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Magnesium (Mg2+) is the most abundant divalent cation in the cell and is critical for numerous cellular processes. Despite its importance, the mechanisms of intracellular Mg2+ transport and its regulation are poorly understood. MgtE is the main Mg2+ transport system in almost half of bacterial species and is an ortholog of mammalian SLC41A1 transporters, which are implicated in neurodegenerative diseases and cancer. To date, only MgtE from Thermus thermophilus (MgtETT) has been extensively characterized, mostly in detergent micelles, and gating-related structural dynamics in biologically relevant membranes are scarce. The MgtE homolog from Bacillus firmus (MgtEBF) is unique since it lacks the entire Mg2+-sensing N-domain but has conserved structural motifs in the TM-domain for Mg2+ transport. In this work, we have successfully purified this novel homolog in a stable and functional form, and ColabFold structure prediction analysis suggests a homodimer. Further, microscale thermophoresis experiments show that MgtEBF binds Mg2+ and ATP, similar to MgtETT. Importantly, we show that, despite lacking the N-domain, MgtEBF mediates Mg2+ transport function in the presence of an inwardly directed Mg2+ gradient in reconstituted proteoliposomes. Furthermore, comparison of the organization and dynamics of Trp residues in the TM-domain of MgtEBF in membrane mimetics, in apo- and Mg2+-bound forms, suggests that the cytoplasmic binding of Mg2+ might involve modest gating-related conformational changes at the TM-domain. Overall, our results show that the gating-related structural dynamics (hydration dynamics, conformational heterogeneity) of the full-length MgtEBF is significantly changed in functionally pertinent membrane environment, emphasizing the importance of lipid-protein interactions in MgtE gating mechanisms.

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Understanding the mechanisms of membrane protein function is critical for biomedical research and drug discovery as membrane proteins constitute ∼30% of the proteins encoded by the genomes of both lower and higher organisms and are targets for two-thirds of approved drugs worldwide. Significant progress has been made in engineering host expression systems for large-scale production of membrane proteins and in determining their three-dimensional high-resolution structures. Despite these efforts, the study of membrane proteins at the atomic level is challenging due to poor expression and extraction, low yields of functional protein, and the complexity and heterogeneity of source membranes. Structural and spectroscopic studies of any membrane protein require that the protein be extracted from its native membranes into a membrane-mimetic stable environment, which is often achieved by the use of detergents. Unfortunately, there is no magic detergent that can extract all membrane proteins and successful extraction often requires a thorough screen of detergents. Furthermore, membrane protein purification in general and the detergents used are very expensive, which puts a financial constraint on sophisticated membrane protein studies. To overcome this hurdle, a dual-detergent strategy has recently been developed and successfully applied to purify various classes of pure, stable, and functionally relevant membrane proteins in a cost-effective manner. This strategy uses an inexpensive detergent for solubilization of the desired protein from membranes and a second detergent during protein purification. In the Basic Protocol, we describe the dual-detergent strategy to significantly reduce the overall purification cost of a bacterial membrane protein using the magnesium ion channel MgtE as an example. Support Protocols are also provided for selecting a suitable E. coli strain for protein expression and the optimal detergent(s) for membrane protein solubilization. © 2022 Wiley Periodicals LLC. Basic Protocol: Expression, membrane solubilization, and cost-effective purification of MgtE Support Protocol 1: Selecting a suitable E. coli strain for optimal protein expression Support Protocol 2: Identification of suitable detergents for membrane protein solubilization.

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The structural organization and dynamic nature of the biomembrane components are important determinants for numerous cellular functions. Particularly, membrane proteins are critically important for various physiological functions and are important drug targets. The mechanistic insights on the complex functionality of membrane lipids and proteins can be elucidated by understanding the interplay between structure and dynamics. In this regard, membrane penetration depth represents an important parameter to obtain the precise depth of membrane-embedded molecules that often define the conformation and topology of membrane probes and proteins. In this review, we discuss about the widely used fluorescence quenching-based methods (parallax method, distribution analysis, and dual-quencher analysis) to accurately determine the membrane penetration depths of fluorescent probes that are either membrane-embedded or attached to lipids and proteins. Further, we also discuss a relatively novel fluorescence quenching method that utilizes tryptophan residue as the quencher, namely the tryptophan-induced quenching, which is sensitive to monitor small-scale conformational changes (short distances of < 15 Å) and useful in mapping distances in proteins. We have provided numerous examples for the benefit of readers to appreciate the importance and applicability of these simple yet powerful methods to study membrane proteins.

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Comprehensive understanding of a protein's function depends on having reliable, sophisticated tools to study protein structural dynamics in physiologically-relevant conditions. Here, we present an effective, robust step-by-step protocol to monitor the structural dynamics (including hydration dynamics) of a protein utilizing various site-directed fluorescence (SDFL) approaches. This protocol should be widely applicable for studying soluble proteins, intrinsically-disordered proteins, and membrane proteins. For complete details on the use and execution of this protocol, please refer to Das et al. (2020), Das and Raghuraman (2021), and Chatterjee et al. (2021).

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Protein hydration dynamics plays an important role in many physiological processes since protein fluctuations, slow solvation, and the dynamics of hydrating water are all intrinsically related. Red edge excitation shift (REES) is a unique and powerful wavelength-selective (i.e. excitation-energy dependent) fluorescence approach that can be used to directly monitor the environment-induced restriction and dynamics around a polar fluorophore in a complex biological system. This review is mainly focused on recent applications of REES and a novel analysis of REES data to monitor the structural dynamics, functionally relevant conformational transitions and to unmask the structural ensembles in proteins. In addition, the novel utility of REES in imaging protein aggregates in a cellular context is discussed. We believe that the enormous potential of REES approach showcased in this review will engage more researchers, particularly from life sciences.

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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 site-specific 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.

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