Gating-related Structural Dynamics of the MgtE Magnesium Channel in Membrane-Mimetics Utilizing Site-Directed Tryptophan Fluorescence

Abstract

Magnesium is the most abundant divalent cation present in the cell, and an abnormal Mg²⁺ homeostasis is associated with several diseases in humans. However, among ion channels, the mechanisms of intracellular regulation and transport of Mg²⁺ are poorly understood. MgtE is a homodimeric Mg²⁺-selective channel and is negatively regulated by high intracellular Mg²⁺ concentration where the cytoplasmic domain of MgtE acts as a Mg²⁺ sensor. Most of the previous biophysical studies on MgtE have been carried out in detergent micelles and the information regarding gating-related structural dynamics of MgtE in physiologically-relevant membrane environment is scarce. In this work, we monitored the changes in gating-related structural dynamics, hydration dynamics and conformational heterogeneity of MgtE in micelles and membranes using the intrinsic site-directed Trp fluorescence. For this purpose, we have engineered six single-Trp mutants in the functional Trp-less background of MgtE to obtain sitespecific information on the gating-related structural dynamics of MgtE in membrane-mimetic systems. Our results indicate that Mg²⁺-induced gating might involve the possibility of a ‘conformational wave’ from the cytosolic N-domain to transmembrane domain of MgtE. Although MgtE is responsive to Mg²⁺-induced gating in both micelles and membranes, the organization and dynamics of MgtE is substantially altered in physiologically important phospholipid membranes compared to micelles. This is accompanied by significant changes in hydration dynamics and conformational heterogeneity. Overall, our results highlight the importance of lipid-protein interactions and are relevant for understanding gating mechanism of magnesium channels in general, and MgtE in particular.

Introduction

Ion channels perform vital physiological functions like ion transport, electrical excitability and cellular signaling, and their dysfunction is often related to several life-threatening diseases. Importantly, ion channel family of membrane proteins are crucial targets for therapeutics [1,2]. In general, the structure, dynamics and associated function of ion channels are affected by its surrounding membrane environment, protein-protein interactions within the structure as well as lipid-protein interactions with the membrane bilayer. Hence, knowledge of the impact of membrane environment on the protein structure and dynamics with respect to change in orientation of amino acid residues, structural transitions between functional states is of vital importance to understand the intricate functioning of ion channels [3].

Magnesium is the most abundant divalent cation present in the cell with a total intracellular concentration ranging between ~20 to 50 mM depending on the cell type, of which 1-2% constitutes the free Mg²⁺ concentration [4–6]. In humans, abnormal Mg²⁺ homeostasis is reported to be associated with pathophysiological conditions, which include heart disease, diabetes, high blood pressure, and cancer [7–9]. Among the ion channels, the mechanism of Mg²⁺ transport and homeostasis are poorly understood.

MgtE is a homodimeric Mg²⁺ ion channel with a monomer molecular weight of ~ 50 KDa (450 amino acids) and contains transmembrane (TM-domain) and cytosolic domains. Importantly, MgtE is an ortholog of the mammalian SLC41A1 transporter [9] which has been implicated in Parkinson’s disease [10] and head/neck cancer [11]. The high-resolution three-dimensional crystal structures of the full-length MgtE in the putative closed state have been solved in detergent micelles [12,13]. It is evident from the full-length structures that the cytosolic domain is divided into two sub-domains namely the N-domain and the cystathione-ß-synthase (CBS) domain, and has several Mg²⁺ binding sites. Consequently, the cytoplasmic domain, particularly the N-domain, is proposed to primarily function as a Mg²⁺ sensor. It has been proposed that while Mg²⁺ binding stabilizes the closed state of the channel, the open state(s) of MgtE channel is favored due to the electrostatic repulsion among many acidic residues in the cytoplasmic domain in the absence of Mg²⁺ [13,14]. This makes MgtE gating interesting since Mg²⁺ is not only a permeating ion but also negatively regulates MgtE function. Although the full-length MgtE structural snapshot is available in one functional state (closed state), the atomic model of TM-domain of MgtE based on the cryo-EM structure of MgtE-Fab complex in Mg²⁺-free conditions using amphipols has been recently posted on the pre-print server for biology, bioRxiv [15]. However, the gating-related structural dynamics in physiologically-relevant membranes is poorly understood and studies focused on MgtE organization and dynamics in membranes are scarce.

In this work, we have compared the organization and dynamics of MgtE in membranemimetic systems such as micelles and membranes. Further, we monitored the changes in gating- related structural dynamics, hydration dynamics and conformational heterogeneity of MgtE in membranes using the intrinsic site-directed Trp fluorescence. For this purpose, we employed various sophisticated fluorescence approaches that include anisotropy, red edge excitation shift (REES), quenching and lifetime distribution analysis by Maximum Entropy Method (MEM) [3]. Intrinsic Trp fluorescence is extensively utilized to monitor the changes in dynamics and local structure of proteins in general and membrane proteins in particular [3,16–23]. Since MgtE is a multi-tryptophan containing ion channel and analysis of ensemble Trp fluorescence is complicated [3], we have engineered six single-Trp mutants in the functional Trp-less background of MgtE to obtain site-specific information on the gating-related structural dynamics of MgtE in membranemimetic systems. Our results show that the organization and dynamics of the full-length MgtE is significantly altered in physiologically-relevant membrane environment compared to micelles. Further, the closed state of MgtE is highly dynamic compared to the open state in membranes, which is in sharp contrast to what has been observed in micellar environment. Furthermore, our MEM results show that the changes in the conformational heterogeneity of the tryptophan residues especially W37 at the Mg²⁺-sensing cytoplasmic domain are dependent on the functional state of MgtE. Based on our results, we propose that the Mg²⁺-induced gating involves ‘conformational wave’ from N-domain to TM-domain of MgtE. Overall, our work highlights the importance of membrane environment and lipid-protein interactions in the gating mechanisms of ion channels in general, and magnesium channels in particular.

Results

Structural integrity of single-Trp MgtE mutants is preserved

The closed state crystal structure of homodimeric MgtE (PDB: 2ZY9) indicating the positions of intrinsic Trp residues [13], and the spatial arrangement of MgtE in membranes with respect to the hydrocarbon core of the lipid bilayer, as obtained from the OPM database [24], are shown in Figure 1a. As seen from the figure, MgtE has six Trp residues per monomer of which four (W282, W288, W352, W380) are present in TM-domain and two (W37 and W79) are present in N-domain. We have engineered six single-Trp mutants (W37only, W79only, W282only, W288only, W352only and W380only) by individually reintroducing each of the native Trp residues in a Trp-less background in such a way that each MgtE construct has only the desired Trp residue. For example, ‘W37only’ mutant of MgtE will contain only one tryptophan residue at position 37 and the remaining Trp residues are replaced by phenylalanine. Importantly, employing the native Trp of MgtE for fluorescence studies involves minimum structural perturbations unlike labelling with the bulky extrinsic fluorophores. Further, the advantage of using single Trp mutants is that the site-directed intrinsic tryptophan fluorescence can be used as a reporter to monitor the structural and gating-related dynamics information in a site-specific manner.

Figure 1. Single-Trp mutants of MgtE.

(a) Shown is the cartoon representation of membraneincorporated closed state of homodimeric MgtE (PDB: 2YVX) obtained from OPM database showing the location of Trp residues (denoted by respective residue numbers). The two subunits of MgtE are colored as magenta and blue, and the colors of Trp residues are given as a visual aid to distinguish between various Trp residues. (b) The relative change in the elution profile with change in pH of W380only is shown by size-exclusion chromatography (SEC). (c) SDS-PAGE of purified wild-type, single-Trp mutants and Trp-less mutant of MgtE. (d) Representative far-UV CD spectra of wild-type, Trp-less and W79only mutants of MgtE. CD spectra were recorded using 2 μM of protein in DDM micelles. See Materials and Methods and Eq. 1 for other details.

