filter of KcsA reconstituted in n-dodecyl-␤-D-maltoside (DDM) 2 micelles undergoes a structural equilibrium between two distinct conformations under the acidic condition that correspond to the permeable and impermeable states (15). Under acidic conditions, where the helix bundle crossing is fully open, the conductivity of the channel in the steady state is determined by the population of the permeable conformation (p perm ) of the selectivity filter. Therefore, identification of factor(s) that determine the populations of the permeable and impermeable conformations of the selectivity filter is important to understand the inactivation mechanism. * This work was supported by grants from the Japan New Energy and Indus- Recently, deletion of a C-terminal intracellular region of a bacterial Na ϩ channel, NavSulP, which forms a tetrameric helix bundle like KcsA, was reported to decrease the tetrameric stability and the inactivation rate (16), suggesting that the stability of the tetramer is correlated with the inactivation rate. It is also reported that deleting the C-terminal intracellular region of KcsA increases both the rate and extent of inactivation (12), although the effect on the inactivation rate is apparently opposite. Because the C-terminal intracellular region of KcsA also stabilizes the tetrameric assembly by forming a bundle in the closed conformation at neutral pH ( Fig. 1A) (2), there is a possibility that the stability of the tetramer under acidic conditions is correlated with the structural equilibrium between the permeable and impermeable conformations of the selectivity filter mentioned above.
In this report, we investigated the effect of the surrounding environment of KcsA on its structural equilibrium and channel function by SDS-PAGE, NMR, and single-channel recording of the K ϩ -current.

EXPERIMENTAL PROCEDURES
Sample Preparation-All KcsA-truncated mutants, except for KcsA125, were constructed by PCR mutagenesis using the wild-type plasmid (15) as the template. The mutations were confirmed by DNA sequencing (supplemental Fig. S1). The wild-type and truncated KcsA proteins were expressed and purified as reported previously (15). KcsA125 was obtained by cleaving the C-terminal intracellular region of wild-type KcsA by chymotrypsin. The plasmid for the expression of the membrane scaffold protein MSP1 was constructed according to the literature (17)(18)(19). The BL21 (DE3) Codon Plus RP Escherichia coli cells were transformed with the plasmid and cultured in Terrific Broth medium at 37°C. Protein expression was induced by the addition of 1 mM isopropyl 1-thio-␤-D-galactopyranoside when the OD 600 reached 1.5. After 3 h of induction, cells were harvested and sonicated in a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, and 150 mM KCl. After centrifugation, the precipitant was solubilized in the buffer containing 10% (v/v) Triton X-100 and a protease inhibitor mixture for 2 h at 4°C. After centrifugation, the supernatant was applied to HIS-Select resin (Sigma). The resin was washed extensively with the buffer containing 1% (v/v) Triton X-100, followed by the buffer containing 50 mM sodium cholate and the buffer containing 20 mM imidazole. The protein was eluted by the buffer containing 500 mM imidazole and was dialyzed against buffer containing 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 1 mM DTT. The His tag was cleaved by Tobacco Etch Virus protease and was removed by passage through HIS-Select resin.
Reconstitution of KcsA in rHDL-Reconstitution of KcsA into rHDL was conducted according to a previous report (19), with some modifications. Delipidations of MSP1 and KcsA were confirmed by 31 P 1D NMR spectroscopy. Dimyristoylphosphatidylcholine and dimyristolyphosphatidylglycerol were mixed at a molecular ratio of 3:1 and dissolved in chloroform. The solvent was dried with nitrogen gas followed by in vacuo to form a lipid film. The film was solubilized with buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 150 mM KCl, and 50 mM sodium cholate. After sonication, MSP1 was added to a final concentration of 200 M. After an incubation for 2 h at room temperature with gentle mixing, KcsA was added to achieve a final molar ratio of KcsA, MSP1, lipids, and sodium cholate of 1:20:800:1600 (19). After incubating the solution at room temperature for 2 h, 50% (w/w) of Biobeads (Bio-Rad), which adsorb detergents, was added in a stepwise manner, and the mixture was incubated at room temperature overnight with gentle mixing. The supernatant was concentrated with an ultrafiltration device (MWCO 10 K), and the KcsA reconstituted in rHDL was purified by size exclusion chromatography using Superdex200 5/50 GL (GE Healthcare).
