Novel Insights in Linking Solvent Relaxation Dynamics and Protein Conformations Utilizing Red Edge Excitation Shift Approach

Abstract

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.

Introduction

Water in biology plays a crucial role in determining the structure and the dynamics of macromolecules such as proteins and can help to fine tune protein functionality in a variety of ways [1,2]. In cells, it is estimated that 10-25% of water molecules have significantly slower reorientational dynamics compared to bulk water [2]. It is widely appreciated that the cellular environment is crowded, and hence, only ~3-4 layers of water molecules are present between proteins [3]. It is well established that a layer of non-bulk water (hydration layer of ~ 1 nm at the physiological ionic strength of about 150 mM) exists around proteins [4], which generally corresponds to ~0.2 grams of water per gram of protein and is the necessary minimum for the proper functioning of proteins [1,3]. This interfacial water molecules around proteins has distinct properties than bulk water [5], and are highly heterogeneous that reflects the roughness of the protein surface [6,7]. Further, the region-specific hydration dynamics surrounding the protein has been shown to be heterogeneous with timescales ranging from few hundred femtoseconds to a few hundred picoseconds [8]. Mechanistic interpretation of the function of a protein is often correlated with its structure without giving much importance to the dynamics of microscopic observables such as water molecules. Several studies suggest that rather than merely acting as solvent, water could be considered as a structural component in modulating protein functions [9–11]. It is therefore necessary to appreciate the dynamics of the constituent molecules as well as that of the system as a whole to understand the structure-function relationship in proteins. In fact, role of water in the protein matrix is now considered as a largely untapped resource for drug discovery [12].

Since protein fluctuations, slow solvation, and the dynamics of hydrating water are all intrinsically related [1,13], hydration dynamics in general plays an important role in several physiological processes such as protein folding, ligand recognition [3], lipid-protein interactions [10,14], and ion channel selectivity [15]. Understanding the functionally-relevant hydration dynamics therefore assumes significance. Although several sophisticated techniques have been utilized to probe protein hydration dynamics, one of the fluorescence approaches, namely the red edge excitation shift (REES) is arguably the most widely used tool to monitor the dynamics of restricted environments in proteins and membranes due to the relatively inexpensive instrumentation. 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, unmasking the structural ensembles in proteins and to detect protein aggregates in cells.

REES: A wavelength-selective fluorescence approach

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. REES is commonly defined as a shift in the wavelength of maximum fluorescence emission toward higher wavelengths (red-shift), caused by a shift in the excitation wavelength toward the red edge of the absorption band. The phenomenon of REES is observed predominantly for polar fluorophores in motionally-restricted polar environments such as viscous solutions, condensed phases or membrane-mimetic systems, where the dipolar relaxation time for the solvent molecules (τ_S) around the excited-state fluorophore is comparable to or longer than its fluorescence lifetime (τ_F) (Figure 1). Therefore, information obtained from REES approach in complex biological systems is valuable since water is the universal solvent and hydration dynamics plays a crucial role in many important cellular processes [2]. Apart from the slow rates of solvent relaxation dynamics, REES also arises due to the change in local dynamics of the protein matrix around the excited state dipole moment of the fluorophore (see later). In addition to the polar nature of fluorophore and surrounding environment, a relatively large change in the dipole moment of the fluorophore upon excitation is required to exhibit REES effects. In case of the most commonly found intrinsic fluorophore in proteins, i.e., the tryptophan (Trp) residue, the dipole moment change of ~6 D upon excitation [16] is sufficient to exhibit red edge effects.

Figure 1. The phenomenon of REES.

Schematic representation of emission profiles of a polar fluorophore in bulk solvent and in motionally-restricted media such as membrane interface, condensed phases etc. upon excitation towards red edge of the absorption band. The colored arrows represent different excitation wavelengths. The emission maximum remains invariant (i.e., no REES) for a polar fluorophore in bulk solvent due to fast solvent relaxation, whereas a significant shift in the wavelength of maximum fluorescence emission is observed when the polar fluorophore experiences the restricted environment due to slow solvent relaxation dynamics. τ_S is the dipolar relaxation time for the solvent molecules, and τ_F is the fluorescence lifetime of the fluorophore. See text for details.