It has earlier been shown that wild-type MgtE in DDM buffer (pH 7.0) elutes as a homodimer in size-exclusion chromatography (SEC) analysis [13,25]. To confirm that the structural integrity of single-Trp mutants is not compromised, we carried out SEC for all the mutants (‘W380only’ is shown as representative in Figure 1b). At pH 7.0, we observe that the gel filtration profiles of purified single-Trp mutants are not homogeneous in the sense that they contain broad doublet peaks. Interestingly, when the purification is carried out at higher pH conditions (pH 8.0), the MgtE-Trp mutants elute at ~10.6 ml with a single homogeneous peak (Figure 1b) similar to wild-type MgtE, indicating the good quality of purified proteins as seen from SDS- PAGE (Figure 1c). To investigate whether the purified single-Trp mutants has a similar secondary structure to that of wild-type MgtE, we carried out far-UV CD spectroscopy. The CD spectra of Trp-less and single-Trp mutants (‘W79only’ as a representative) along with wild-type channel in

DDM micelles are shown in Figure 1d, which show features of a-helical conformation, which indicates that the mutants are extracted and purified in a properly folded form. Taken together, our results show that the structural integrity of MgtE dimer conformation of the MgtE-Trp mutants is well maintained at pH 8.0, and we have used this modified purification conditions for further experiments to monitor the structural dynamics of MgtE.

Transport activity and ligand-induced conformational changes of Trp-less mutant of MgtE

To monitor whether the Trp-less mutant retains the functional activity similar to wild-type MgtE, we have utilized the fluorescence assay for monitoring Mg²⁺ transport using the Mg²⁺- sensitive membrane-impermeable fluorophore Mag-Fura-2 (Figure 2a) and the limited protease protection assay to observe the ligand-induced conformational changes upon gating (Figure 2b) in a properly folded purified protein (see Figures 1c,d). In Mg²⁺ transport experiments, the fluorescence intensity of Mag-Fura-2 encapsulated in liposomes and proteoliposomes remains stable in the absence of Mg²⁺. Interestingly, upon addition of 20 mM Mg²⁺, both the wild-type and Trp-less mutant of MgtE reconstituted in Mag-Fura-2 encapsulated liposomes show a significant increase in the fluorescence intensity of the fluorescent dye over time. However, the control liposomes in which no protein is reconstituted, there is no increase in Mag-Fura-2 intensity upon addition of Mg²⁺ (Figure 2a). This clearly indicates that both the wild-type and the Trp-less mutant of MgtE mediates the Mg²⁺ influx in the presence of inwardly directed Mg²⁺ gradient.

Figure 2. Fluorescence transport assay and ligand-induced conformational changes.

(a) Representative experiments show the change in fluorescence intensity of Mag-Fura-2 encapsulated in PC/PG liposomes, reconstituted with either wild-type or Trp-less mutant of MgtE, monitored in a time-scan mode upon addition of 20 mM MgCl2 (indicated by the arrow). Liposomes loaded with Mag-Fura-2 (but without protein) serve as control. This assay was carried out in triplicates and similar results were obtained in all cases. (b) Protease protection assay of Trp-less MgtE in DDM micelles in the absence and presence of trypsin (16 μg/ml) with increasing concentration of Mg²⁺ as indicated. The ΔN (~37 KDa band) represents MgtE lacking N-domain. Control sample does not contain trypsin and MgCl2. (c,d) Changes in ensemble Trp fluorescence emission maximum and (e,f) anisotropy of wild-type MgtE as a function of increasing Mg²⁺ concentration in DDM micelles (black) and PC/PG liposomes (red) are shown. The solid line represents the best fit curves of Hill equation to the data using OriginPro 8.0. Values represent mean ± SE of three independent measurements, and the emission maximum values contain negligible errors. The estimated Mg²⁺ concentrations reaching the half-maximal values of changes (EC50) are shown in each graph. See Materials and Methods for other details.

Since MgtE transport activity must be preceded by the ligand-induced conformational changes, we have carried out limited proteolysis assay using trypsin (Figure 2b). Since MgtE is negatively regulated by its permeating ion Mg²⁺, and the cytosolic domain is hypothesized to act as a Mg²⁺ sensor [12,13], MgtE shuttles from the open to closed conformation in the presence of Mg²⁺. Consequently, MgtE is protected from the protease trypsin upon increasing the concentration of Mg²⁺. Figure 2b shows the appearance of full-length MgtE band at high concentrations of Mg²⁺, which shows that the Trp-less mutant of MgtE is sufficiently protected from protease at higher concentration of Mg²⁺ whereas it is sensitive to proteolysis in the absence of Mg²⁺. This is indicative of the ligand-induced conformational changes during gating. Taken together, the Trp-less mutant functions similar to wild-type MgtE as shown previously [13,25]. Since Trp-less mutant of MgtE is stably folded (see Figure 1d) and functional, i.e., it retains both its transport activity (Figure 2a) and exhibits ligand-induced conformational changes (Figure 2b), our results suggest that the Trp residues may not be a predominant factor in stability and function of MgtE, which is in agreement with Trp-less mutants of other membrane proteins [20,21,26,27].

It has been earlier shown, in patch-clamp electrophysiology experiments, that MgtE is fully closed at a concentration of 10 mM Mg²⁺ and above [13]. We have monitored the changes in fluorescence emission maximum¹ of Trp residues in wild-type MgtE as a function of increasing Mg²⁺ concentrations to check whether the changes in ensemble Trp fluorescence is sensitive to the conformational changes associated with closing of the channel in micelles (Figures 2c, e) and membranes (Figures 2d, f). The emission maximum of Trp residues increases significantly (red shifted) until ~8 mM Mg²⁺ in micelles (Figure 2c) and ~6 mM in membranes (Figure 2d), beyond which there is no change. The half maximal effective concentration (EC50) values associated with these changes are 3.26 mM and 2.37 mM Mg²⁺in micelles and membranes, respectively. Interestingly, this is supported by changes in the rotational mobility of Trp residues as monitored by fluorescence anisotropy, which is a robust method to monitor the rotational dynamics and flexibility of a fluorophore and has been employed to obtain dynamic information of ion channels in different functional states [28–30]. The EC50 values obtained from anisotropy measurements are very similar to the values obtained using emission maximum changes (Figures 2e, f).

These results are very interesting since these spectroscopic changes are strongly correlated with the gating-induced conformational changes in MgtE. Importantly, this also suggests that the local dynamic changes (both the environment as well as segmental mobility) around the Trp residues faithfully report the global conformational changes during gating. Overall, these results conclusively show that the Trp fluorescence of MgtE could be effectively used as a sensitive reporter of conformational changes when the channel shuttles between open and closed states. It should be noted that ensemble open conformations of MgtE are possible in the absence or low concentrations of Mg²⁺. Based on this result, we use 20 mM Mg²⁺ in our single-Trp MgtE experiments to ensure that the channel is predominantly stabilized in the closed state.

Microenvironment of single-Trp residues of MgtE in micelles and membranes

We monitored the fluorescence emission maximum of the Trp-less mutant of MgtE in micelles upon excitation at 280 nm to ensure that the fluorescence emission spectrum is devoid of any characteristic Trp fluorescence (Figure 3a). As expected, the fluorescence emission maximum of this mutant, which does not contain any Trp residues, is 308 nm, which is the characteristic emission maximum for Tyr fluorescence [31]. Figure 3b shows the representative emission scans of the wild-type and single-Trp mutants of MgtE in the open state in detergent micelles when excited at 295 nm. The ‘average’ tryptophan emission maximum of wild-type MgtE in open state is 334 nm as shown previously [25]. On the other hand, the Trp-less mutant of MgtE lacks the Trp fluorescence emission characteristic of the wild-type, thereby providing a background for monitoring the structural dynamics of MgtE. Interestingly, it appears that the W282 in the TM- domain is the dominant contributor to the wild-type fluorescence since the fluorescence intensity of ‘W282only’ mutant is ~80% of the wild-type. In contrast, the fluorescence intensity of the cytoplasmic N-domain W37 is the lowest among all the single-Trp mutants (Figure 3b) although the fluorescence emission maximum of this mutant (333 nm) indicates the nonpolar environment around W37. Since W37 is present in the soluble domain of MgtE, the unique combination of nonpolar localization and reduced fluorescence intensity of ‘W37only’ mutant could be attributed to the presence of several nonpolar and charged amino acids - efficient quenchers of Trp fluorescence [17] - within 4Å of this residue, respectively (see Figure 3d). Interestingly, summation of the individual spectra from the single-Trp mutants gives a spectrum that is much higher than the wild-type spectrum (Figure 3c), suggesting a significantly quenched ensemble Trp fluorescence in wild-type MgtE. This could be due to the presence of neighboring charged amino acids (Figure S1) and homoFRET involving the Trp residues in the TM-domain (particularly W282 and W288 whose indole ring distances in the closed state structure are ~15 Å) that form the ‘aromatic ring’ at the membrane interface along with Tyr residues (see Figure 1a and ref. [3]).