Stability of the Tetramer-The stabilities of the tetramers were compared by an SDS-PAGE analysis. Samples with and without an incubation at 90°C for 30 min at pH 3.2 were centrifuged and applied to the SDS-PAGE gel (15%). After electrophoresis and Coomassie Brilliant Blue staining, the intensities of the bands were measured by densitometry using ImageJ software (20). The stability of each tetramer was quantified as the ratio of the intensity of the band obtained with the incubation over that of the band without the incubation.
NMR Spectroscopy-All NMR spectra were recorded in buffer containing 10 mM K 2 HPO 4 and 180 mM KCl in 100% D 2 O at the indicated temperatures and pHs. All experiments were performed on a Bruker Avance 800 MHz spectrometer equipped with a cryogenic probe. Methyl-TROSY spectra were acquired as reported previously (15). 13 C ZZ exchange spectra were acquired and analyzed as described previously (21). The signal intensities of the four peaks from V76 ␥(B) (namely, the auto peak of the permeable conformation, I pp ; that of the impermeable conformation, I ii ; the cross peak from the permeable to the impermeable conformations, I pi ; and that from the impermeable to the permeable conformations, I ip ) were acquired with eight or nine mixing times, T. All data points were simultaneously fitted to the theoretical equation where 1,2 , x p and x i are defined according to the following relationships k ip , the longitudinal relaxation rate in the permeable conformation, R 1p , and that in the impermeable conformation, R 1i , were set as variants. I p (0) and I i (0) denote the amounts of longitudinal carbon magnetization associated with the permeable and impermeable conformations at the start of the mixing period. Errors were estimated from 500 Monte Carlo simulations using the uncertainties in the peak intensities.
Single-channel Current Recording-Single-channel currents were measured by the planar lipid bilayer technique. KcsA was reconstituted into liposomes as described previously (22). Two chambers (cis and trans), divided with a plate with a hole (50 -100 m in diameter), were used for the analyses. The cis and trans chambers were asymmetrically buffered by 10 mM HEPES (pH 7.5) and 10 mM succinic acid (pH 4.0), respectively, in the presence of 200 mM KCl. The reference electrode was placed in the cis chamber, to which the reconstituted channels were added. The current data were recorded through the low-pass filter (2 kHz for the cutoff frequency, Axopatch 200B amplifier, Molecular Devices, Sunnyvale, CA), and the data were sampled at 5 kHz (Digidata 1322A digitizer and pCLAMP software, Molecular Devices, Sunnyvale, CA). To investigate the effect of TFE, equivalent amounts of TFE were added to the cis and trans chambers to the desired final concentrations.
The open probability (p open ) was evaluated by the following method. The all point histogram was drawn from a current trace at the steady state and the Gaussian fit was performed to get the relative frequency of each conductance level. In the case of multiple channel-containing membranes, the open probability was calculated from the relative frequencies by using the binomial distribution function. We prepared the full-length KcsA (hereafter referred to as KcsA160, after the residue number at the C terminus) and the KcsA variants with truncated C-terminal intracellular regions (KcsA134, KcsA132, KcsA130, KcsA128, and KcsA125) (Fig.  1A). All of the KcsA variants migrated as tetramers in the SDS-polyacrylamide gel, indicating that they formed stable tetramers at room temperature under acidic conditions (Fig.  1B). After an incubation at pH 3.2 and 90.0°C for 30 min, more than 70% of the KcsA160 tetramer survived, whereas about 50% of the KcsA134, KcsA132, KcsA130, and KcsA128 tetramers and 20% of the KcsA125 tetramer survived, indicating that the C-terminal intracellular region is responsible for the stabilization of the tetrameric assembly under acidic conditions (Fig. 1C).

Correlation of the Population of the Permeable
The methyl-TROSY spectra of these KcsA variants with {u-2 H, Leu/Val-[ 13 CH 3 , 12 CD 3 ]} labeling were acquired at pH 3.2 and 40.0°C in the presence of 50 mM K ϩ ( Fig. 2A), in which the populations of the permeable and impermeable conformations are reflected as the relative intensities of the separate signals. Although the chemical shifts for the signals from one of the prochiral methyl groups of Val-76 in the selectivity filter, V76 ␥(B) (15), were identical among these variants, the p perm values largely varied. The p perm values were 67% for KcsA160, 48% for KcsA134, 32% for KcsA132, 27% for KcsA130, 30% for KcsA128, and 12% for KcsA125 ( Fig. 2A).
Plots of the tetrameric stability of each variant (Fig. 1C) against p perm ( Fig. 2A) indicated that the tetrameric stability positively correlates with the p perm values under acidic conditions (Fig. 2B). The coefficient of determination, R 2 , value of the correlation was 0.82.