Figure 3. Steady-state fluorescence of MgtE-Trp mutants.

Representative fluorescence emission spectra of (a) Trp-less mutant excited at 280 nm; (b) wild-type, Trp-less and the indicated single-Trp mutants of MgtE in DDM micelles upon 295 nm excitation are shown. (c) Comparison of the wild-type spectrum of MgtE with the sum of spectra of single-Trp mutants is shown. (d) Neighboring residues within 4 Å of W37 based on the crystal structure of MgtE (PDB: 2ZY9) and the charged residues are colored red. The concentration of MgtE was 1.6 μM in all cases. See Materials and Methods and text for other details.

The well-established sensitivity of Trp fluorescence to environment factors makes an ideal choice of natural fluorophore to monitor structural dynamics and conformational changes of proteins and to characterize membrane partitioning, folding and lipid-protein interactions [3,16,19–22,32,33]. Figure 4a shows the representative emission scans of a single-Trp mutant (‘W37only’) of MgtE in micelles and membranes to highlight the difference in the microenvironment. The fluorescence emission maximum of the Trp residues in the cytoplasmic N-domain (W37 and W79) ranges from 332 to 335 nm in micelles and membranes (Figure 4b). This suggests that the microenvironment experienced by these residues are nonpolar despite the fact that they are localized at the soluble domain of MgtE. In case of Trp residues in TM-domain, the emission maximum ranges from 332 to 336 nm in micelles and 331 to 337 nm when reconstituted in membranes, indicating a preferential localization of Trp residues at the membrane interface as shown previously for channel-forming peptides and proteins in membrane-mimetic systems (19,34-36). These results suggest that the Trp environment is similar in micelles and membranes for all single-Trp mutants (±1 nm change) whether the Trp residue is present in soluble N-domain or TM-domain (see Figure 4b). Although the emission maximum for single-Trp mutants is similar in membrane-mimetic systems, the fluorescence intensity is much higher for all the Trp mutants in micellar environment (Figure 4c), suggesting that Trp residues experience a relatively hydrophobic/nonpolar environment in micelles compared to membranes. Interestingly, the Trp residues in the cytoplasmic part (N-domain) of MgtE show the maximum intensity changes (~2 fold) suggesting that the organization of the Trp residues in MgtE is altered in general, and the Mg²⁺-sensing N-domain in particular in micelles and membranes.

Figure 4. Tryptophan fluorescence emission maximum of the MgtE single-Trp mutants in membrane-mimetic systems.

(a) Representative fluorescence emission spectra of W37only in DDM micelles and POPC/POPG (3:1 mol/mol) membranes are shown. (b) Shown are the fluorescence emission maximum of MgtE-Trp mutants in the open state in micelles (black) and membranes (red) at a protein/lipid molar ratio of 1:100. (c) Changes in emission intensity of Trps (Fmicelle/Fmembrane) for single-Trp mutants observed at respective emission maximum are shown. The excitation wavelength used was 295 nm and the concentration of MgtE was 1.6 μM in all cases. See Materials and Methods for other details.

Fluorescence emission intensity changes are not always reliable for monitoring probe localization due to its dependence on multifactorial property of emission phenomenon [3,37]. In contrast, fluorescence lifetime is a faithful indicator of local environment and is an intrinsic property of the probe [3,38,39]. Therefore, we measured Trp fluorescence lifetimes of the single- Trp mutants of MgtE in membrane-mimetic systems (Figure 5). The fluorescence lifetime of Trp has been well established to be sensitive to solvent and excited-state interactions, and can be used to directly monitor the environment of the probe especially its water accessibility due to fast deactivating processes in polar environment [3,38,40].

Figure 5. Fluorescence lifetimes of MgtE single-Trp residues in micelles and membranes.

(a) Time-resolved Trp fluorescence intensity decay of W37only in the open state (red) in DDM micelles is shown. Excitation wavelength was 285 nm, and emission was monitored at 333 nm. The sharp peak on the left (black) is the lamp profile, and the relatively broad peak on the right is the decay profile (red), fitted to a triexponential function. The plot in the inset shows the weighted residuals of the decay fit. (b) Mean fluorescence lifetimes of Trps for single-Trp mutants in DDM micelles (black) and POPC/POPG membranes (red) in the open state are shown. Values represent mean ± SE of three independent measurements. (c) The effect of membrane environment on the lifetimes of single-Trp mutants depicted as percent of lifetime changes is shown. All other conditions are as in Figure 4. See Materials and Methods and Table 1 for other details.

A typical fluorescence decay profile of ‘W37only’ with its triexponential fitting and the associated residuals are shown in Figure 5a. The intensity-weighted mean fluorescence lifetimes of the single-Trp residues, <τ>, in membrane-mimetic systems are shown in Figure 5b and Table 1. It is obvious that the mean fluorescence lifetime of single-Trp residues in micelles are higher (up to ~12%) than in membranes, which shows that the residues face a relatively hydrophobic environment in micelles than in membranes (Figures 5b,c). Further, the lifetime changes in membrane-mimetic systems are more for N-domain Trp residues (‘W37only’ and ‘W79only’) compared to TM-domain with the exception of ‘W352only’. This is in agreement with the significant increase in emission intensity (see Figure 4c) for Trp residues in micelles. The shortening of lifetimes for both the cytosolic as well as TM-domain Trp residues of MgtE upon membrane reconstitution could not only be due to changes in immediate polarity, but also due to cation-π interactions [41] with the neighboring positively charged residues (see Figure S1). These results clearly indicate that the overall organization of MgtE is altered in physiologically-relevant membrane environment.

Table 1. Fluorescence lifetimes of Single-Trp mutants of MgtE in different functional states.

		α1	τ1	α2	τ2	α3	τ3	<τ>(ns)a	τH(ns)b
DDM micelles
W37only	Open	0.51	3.45	0.17	7.32	0.32	1.26	4.60	3.59
	Closed	0.49	3.07	0.23	6.65	0.28	1.02	4.54	3.63
W79only	Open	0.37	2.89	0.45	6.25	0.18	1.22	5.10	4.28
	Closed	0.41	2.64	0.44	6.27	0.15	1.05	5.08	4.19
W282only	Open	0.19	3.01	0.72	7.08	0.09	1.33	6.56	5.79
	Closed	0.20	3.02	0.73	7.10	0.07	1.24	6.59	5.87
W288only	Open	0.64	4.51	0.22	8.45	0.14	1.54	5.86	5.76
	Closed	0.52	4.22	0.37	7.77	0.11	1.67	6.07	5.24
W352only	Open	0.63	3.76	0.21	7.28	0.16	1.44	4.93	4.24
	Closed	0.58	3.64	0.28	6.94	0.14	1.23	5.06	4.40
W380only	Open	0.53	3.40	0.35	6.58	0.12	0.87	5.08	4.44
	Closed	0.49	3.32	0.39	6.36	0.12	0.92	5.05	4.44
PC/PG membranesc
W37only	Open	0.40	2.27	0.37	5.69	0.24	0.752	4.44	3.78
	Closed	0.42	1.92	0.41	5.51	0.17	0.521	4.45	3.73
W79only	Open	0.44	1.94	0.43	5.56	0.13	0.331	4.55	3.86
	Closed	0.21	7.36	0.50	3.28	0.29	1.04	4.89	3.80
W282only	Open	0.39	2.51	0.51	6.92	0.10	0.439	5.91	4.91
	Closed	0.38	2.32	0.51	6.44	0.11	0.421	5.51	4.64
W288only	Open	0.35	2.37	0.53	5.9	0.12	0.307	5.12	4.48
	Closed	0.36	2.49	0.52	6.91	0.12	0.347	5.98	5.03
W352only	Open	0.43	2.2	0.39	5.88	0.18	0.393	4.71	3.96
	Closed	0.42	1.92	0.41	5.5	0.17	0.389	4.47	3.68
W380only	Open	0.40	2.75	0.45	5.95	0.15	0.782	4.89	4.29
	Closed	0.45	3.09	0.37	6.25	0.19	0.912	4.88	4.23

The concentration of protein was 1.6 μM in all cases. The excitation wavelength was 285 nm and the emission was monitored at respective emission maximum. See Materials and methods for other details.

^aMean fluorescence lifetime (<τ>) calculated using Eq. 6;

^bCalculated using Eq. 7;

^cThe ratio of single Trp MgtE/total lipid is 1:100.

Apart from the model-dependent mean fluorescence lifetime calculation, the Trp fluorescence lifetimes from the histogram of photons counted during the time-resolved decay measurement (see Eq. 7 and Table 1) has also been calculated in a model-independent manner as shown previously [25,28,42]. Understandably, model-dependent and model-independent approaches give slightly different fluorescence lifetimes. Irrespective of the methods used to calculate Trp lifetimes, the conclusions resulting from the time-resolved experiments are consistent.

Gating-related rotational dynamics of MgtE-Trp residues in micelles and membranes

It is well known that the rapidly tumbling Trp residue displays negligible polarization [34] whereas its limiting anisotropy is ~0.16 [3,43]. In this regard, the steady-state fluorescence anisotropy values of Trp residues in the open and closed states of MgtE in both DDM micelles and PC/PG membranes generally indicate a considerably restricted mobility (Figure 6a,b). This is suggestive of motional restriction experienced by Trp residues in MgtE irrespective of whether the Trp residues are localized in the cytosolic or TM-domain. In the open state, for which high- resolution atomic structure is not available yet, most of the MgtE-Trp residues in DDM micelle environment display lower anisotropy (i.e., increased rotational dynamics) values than PC/PG membranes. It is interesting to note that the hydrodynamic diameter of DDM micelles [44] and small unilamellar liposomes [45] is ~7 and 40 nm, respectively. Since micelles are smaller than membranes, the curvature will be more in micelles and might have reduced order parameter due to interfacial packing defects as compared to liposomes [46].

Figure 6. Rotational mobility of MgtE single-Trp mutants upon gating in micelles and membranes.

Steady-state anisotropy of Trp fluorescence measured for single-Trp mutants in the (a) open (absence of Mg²⁺) and (b) closed (20 mM Mg²⁺) states of MgtE in DDM micelles (black) and reconstituted in POPC/POPG (3:1 mol/mol) liposomes (red) at a protein/lipid molar ratio of 1:100. The excitation wavelength used was 295 nm; emission was monitored at their respective emission maxima in all case. Values represent mean ± SE of three independent measurements. (c) The difference in anisotropy values (Δr) between closed and open states in micelles (black) and membranes (red) is shown. The excitation wavelength used was 295 nm and the concentration of MgtE was 1.6 μM in all cases. See Materials and Methods for other details.

Interestingly, the changes in rotational dynamics of Trp residues appear to be dependent on the functional state of MgtE. For instance, the anisotropy changes are modest in the closed state for MgtE in micelles and membranes (Figure 6b). Interestingly, significant changes in anisotropy values are observed for Trp residues when MgtE is stabilized in the open state (Figure 6a). The membrane-induced restricted dynamics (low mobility) of Trp-residues, particularly in the open state, clearly suggests the differential MgtE dynamics upon gating. The differential dynamics of MgtE in membrane-mimetic systems is reflected in difference plot (Figure 6c) which shows that, although gating-related dynamic changes are observed in both micelles and membranes, the trend is quite opposite.

To ensure that the anisotropy values measured for the MgtE-Trp mutants do not suffer from lifetime-induced artifacts, the apparent (average) rotational correlation times were calculated using Eq. 3. The gating-related changes in apparent rotational correlation times for MgtE-Trp residues in membranes (Figure 7) and micelles (Figure S2) are in excellent agreement with the anisotropy results (Figure 6). Upon channel opening in the membrane environment, MgtE-Trp residues undergo decreased/restricted rotational dynamics with pronounced dynamic variability, particularly the W37, W282 and W352 residues (Figure 7). Since both cytoplasmic N-domain (W37) and the TM-domain (W282 and W352) Trp residues are sensitive to gating-induced dynamic changes in membranes, this suggests that gating-related conformational dynamics might involve a ‘conformational wave’ from the Mg²⁺-sensing cytoplasmic domain to TM-domain. However, unlike in membranes, MgtE in the micellar environment does not exhibit dynamic variability and only W288 residue shows a significantly increased dynamics upon opening (Figure S2). Taken together, our data suggest that the structural dynamics of MgtE is significantly altered in physiologically-relevant membrane environment, and is supportive of the importance of side chain dynamics in channel gating [30,47].

Figure 7. Apparent rotational correlation times of MgtE single-Trp mutants in membranes.

Shown are the (a) apparent rotational correlation times in the open (white bars) and closed (grey bars) states of MgtE-Trp mutants in membranes; and (b) difference in apparent rotational correlation times (Δτ_c) between closed and open states. All other conditions are as in Figure 6. See Materials and Methods for other details.

Extent of water penetration for single-Trp mutants of MgtE

The above results show significant differences in the structural dynamics of MgtE in micelles and membranes. To examine the accessibility and changes in localization of the Trp residues upon gating, we probed collisional quenching of Trp fluorescence. Acrylamide is a well- known neutral aqueous quencher [48], which has been widely used to study protein folding [49] and partitioning of membrane peptides and proteins in membrane-mimetic systems [25,50]. Figure 8a shows the representative Stern-Volmer plots for quenching of ‘W380only’ in the open and closed states in PC/PG membranes by acrylamide. The Stern-Volmer quenching constant (Ksv) of the single-Trp MgtE mutants in membranes is shown in Figure 8b. The K_SV values, which are related to water accessibility, of Trp residues in the TM-domain of MgtE (W282, W288, W352 and W380) are in the range of ~ 4.5 M^-1-6 M^-1, which is consistent with the localization of Trp residues at the interface [34]. Considering the complete exposure of Trp residue to aqueous environment has a K_SV value of ~ 18 M^-1 [3,34], the low K_SV values for the cytoplasmic Trp residues (W37 and W79) suggests that the N-domain of MgtE is significantly shielded from the aqueous phase in the open and closed states of MgtE incorporated in micelles (Figure S3) and membranes (Figure 8) which agrees well with our previous results (see Figure 4) and the recently reported quenching results from wild-type MgtE [25]. Since the K _SV values are higher in the closed state of MgtE, it appears that the water penetration accessing the Trp residues is more in the closed state than the open state of MgtE (Figure 8b). Because K _SV values are intrinsically dependent on fluorescence lifetime (see Eq. 12), we calculated the bimolecular quenching constant (kq), which offers more precise information regarding the degree of aqueous exposure since it considers the differences in fluorescence lifetimes. Our results show that there is a significant increase in water accessibility for most of the Trp residues (W37, W79, W352 and W380) in the closed state of MgtE than open state in membranes (Figure 8c) which is in excellent agreement with K_SV values, and supports the notion of gating-induced ‘conformational-wave’. Interestingly, the water accessibility profiles for MgtE-Trp residues in micelles (see Figure S3) are almost identical to membranes except for W380 residue, which could be due to its localization in the lipid-protein interface at the upper leaflet of the membranes. Importantly, the low water accessibility for N- domain Trp residues in the open state of MgtE also suggest that the overall structural integrity of the cytoplasmic Mg²⁺-sensing N-domain is preserved during gating [12].