The C-terminal Intracellular Region Stabilizes the Permeable Conformation of the Selectivity Filter under Acidic Conditions-Despite the positive correlation between the tetrameric stability of the C-terminally truncated variants and their p perm values, it is not clear whether the role of the C-terminal region is to enhance the transition from the impermeable to the permeable conformation of the selectivity filter or suppress the opposite transition or both. Here, by using KcsA160 and KcsA125, we investigated the transition rates between the permeable and the impermeable conformations, which correspond to the inverse of the averaged life times of each conformation.
First, a 13 C ZZ exchange spectrum (21) for {u-2 H, Leu/Val-[ 13 CH 3 , 12 CD 3 ]}-KcsA160 was acquired with a mixing time of 250 ms, at pH 3.2 and 40.0°C, in the presence of 50 mM K ϩ (Fig.  3A), where the populations of the permeable and impermeable conformations are comparable. The cross peaks that are derived from the exchange process between the permeable and impermeable conformations during the mixing time were clearly observed for V76 ␥(B). The dependence of the signal intensities on the mixing time was fitted to the theoretical equation (see "Experimental Procedures" for details), resulting in the transition rate from the permeable to the impermeable conformation, k pi , of 0.46 Ϯ 0.02 s Ϫ1 , and the transition rate in the opposite direction, k ip , of 0.94 Ϯ 0.04 s Ϫ1 , respectively (Fig. 3B).
The 13 C ZZ exchange experiments were then performed for {u-2 H, Leu/Val-[ 13 CH 3 , 12 CD 3 ]}-KcsA125. Under the same conditions as those for KcsA160, k pi and k ip were 7.1 Ϯ 0.1 and 0.96 Ϯ 0.02 s Ϫ1 , respectively (Fig. 3B). The k pi value of KcsA125 is more than 10 times higher than that of KcsA160, whereas the k ip values are comparable for the two variants. Because the transition rate, k pi , is the inverse of the lifetime of the permeable conformations of the selectivity filter, the smaller k pi value of KcsA160 indicates that the C-terminal intracellular region indirectly stabilizes the permeable conformation of the selectivity filter under acidic conditions, where the selectivity filter is in the structural equilibrium between the permeable and impermeable conformations, and the helix bundle crossing is fully open.
Membrane Environments Affect the Structural Equilibrium of the Selectivity Filter of KcsA-At neutral pH, the tetrameric assembly of KcsA is influenced by whether it is reconstituted in   NOVEMBER 16, 2012 • VOLUME 287 • NUMBER 47 detergent micelles or in the lipid bilayer (23). We then investigated the p perm values in the lipid bilayer under acidic conditions by embedding KcsA in rHDL, in which the KcsA reconstituted in the lipid bilayer is surrounded by membrane scaffold proteins to form a disc-shaped particle (17,19).

Functional Equilibrium of the KcsA Structure Revealed by NMR
We acquired the methyl-TROSY spectrum of {u-2 H, Leu/ Val-[ 13 CH 3 , 12 CD 3 ]}-KcsA160 reconstituted in rHDL at pH 3.2 and 45.0°C in the presence of 200 mM K ϩ (supplemental Fig.  S2). Compared with the spectra recorded in the DDM micelles, small but significant differences in the chemical shifts were observed, not only for the residues exposed to lipid bilayer in rHDL but also for the residue of the selectivity filter, Val-76, and those peripheral to the selectivity filter, Leu-59 and Leu-81 (supplemental Fig. S2). Furthermore, the signals from the permeable and impermeable conformations were simultaneously observed for Val-76 and Leu-59. These results indicate that the overall folds and/or the tetrameric assembly in rHDL and DDM micelles are slightly different. The most striking differences are the p perm values. In rHDL, the p perm value is about 20%, whereas it is almost 100% in DDM micelles under the same conditions (15). These results clearly indicate that the surrounding environments affect the conformations and their equilibria of KcsA.