Figure 8. Water accessibility probed by acrylamide quenching of Trp fluorescence in membranes.

Shown are (a) Representative data for Stern-Volmer analysis of acrylamide quenching of tryptophan fluorescence of ‘W380only’ mutant in the open (magenta) and closed (blue) states, where Fo is the fluorescence in the absence of quencher and F is the corrected fluorescence in the presence of quencher, (b) Stern-Volmer constants (K_SV) and (c) bimolecular quenching constants (kq) for acrylamide quenching of Trp fluorescence for the single-Trp mutants in open (white bars) and closed (grey bars) states of MgtE in PC/PG membranes. The KSV values represent mean ± SE of three independent measurements. The excitation wavelength used was 295 nm, and the emission was monitored at respective emission maximum. All other conditions are as in Figure 6. See Materials and Methods for other details.

Hydration dynamics and conformational substates of MgtE-Trps as probed by REES

It is well known that that protein dynamics is intrinsically related to hydrating solvent molecules and slow solvation [51]. Further, hydration dynamics has been shown to play crucial roles in lipid-protein interactions [52], mediating ion channel functional states [30,53] and ion channel selectivity [54]. Red edge excitation shift (REES) is a well-established fluorescence approach, which offers valuable information on the relative rates of water relaxation dynamics and is sensitive to changes in local hydration dynamics [38,55]. Therefore, in complex biological systems, REES is a robust tool to directly probe the environment-induced restriction and dynamics in the immediate environment of a fluorophore (for reviews see refs [3,33,56,57]). Further, REES has been shown to be sensitive to changes in restricted motions of the surrounding protein matrix due to rearrangements in solvating polar side chains around the fluorophore [57,58]. In this way, REES is a powerful tool to probe the presence of restricted/bound water molecules as well as side chain rearrangements in a protein core.

REES is operationally defined as the shift in the wavelength of maximum fluorescence emission toward higher wavelengths, caused by a shift in the excitation wavelength toward the red edge of the absorption band [33]. The magnitude of REES, i.e., the total shift in emission maximum upon changing the excitation wavelength from 295 to 305nm, for the single-Trp mutants of the open state of MgtE in DDM micelles and PC/PG membranes is shown in the left panel of Figure 8a. In general, all single-Trp mutants exhibit significant REES, which is an indication of motionally restricted environments around Trp residues in both micelles and membranes, and the presence of restricted/bound water molecules (Figure 9a, left). Using REES, it has been shown that restricted or bound water molecules have substantial contribution in ion channel gating mechanism [30]. Interestingly, the magnitude of REES for MgtE-Trp ranges from 4-6 nm in micelles, whereas the corresponding values are 1-3 nm in membranes. This significant reduction in the magnitude of REES for all membrane reconstituted MgtE-Trp mutants clearly suggests faster solvent (water) relaxation around the excited-state Trp residues in membranes. In other words, the dynamics of hydration is considerably increased around MgtE-Trp residues in membranes compared to micelles, which is similar to what has been recently observed for the interaction of voltage sensor loop of KvAP with membranes [28]. It is important to note that the changes in the hydration dynamics for MgtE-Trp residues in micelles and membrane bilayer are not only due to polarity changes in the immediate environment since emission maxima and water accessibility is similar in both membrane-mimetic systems (see Figures 4b and S3). The differential magnitude of REES in micelles and membranes also indicates an altered organization of the side chains around the Trp residues. Interestingly, gating-induced REES changes in membranes are observed only in Trp residues in the TM-domain and not in the N-domain of MgtE (Figure 9b, left). The magnitude of REES of single-Trp residues in the TM-domain of MgtE (except ‘W288only’) slightly increase in the closed state (Figure 9b, left) suggesting a role of differential hydration dynamics during MgtE gating. Interestingly, the identical magnitude of REES for N-domain Trp residues while shuttling between open and closed states in membranes supports our earlier observation that the core packing of N-domain is not significantly altered during gating (see Figure 8).

Figure 9. REES of single-Trp mutants of MgtE.

Comparison of the magnitude of REES (left) and the associated area (right) for (a) MgtE-Trp mutants in the open state in micelles (black) and membranes (red) and (b) during gating in membranes. The relative area was calculated by fitting the REES data using Eq. 4. All other conditions are as in Figure 6. See Materials and Methods for other details.

As shown above, the REES magnitude is suitable to monitor relative solvent relaxation dynamics and the corresponding changes in the bulk and restricted water molecules in a qualitative manner. Recently, a novel analysis of REES data has been demonstrated to offer unique insights on conformational changes in proteins and the equilibrium of conformational states [59], and this approach has recently been utilized to the voltage sensor of potassium channels [28]. Particularly, the area extracted from fitting the REES data using Gaussian probability distribution is very useful to monitor changes in protein conformational states [59]. Our REES data was fitted with Eq. 4 to extract the relative area of the distribution for MgtE-Trp residues (open state) in micelles and membranes (Figure 9a, right) and upon gating in membranes (Figure 9b, right). Our results show that the relative area, which is directly proportional to the number of conformational states, for membranes is significantly low (Figure 9a, right), which indicates that the MgtE-Trp residues might have altered conformational states in membranes. Gating-induced changes in the obtained area is significantly different only for TM-domain Trp residues of MgtE (Figure 9b, right) in which the closed state shows increased area values. Taken together, these results show that not only the dynamics of hydration is significantly different between membrane-mimetic systems, but also indicate the altered conformational states of MgtE during gating.

Conformational heterogeneity of MgtE-Trp by Maximum Entropy Method (MEM)

As mentioned above, the REES results have indicated that gating-related changes might involve altered conformational states of MgtE. However, information on the conformational states obtained using REES approach is indirect in nature and depends on the kinetics of solvent reorientation around the fluorophore. This means that this approach cannot be reliably used for obtaining information on protein conformation if the fluorophore (Trp residue in our case) does not exhibit REES or when the magnitude of REES is very small as in the case of ‘W37only’ mutant of MgtE. In contrast, MEM-lifetime distribution is a direct read-out of structural heterogeneity irrespective of the solvent (water) relaxation dynamics around the fluorophore since fluorescence lifetime distribution gives an ultrafast snapshot of the protein population distribution [60,61]. MEM analysis of fluorescence decay is robust and involves resolving the lifetime components in a model-independent manner, which has been widely used to monitor unfolding transitions in soluble proteins [61–65], and membrane heterogeneity [66]. However, this robust MEM approach has rarely been used to decipher conformational heterogeneity of membrane proteins.

Representative MEM lifetime distributions of MgtE-Trp residues in the absence (open) and presence (closed) of magnesium ions in membranes is shown in Figure 10. It should be noted that although the model-independent MEM analysis has been performed assuming the equal probability of the existence of lifetimes in the range of 0.1 to 100 ns in logscale, we have not observed any lifetime distribution beyond 10 ns. In general, irrespective of the functional state of MgtE (Figure 10) and the membrane-mimetic system used (Figure S4), the fluorescence lifetime distribution profiles for all MgtE-Trp mutants contain multiple peaks and are complex. This is not surprising considering the complex nature of organized molecular assemblies such as membranemimetics and proteins [65,66]. Monitoring the changes in the conformational heterogeneity within various functional states of the channel is therefore not trivial. However, change in distribution profiles can offer significant insights into the nature of altered conformational substates. Most of the single Trp mutants of MgtE embedded in micelles display relatively broad peaks compared to MgtE-Trps reconstituted in membranes (Figure S4), which suggests an increased conformational heterogeneity of MgtE in DDM micelles compared to PC/PG membranes. This is well supported by the observation of increased area associated with micellar environment (see Figure 9a, right).

Figure 10. Conformational heterogeneity of MgtE single-Trp mutants during gating in membranes.