TFE Shifts the Structural Equilibrium of KcsA-In the lipid bilayer, the tetrameric stability of KcsA is reportedly perturbed by the surrounding membrane environments such as lipid composition (23) and partition of alcohol, such as TFE (24,25). We then acquired the methyl-TROSY spectra of KcsA160 in rHDL upon sequential additions of TFE (Fig. 4) to investigate the effect of modulating the surrounding membrane environment on the structural equilibrium under acidic conditions. When TFE was added to final concentrations of 1 and 2%, the intensities of the impermeable conformation signals that are predominant in the absence of TFE decreased, whereas those of the permeable conformation increased, clearly indicating the shift of the structural equilibrium from the impermeable to the permeable conformation. Surprisingly, upon further addition of TFE to final concentrations of 3 and 4%, the signals of the permeable conformation of the selectivity filter decreased, and the signals that correspond to the closed conformation, which are observed only under the neutral condition without TFE, emerged, although the pH of the sample was kept unchanged at pH 3.2. The other signals did not exhibit large chemical shift changes upon the addition of TFE, suggesting that TFE does not directly interact with KcsA and that the global fold of KcsA is not affected by TFE in the concentration range below 4%.
The Structural Equilibrium Shift Induced by TFE Is Reflected in the Channel Activity-We performed electrophysiological experiments to investigate whether the addition of TFE affects the open probability of KcsA. Here, KcsA160 was reconstituted into a planar lipid bilayer, and single channel currents were recorded in symmetrical 200 mM KCl solutions. The bath solutions were made asymmetric in terms of pH (pH 7.5 for the cis side, pH 4.0 for the trans side). Because one of the two gates, the helix bundle crossing on the intracellular side opens when the intracellular pH is acidic, the gate of the channels oriented with the intracellular region facing the trans side is constitutively open, and the gate of the channels oppositely oriented is closed (22). Therefore, the currents observed here are caused by opening of another gate, the selectivity filter, of the channels oriented with the intracellular region facing the trans side.
As reported previously, KcsA160 exhibited the K ϩ current at ϩ200 mV in the planar lipid bilayer of 1-palmitoyl-2oleoylphosphatidylethanolamine:1-palmitoyl-2-oleoylphosphatidylglycerol ϭ 3:1, where the open probability (p open ) was very low (ϳ1%) (Fig. 5A, top panel) (22). When equal amounts of TFE were added to the cis and trans sides to a final concentration of 3%, by 0.5 per step, the channel gradually increased its activity (Fig. 5A). The current trace became noisy, and spikes were frequently observed for which the defined conductance level could not be resolved even with the expanded traces (Fig. 5A, right panels). At 3% TFE, the channel stayed at the open state for a longer time, and the open probability increased dramatically up to 60% (Fig. 5, A  and C). Under acidic pH conditions, the activation gate of the helix bundle crossing fully opened, and the low p open value in the absence of TFE represents the infrequent opening of the selectivity filter. Thus, these results indicate that the conformational equilibrium of the selectivity filter is shifted toward the conductive form by TFE.
The single-channel amplitude was slightly but substantially decreased as the concentration increased (Fig. 5B). It is likely that TFE is located at the membrane interface where the dipole potential of the membrane is modified so that the local K ϩ concentration is depleted.
Although the analysis could not be conducted with a TFE concentration higher than 3% because of the instability of the planar lipid bilayer, these results strongly support the NMR results, in which a comparable concentration of TFE increased the population of the permeable conformation (p perm ).  (Fig. 1A) are shown. P, I, and C stand for the signals from the permeable, impermeable, and closed conformations, respectively.

DISCUSSION
In this study, we first demonstrated that the C-terminal intracellular region of KcsA increases the thermal stability of the tetrameric assembly (Fig. 1, B and C), suggesting that the C-terminal intracellular region also forms a helix bundle under acidic conditions, which is probably similar to the crystal structure in the closed state under neutral conditions (A).
The C-terminal intracellular region also contributes to the increase in the population of the permeable conformation, p perm ( Fig. 2A), of the selectivity filter under acidic conditions, which positively correlates with the increase in the thermal stability of the tetramer (B). The transition rates between the permeable and impermeable conformations indicated that the increased p perm by the C-terminal intracellular region is due to the stabilization of the permeable conformation and not to the destabilization of the imperme-able conformation (Fig. 3). Conversely, the truncation of the C-terminal intracellular region destabilizes the permeable conformation, increasing the population of the impermeable conformation. These results suggest that the population of the permeable conformation is increased by the "bundling" effect of the C-terminal intracellular region, which would make the transmembrane region more compact and tightly associated, and that the transmembrane region is relatively loose in the impermeable conformation as compared with that in the permeable conformation.