MEM tryptophan fluorescence lifetime distributions for (a) W37only, (b) W79only, (c) W282only, (d) W288only, (e) W353only and (f) W380only mutants in the open (cyan) and closed (magenta) states of MgtE in membranes are shown. The normalized probability amplitudes are plotted against their corresponding lifetime on a linear scale. All other conditions are as in Figure 6. See Materials and Methods for details.

As seen from the Figure 10, the cytoplasmic W37 residue has three discrete peaks in the open state. However, upon closing the channel, the discrete nature of the peaks is lost in such a way that the lifetime distribution is spread from ~1 to 8 ns, indicating an increase in conformational heterogeneity for this residue in the closed state (Figure 10a). In case of cytoplasmic W79 residue, we observe a lesser number of discrete lifetime distributions in the closed state. However, the two lifetime peaks which appear in 2-6 ns region for the open state of MgtE are merged into a single broad lifetime peak in the closed state, supporting the altered sidechain conformational heterogeneity (Figure 10b). The fact that the lifetime distribution profiles of W37 and W79 residues do not drastically differ between the functional states suggests that N-domain is structurally preserved during gating. Since the N-domain of MgtE has been implicated in Mg²⁺- sensing and regulation of MgtE gating, we therefore attribute the change in conformational heterogeneity of these Trp residues to gating-related structural changes of the N-domain. In case of TM-domain Trp residues (W282 and W288), the distribution profiles are complex in both the closed and open states of MgtE (Figure 10c, d). Interestingly, despite retaining their complicated distribution profiles, the closed state of the channel has more peaks than the open state suggesting the changes in conformational microheterogeneity during gating, which is in excellent agreement with the above-mentioned REES data (see Figure 9b, right). However, the profiles of TM-domain W352 and W380 residues of MgtE do not show a significant change upon gating (Figure 10e,f). Taken together, MEM-lifetime distribution analysis supports the notion of ligand-induced ‘conformational wave’ from the Mg²⁺-sensing N-domain to TM-domain of MgtE, and the structural integrity of N-domain is preserved during the gating process.

Discussion

Biomembranes are complex assemblies of lipids and proteins, which provide a unique heterogeneous microenvironment for the optimum performance of membrane proteins and hence modulate various cellular signaling pathways [67]. The critical dependence of membrane proteins on lipid constituents of membranes suggests a coevolution of lipids and proteins [68]. Further, membrane lipids have been shown to play an important role in stabilizing membrane protein structure and function, and ion channels in particular [69–74]. Further, local lipid composition influences the topology, structural dynamics and conformation of membrane proteins and peptides [28,35,70,71], highlighting their functional dependence on lipid-protein interactions in membranes. Although the importance of lipid-protein interactions is well established, the high- resolution structures of membrane proteins is often solved in detergent micelles or amphipols using monoclonal antibodies due to technical difficulties [15, 75].

Among ion channels, magnesium ion channels are arguably the least understood despite Mg²⁺ being the most abundant divalent cation and is crucial for many cellular processes [76,77]. Despite its significance, the mechanisms governing the transport and regulation of magnesium channels are poorly understood. Our present understanding of the molecular basis of Mg²⁺ transport comes from studies on prokaryotic magnesium ion channels (CorA and MgtE) [78,79]. It is known that MgtE, like CorA, is the main Mg²⁺ transport system in almost half of all prokaryotes. Interestingly, unlike potassium and sodium ion channels, the subunit organization (i.e., molecular architecture) of magnesium channels is not conserved as can be seen from the three-dimensional X-ray crystallographic structures of magnesium ion channels. For instance, while CorA magnesium channel functions as a homopentamer [78,79], MgtE from Thermus thermophilus functions as a homodimer [13,80]. Although CorA and MgtE channels are evolutionarily distinct and have different architecture, they have conceptually similar gating mechanism. It is therefore important to understand the events associated with Mg²⁺-dependent gating.

Interestingly, MgtE from T. thermophilus is distantly related to mammalian SLC41A1 transporter [81] and can functionally compensate TRPM7-deficiency in vertebrate B-cells [82]. Importantly, MgtE is a dual-function protein in Pseudomonas aeruginosa, i.e., it functions both as a Mg²⁺ transporter and a virulence modulator and thus playing an important role in linking magnesium availability to pathogenesis [83], and thus a potential antibiotic target. Because the MgtE transport mechanisms across membranes at the molecular level are poorly understood due to unavailability of structural information in different functional states of full-length MgtE, the study of gating-related structural dynamics of MgtE assumes significance.

As mentioned earlier, the high-resolution closed state crystal structures of full-length MgtE has been obtained in detergent micelles [12,13,80]. Further, structural and dynamic information regarding the mechanism of MgtE function has been obtained for full-length MgtE in micelles [84] and the isolated cytoplasmic domain in solution [12,85] in the presence and absence of Mg²⁺. Furthermore, the TM-domain structure of MgtE bound to Mg²⁺ have been solved using lipidic cubic phase crystallization in a monoolein lipid environment [86]. Very recently, a preprint on the 3.7 Å cryo-EM structure of MgtE-Fab complex in Mg²⁺-free conditions is available in which the cryo-EM density is only detectable for the TM-domain [15]. It is obvious from this atomic model of TM-domain structure of MgtE that the ion-conducting pore is opened on the cytoplasmic side, but closed on the periplasmic side indicating that the pore is in non-conducting configuration. This structure therefore might represent one of the partially-open (‘pre-open state’) conformations of MgtE under Mg²⁺-free conditions.

To properly understand the structure-function relationship of MgtE, knowledge of changes in the structural organization and dynamics of MgtE in membrane-mimetic systems, and monitoring gating-related structural dynamic changes in membranes is therefore crucial, and this has been the focus of this work. For this purpose, we have engineered single-Trp mutants of MgtE in the functional Trp-less background as MgtE is a multi-tryptophan protein, and utilized various sophisticated fluorescence approaches to obtain site-specific information on structural dynamics. Our results utilizing the site-specific Trp fluorescence of MgtE clearly demonstrates, for the first time, the altered organization, dynamics and conformational heterogeneity of MgtE-Trp residues in physiologically-relevant PC/PG membranes compared to DDM micelles. This observation is supported by the fact that micelles, despite being widely used as membrane-mimetic systems, have been shown to drastically affect the function, dynamics, structural integrity in several membrane proteins [28,87–90].

From our results, we propose a model that highlights significant differences in the gating-related structural dynamics of MgtE in micelles and membranes (Figure 11). In the Mg²⁺-bound (closed state) conditions, most of the MgtE-Trp residues in micelles experience decreased rotational motion (i.e., relatively restricted) compared to membranes. Importantly, although MgtE is responsive to Mg²⁺-induced gating in both micelles and membranes, the trend is quite opposite in the sense that the MgtE is not only more dynamic but also displays dynamic variability in membranes than micelles upon closing (see Figure 6). This is accompanied by significant changes in environmental motional restriction (hydration dynamics and protein matrix), due to altered ratio of restricted (bound) to free (bulk) water molecules and possible polar sidechain rearrangements (see Figure 10). Interestingly, the functional correlation of hydration and structural dynamics is well established in different functional states of K⁺ channels [30,91]. In this regard, the observed changes in hydration dynamics might be important for gating and permeation mechanisms of MgtE. Interestingly, our model predicts that the structural integrity of Mg²⁺-sensing N-domain in full-length MgtE is preserved in both open and closed states of MgtE (see Figures 8 and 10), which is in agreement with the crystal structures of the soluble cytosolic domain in the presence and absence of Mg²⁺ [12]. In other words, the N-domain does not undergo conformational collapse in the apo/open state (i.e., in the absence of Mg²⁺). Importantly, our model also indicates the possibility of ligand-induced ‘conformational wave’ from the Mg²⁺-sensing N-domain to TM- domain of MgtE during gating. This is supported by the recent cryo-EM structure of TM-domain of MgtE solved in Mg²⁺-free conditions in which the side chain of Trp residues in the TM-domain have undergone significant change in orientation compared to the closed state of MgtE (see Figure S5). Considering that the cryo-EM structure might represent the ‘pre-opening state’ [15], the magnitude of this structural change could be much larger in the fully open state of MgtE. Overall, our results are relevant to understand the importance of physiologically-relevant membrane environment and lipid-protein interactions in the gating mechanisms of magnesium channels in general, and MgtE in particular.