We revealed previously that the selectivity filter with the permeable conformation is in the K ϩ -bound, H 2 O-unbound state, whereas that with the impermeable conformation is in the K ϩ -unbound, H 2 O-bound state (15). The desolvated K ϩ is spherically symmetric, and its size is smaller than that of H 2 O, whereas H 2 O is spherically asymmetric in its shape and polarity, suggesting that the selectivity filter in the K ϩ -bound permeable conformation is more compact and 4-fold symmetric as compared with that in the H 2 O-bound impermeable conformation.
At neutral pH, the tetrameric assembly of KcsA is reportedly influenced, whether it is reconstituted in the detergent micelles or in the lipid bilayers (23). We then reconstituted KcsA into rHDL, where KcsA is surrounded by the lipid bilayer, to investigate the effect of the surrounding environment on the structural equilibrium under acidic conditions. The methyl-TROSY spectra of KcsA in rHDL in the absence of TFE provided signals from the permeable and impermeable conformations simultaneously, indicating that the structural equilibrium identified in DDM micelles also exists in the lipid bilayer (supplemental Fig.  S2).
Interestingly, the p perm value at pH 3.2 and 45.0°C in the presence of 200 mM KCl in rHDL (20%) is quite different from that in DDM micelles (100%), indicating that the transition between the permeable and impermeable conformations are different in these two media. Indeed, the small but significant chemical shift differences in DDM micelles and in rHDL were observed for the methyl groups not only in the region exposed to the surrounding environment but also in internal region close to the selectivity filter (supplemental Fig. S2). These results strongly indicate that the surrounding environments affect the structural equilibrium of KcsA, although the overall structures of KcsA are similar to each other in DDM micelles and rHDL.
As reported previously (15), the p perm value in DDM micelles decreases to 60% as the temperature decreases to 25.0°C, at pH 3.2. However, this p perm value is higher than the open probabilities observed in the electrophysiological assays of 5-15% at pH 4.0 (3), indicating that KcsA in DDM micelles is not functionally identical to that in lipid bilayers. On the other hand, the p perm value in rHDL would be lower than 20% at room temperature, corresponding well with the open probabilities of 5-15%, although the temperature dependence of p perm values could not be analyzed in rHDL because of low sensitivity of the NMR signals at room temperature.
We then demonstrated that the spectral changes in the methyl-TROSY spectra of KcsA in rHDL upon the addition of only a few percent of TFE shifted the structural equilibrium to the permeable conformation (Fig. 4). The addition of 4% or more of TFE shifted the equilibrium to the closed state, which should be observed only under neutral conditions without TFE. The single-channel recording of KcsA also showed that the addition of a similar amount of TFE increases the open probability of KcsA in the planar lipid bilayer (Fig. 5), indicating that the increase in the p perm value observed in the NMR analyses is reflected in the actual channel activity as an increase of the open probability. Although we could not examine whether higher TFE concentrations shift the equilibrium to the closed state as observed by NMR because of destabilization of the planar bilayer at the higher TFE concentrations, these results strongly suggest that the addition of TFE modulates both the structural equilibrium and the open probability of KcsA.
The effect of TFE on the structural equilibrium is seemingly inconsistent with the reported effect of TFE to destabilize the tetramer assembly of KcsA (25), which would lead to the decrease in the p perm value. However, the shift of the structural equilibrium was observed at the lower TFE concentrations (2-3%) as compared with the disruption of the tetrameric assembly (20%) (25), suggesting that the mechanism of the structural equilibrium shift by TFE may not be due to the destabilization of KcsA.
TFE is known to stabilize the secondary structures of proteins, especially ␣-helices (26). However, this effect is significant at TFE concentrations higher than 20% (27), indicating that the stabilization of ␣-helices might not be the source of the shift of the structural equilibrium. TFE is also known to increase the pK a values of p-or o-hydroxybenzoic acids (28). Because the activation of KcsA is triggered by the protonation of H25 near the helix bundle crossing (10,15), changes in the pK a values of the acidic residues by the addition of TFE can potentially modulate the structural equilibrium of KcsA. However, the increase in pK a by 25% TFE is only 0.5 points for hydroxybenzoic acids, and the effect would be smaller in the TFE concentration range used here. If TFE increased the pK a values of the acidic residues in KcsA, then the protonation states of these residues would not change because these residues have already been completely protonated in the absence of TFE at pH 3.2. The effects of direct interactions of TFE with KcsA may also be negligible because the chemical shifts of the methyl groups of KcsA did not change upon the addition of TFE. Therefore, the shift of the structural and functional equilibrium of KcsA is most likely caused by the modulation of the surrounding membrane environment.