Figure 11. Gating-related conformational dynamics of MgtE in micelles and membranes.

Schematic representations of the events associated with Mg²⁺-induced closing of MgtE in membrane-mimetic systems are shown. One subunit of the homodimeric MgtE is shaded grey to show the organization of the channel, and the transmembrane (TM) and cytoplasmic (N and CBS) domains are indicated. The open state representation shown is based on previous studies [13,81,85]. The respective residue numbers of MgtE-Trps (denoted by squares) is shown. Gating- induced changes in motional dynamics of MgtE-Trp residues are shown with blue and red to represent increased (dynamic) and restricted mobilities, respectively. The changes in hydration dynamics upon Mg²⁺ (yellow spheres) mediated closing is represented by the change in ratio of bulk/free (cyan spheres) vs. restricted/bound (blue spheres) water molecules. Mg²⁺-binding induces a ‘conformational wave’ from N-domain to TM-domain, which is indicated by a curved broken arrow. See Discussion for details.

Materials And Methods

Materials

E. coli C41(DE3) strain was purchased from Agilent (Santa Clara, CA). n-dodecyl-ß-D-maltopyranoside (DDM) and Triton X-100 were obtained from Anatrace (Maumee, OH). Protease inhibitors were obtained from GoldBio (St. Louis, MO). 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) were obtained from Avanti Polar Lipids (Alabaster, AL). All other chemicals used were of the highest purity available from either Merck (Kenilworth, NJ) or Amresco (Radnor, PA).

Mutagenesis, channel expression and purification

The gene encoding MgtE from Thermus thermophilus genomic DNA from strain HB8 (ATCC 27634) was cloned into a pET28a vector with a N-terminal His-tag (Qiagen, Hilden, Germany). To generate single-Trp mutants of MgtE, we made a Trp-less mutant, where all the native Trps have been converted to phenylalanine (W37F/W79F/W282F/W288F/W352F/W380F). Then one Trp at a time was introduced back to its native site making six ‘Trp-only’ mutants. For instance, reintroducing Trp37 to the Trp-less construct would result in a construct (‘W37only’) with only one tryptophan residue in MgtE. The mutations were confirmed by DNA sequencing. Trpless and single-Trp mutants of MgtE were expressed and purified using the ‘dual-detergent strategy’ as described previously [25] except that 10% glycerol was added during solubilization step for Trp-less and Single-Trp mutants. The concentration of the protein was checked in a DS- 11+ microvolume spectrophotometer (DeNovix, Wilmington, DE). To analyze whether the channel is folded properly, the purified protein was applied onto a Superdex 75 10/300 (GE Healthcare, Chicago, IL) size-exclusion column equilibrated with 20 mM HEPES, 150 mM NaCl, 1 mM DDM (pH 8.0) buffer. The purified MgtE in the absence of added Mg²⁺ represents the open state and the channel is closed by incubating in presence of 20 mM MgCl₂ for 30-40 minutes [13,25].

Membrane reconstitution of MgtE single-Trp mutants

The single-Trp mutants of MgtE were reconstituted at a lipid-to-protein molar ratio of 100:1 in POPC:POPG (3:1) liposomes. Briefly, 120 nmoles POPC and 40 nmoles of POPG (160 nmoles of total lipids) in chloroform were mixed well and dried under a stream of nitrogen while being warmed gently (~35 °C). After the lipids were dried further under a high vacuum for at least 3 hr, they were hydrated (swelled) by adding 1 ml of 20 mM HEPES, 150 mM NaCl (pH 8.0) buffer and vortexed vigorously for 2 min to disperse the lipids and sonicated to clarity. Protein was then added to give a molar ratio of 100:1 lipid:MgtE. The sample was left at room temperature for 30 minutes on a rotator and 200 mg of pre-washed biobeads (SM-2, Bio-Rad, Hercules, CA) were then added and the mixture was incubated on a rotator overnight at 4 °C to remove the detergent. The biobeads were removed by filtering using a Bio-Rad 5 ml column filter before use.

Circular dichroism (CD) measurements

CD measurements were carried out at room temperature in a Jasco J-815 spectropolarimeter purged with a nitrogen flow of 15 L/min. Wild-type MgtE, Trpless and single- Trp mutants were measured at a concentration of 2 μM in 20 mM HEPES, 150 mM KCl, 1 mM DDM (pH 8.0) buffer to obtain a good signal-to-noise ratio. The spectra were scanned with a quartz optical cuvette with a pathlength of 0.1 cm. All spectra were recorded with a bandwidth of 1 nm and integration time of 0.5 s with a scan rate of 50 nm/min. Each spectrum is the average of 10 scans. All spectra were appropriately blank subtracted and smoothed so as to ensure that the overall shape of the spectra remains unaltered. The ellipticity data obtained in millidegree was converted to molar ellipticity ([θ]) by using the following equation:

$$[\theta ]={\theta }_{\text{obs }}/(10Cl)$$ (Eq. 1)

where θ_obs is the observed ellipticity in millidegree, C is the concentration in mol/L and l is the pathlength in cm.

Limited protease protection assay

The limited protease protection assay with purified Trp-less mutant of MgtE was carried out as previously described [25]. Briefly, 5 μl of purified protein (2 mg/ml) was equilibrated with 0 to 32 mM MgCl2 for 30 minutes, and 2 μl of trypsin (15.6 μg/ml) was added to each reaction mix and incubated at 4 °C for 17 hrs. Trp-less MgtE with no added Mg²⁺ and trypsin served as a control. After the addition of 4X laemmli buffer, the samples were run on a 10% SDS-PAGE.

Mg²⁺ transport assay

The fluorescence assay for Mg²⁺ transport using the Mg²⁺-sensitive fluorophore Mag-Fura-2 was carried out as described previously [92]. The wild-type and Trp-less mutant of MgtE were reconstituted in POPC/POPG (3:1 mol/mol) liposomes as described above with few modifications. Briefly, the dried lipids were hydrated (swelled) by adding 1 ml of 20 mM HEPES, 150 mM KCl, pH 8.0 buffer and vortexed vigorously for 2 min to disperse the lipids. Membrane-impermeable Mag-Fura-2 (Invitrogen) was added to a final concentration of 60 μM to the lipid suspension and sonicated to clarity to form small unilamellar vesicles loaded with Mag-fura-2. The total lipid used was 2560 nmoles and the protein reconstitution was done at a lipid-to-protein molar ratio of 1000:1. Mag-Fura-2-loaded proteoliposomes were subsequently separated from nonencapsulated (free) Mag-Fura-2 by gel filtration on a Sephadex G-50 column (GE Healthcare, Chicago, IL) using a potassium-free buffer (150 mM N-methyl-D-glucamine chloride (NMDG-Cl), 20 mM HEPES, pH 8.0) to remove extraliposomal potassium and generate an outward potassium gradient. Fluorescence intensity measurements in a time-scan mode were made at room temperature in a PTI Quantamaster 8000 (HORIBA) spectrofluorometer in 3 ml quartz cuvette with constant stirring and a nominal bandpass of 5 by 5. Magnesium influx was initiated by adding 20 mM MgCl2 after making sure that the baseline for the Mag-Fura-2 loaded proteoliposomes is flat. Continuous recording of fluorescence emission intensity of Mag-Fura-2 at 509 nm was used to monitor the rate of magnesium influx while exciting at 329 nm. Experiments with protein-free Mag-fura-2 liposomes served as control.

Steady-state fluorescence measurements

Steady-state fluorescence measurements were performed with a Hitachi F-7000 spectrofluorometer using 1 cm path length quartz cuvettes. Excitation and emission slits with a nominal bandpass of 5 nm were used for all measurements except in Mg²⁺ titration experiments in which excitation and emission slits used were 1 nm for emission maximum measurements. Background intensities were appropriately subtracted from each sample spectrum to cancel out any contribution due to the solvent Raman peak and other scattering artefacts. Corrected spectra were recorded for measuring tryptophan fluorescence of MgtE in micelles and when reconstituted in membranes. Fluorescence anisotropy measurements were performed at room temperature using Hitachi polarization accessory. Anisotropy values were calculated from the equation [37]:

$$r=\frac{I_{VV}-G{I}_{VH}}{I_{VV}+2G{I}_{VH}}$$ (Eq. 2)

where, I_VV and I_VH are the measured fluorescence intensities (after appropriate background subtraction) with the excitation polarizer vertically oriented and emission polarizer vertically and horizontally oriented, respectively. G is the grating correction factor and is the ratio of the efficiencies of the detection system for vertically and horizontally polarized light, and is equal to I_HV/I_HH. The apparent (average) rotational correlation times were calculated using Perrin's equation [37]:

$${\tau }_c=\tau >r/\left(r_o-r\right)$$ (Eq. 3)

where r_o is the limiting anisotropy of Trp (0.16), r is the steady-state anisotropy, and <τ> is the mean fluorescence lifetime taken from Table 1.

Red edge excitation shift (REES) measurements were done by measuring the emission maximum as a function of increasing excitation wavelength from 295 to 305 nm. The magnitude of REES represents the total shift in emission maximum upon the indicated change in the excitation wavelength. The REES data was fitted by a Gaussian probability distribution of the form [59]:

$$f(x)=R_0+\frac{A\sqrt{2/\pi }}{w}\exp \left(-2{\left(\frac{x-m}{w}\right)}^2\right)$$ (Eq. 4)

where A is the area, w is the full width at half-maximum (fwhm), m is the midpoint and R ₀ is the y-intercept and m is the excitation wavelength that gives the largest change in the emission peak wavelength.

Time-resolved fluorescence measurements

Fluorescence lifetimes were calculated from time-resolved fluorescence intensity decays using HORIBA Fluoromax-3 in time-correlated single-photon counting (TCSPC) mode with a picosecond pulsed 285 nm Nano-LED as the light source. Lamp profiles were measured at the excitation wavelength using Ludox (colloidal silica) as the scatterer. To optimize the signal/noise ratio, 10,000 photon counts were collected in the peak channel. All experiments were performed with a bandpass of 5-8 nm. Fluorescence intensity decay curves so obtained were deconvoluted with the instrument response function and analysed as a sum of exponential terms:

$$F(t)=\sum _i{\alpha }_i\exp \left(-t/{\tau }_i\right)$$ (Eq. 5)

where F(t) is the fluorescence intensity at time t and α_i is a pre-exponential factor representing the fractional contribution to the time-resolved decay of the component with a lifetime τ _i such that ∑_i α _i = 1. Mean (average) lifetimes <τ> for triexponential decays of fluorescence were calculated from the decay times and preexponential factors using the following equation [37]:

$$<\tau >=\frac{{\alpha }_1{\tau }_1^2+{\alpha }_2{\tau }_2^2+{\alpha }_3{\tau }_3^2}{{\alpha }_1{\tau }_1+{\alpha }_2{\tau }_2+{\alpha }_3{\tau }_3}$$ (Eq. 6)

The mean fluorescence lifetime, τ_H, can be directly calculated using a model-independent approach from the histogram of photons obtained during fluorescence lifetime measurements using the following equation [25,42]:

$${\tau }_H=\frac{\sum _{i=p}^n\left(N_i-\text{ noise }\right)t_i}{\sum _{i=p}^n\left(N_i-\text{ noise }\right)}-t_p$$ (Eq. 7)

where N_i and t_i denote the number of detected photons in the i-th channel and the corresponding value on the time axis, respectively, n is the total number of channels in the histogram, p is the channel with the highest number of detected photons (peak of the decay) and t_p is the corresponding time.

Maximum Entropy Method (MEM) analysis of fluorescence intensity decay

The fluorescence decay data analysis by MEM represents a convenient, robust, model-free and realistic approach of data analysis [93–95]. In MEM, the fluorescence intensity decay [I(t)] is analysed using the model of continuous distribution of lifetimes:

$$I(t)={\int }_0^{\infty }\alpha (\tau )\exp (-t/\tau )dt$$ (Eq. 8)

where α(τ) represents the amplitude corresponding the lifetime τ in the intensity decay. In practice, the limits on the above integration are set based on the information regarding the system under study and the detection limit of the instrument. In our case, the lower and upper limits are set to 0.1 ns and 10 ns, respectively. For practical purposes, the above equation can be written in terms of a discrete sum of exponentials as

$$I(t)=\sum _{i=1}^N{\alpha }_i\exp \left(-t/{\tau }_i\right)$$ (Eq. 9)

where N represents the total number of exponentials. In our analysis, N is taken as 100 exponentials equally spaced in the log(τ) space between the lower and upper limits. MEM initially starts with a flat distribution of amplitudes a(τ), i.e., each lifetime has equal contribution in the beginning and arrives at the amplitude distribution which best describes the observed experimental fluorescence intensity decay. The optimization of the amplitude distribution α(τ) is carried out in successive iterations controlled by a regularization parameter γ (set to a value of 0.001) such that the χ² is minimized while maximizing the entropy (S). The expression used for S is the Shannon- Jaynes entropy function, which is:

$$S(\alpha )=-\sum _{i=1}^N{\alpha }_i\log \left(\frac{{\alpha }_i}{b_i}\right)$$ (Eq. 10)

where the set of values b represent a default model for the system. However, in the absence of a default model for our system, b_i are generally set to a constant value. Using constant b_i value favours equal contribution from all lifetimes which means that the introduction of structure into the distribution is discouraged. The analysis is terminated when χ² reaches the specified lower limit or when χ² and α(τ) show no change in successive iterations. All MEM fits were performed on a standard PC using the open access AnalyseDistribution MATLAB code (see ref. [95] for further details).

Fluorescence quenching measurements

Acrylamide quenching experiments were carried out by measurement of fluorescence intensity of MgtE in 20 mM HEPES, 150 mM NaCl, 1 mM DDM (pH 8.0) buffer by sequential addition of freshly prepared stock solution (1 M) of acrylamide in water to each sample. After addition of acrylamide, the sample was incubated in dark for about 5 minutes before taking the measurement. The excitation wavelength used was 295 nm and emission was monitored at the respective emission maxima. After dilution correction, the inner filter effect correction was made using the following equation [37]:

$$F=F_{\text{obs }}\text{antilog}\left[\left(A_{ex}+A_{em}\right)/2\right]$$ (Eq. 11)

where F is the corrected fluorescence intensity and F_obs is the background subtracted fluorescence intensity of the sample, A_ex and A_em are the measured absorbance at the excitation and emission wavelengths. The absorbance of the samples was measured using a Jasco V-650 ultraviolet (UV)- visible spectrophotometer. Quenching data were analysed by fitting to the Stern-Volmer equation [37]:

$$F_{\text{o}}/F=1+K_{\text{SV}}[Q]=1+k_{\text{q}}{\tau }_{\text{o}}[Q]$$ (Eq. 12)

where F _o and F are the fluorescence intensities in the absence and presence of the quencher, respectively, K _SV is the Stern-Volmer quenching constant, and [Q] is the molar quencher concentration. The Stern-Volmer quenching constant K _SV is equal to k_qτ_o, where k_q is the bimolecular quenching constant and τ_o is the lifetime of the fluorophore in the absence of quencher.

Abbreviations

CBS

cystathione-β-synthase

CD

circular dichroism

DDM

n-dodecyl-β-D-maltopyranoside

HEPES

hydroxyethylpiperazine ethane sulfonic acid

IPTG

isopropylthiogalactoside

MEM

maximum entropy method

NMDG-Cl

N-methyl-D-glucamine chloride

PDB

protein data bank

POPC

1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

POPG

1-palmitoyl-2-oleoyl-sn- glycero-3-phospho-(1'-rac-glycerol)

REES

red edge excitation shift

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SEC

size exclusion chromatography

TCSPC

time-correlated single photon counting

Trp

tryptophan