The red edge effects have been discovered in 1970 (see [17] review for details) and, since then, have been extensively used in fluorescence studies to monitor complex micro-heterogeneous systems. In proteins, REES was first documented for the multidomain protein, human serum albumin (HSA), using intrinsic Trp fluorescence [18]. Several excellent reviews are available in literature that extensively discusses the progress in the field of red-edge effects and its physical origin [17,19], various models of solvent relaxation and criteria for obtaining REES [20,21], its application to membrane organization [22] and structural dynamics of soluble and membrane peptides/proteins [19–21,23,24]. The myriad applications of REES approach for protein and membrane studies are summarized in Figure 2.

Figure 2. Applications of REES in protein and membrane studies.

Schematic representation of REES applications is shown for soluble and membrane proteins to monitor (a) folding and aggregation; (b) intrinsically disordered proteins; (c) protein matrix; (e) membrane partitioning, folding and architecture; (f) lipid-protein interactions; and (g) hydration dynamics associated with different functional states. The random coil state of the protein is denoted by curved lines in (a) and (e), whereas the α-helical conformation is denoted by cylinders in (a), (b) and (e). In (c), the labelled fluorophore in the middle of the protein core is depicted in red, and the ‘Y’ shapes represent the relaxing polar side chains of the protein (green). The restricted/bound and bulk/free water molecules in (g) are represented by purple and blue spheres, respectively, around the protein conformers. Further, REES is a powerful approach to monitor membrane dynamics and heterogeneity (d). The colored spheres in (d) and (f) represent different types of lipid headgroups, and the intrinsic membrane protein and cholesterol are denoted by irregular shaped block and diamonds, respectively in (f). The recently developed novel application of REES in determining the discrete protein conformational states (free energy landscape) is depicted (h). The energy (E) barrier (left) represents a small number of discrete conformational states, tending towards a predominantly single conformation (purple circle), while the energy barrier (right) represents an increased number of discrete conformational states (colored circles). See text for details.

REES as a tool to monitor structural dynamics and conformational transitions in proteins

In proteins, the intrinsic fluorescence is often contributed by Trp residue(s) due to its dominant fluorescence characteristics. In addition, compared to other aromatic amino acids, Trp residues comprise only ~1% and ~3-7% in most soluble and membrane proteins, respectively, that allows site-specific probing of structural changes [24]. Importantly, the Trp fluorescence is highly sensitive to local environmental changes unlike Tyr residues, and hence the term ‘natural protein fluorescence’ is almost always associated with tryptophan fluorescence [21,24]. Trp fluorescence has therefore been widely used to monitor the local structure and dynamics in proteins [25], protein-protein interactions and lipid-protein interactions in membrane-mimetic systems [16,24,26,27]. Particularly, most of the REES studies in proteins have utilized the Trp fluorescence to probe the environmental restriction and slowly relaxing protein matrix [21,23,28]. Further, extrinsic fluorophores have also been used for REES studies of which 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group is the most extensively used for monitoring membrane dynamics [29–31], lipid-protein interactions [14,32] and conformational dynamics in proteins [10,24,32].

Since water plays a key role in structure and dynamics of proteins [1,3,8,12,33], REES has been effectively used to monitor the conformational changes of proteins during various stages of folding process. The first report in which a systematic characterization from an unstructured random coil state to the folded and aggregated state of a protein utilizing REES of the sole Trp fluorescence has been demonstrated for melittin [34], which is a membrane-active, hemolytic peptide with diverse cellular functions [35]. Further, the conversion of non-channel to channel conformations through the intermediates during folding has been studied using REES in the ion channel peptide, gramicidin [23]. The role of intramolecular dynamics in pH and temperature-induced changes in conformational stability of IgG1 and IgG2 antibodies has also been monitored using REES [36,37]. Importantly, REES data has been shown to be quite useful to distinguish between structurally identical therapeutic monoclonal antibodies, and are sensitive to aggregation and unfolding [38]. REES can therefore be effectively used as a powerful tool to provide a fingerprint of the structural stability of the protein.

Almost all native proteins exhibit REES with a possible exception of bovine serum albumin (BSA) whose tryptophan emission maximum is independent of the excitation wavelength [21]. It is generally assumed that unstructured peptides such as monomeric melittin in aqueous solution [21,34] and denatured proteins exhibit no or negligible REES since the native Trp residue(s) or the covalently labelled extrinsic probes are fully exposed to bulk water undergoing much faster solvent relaxation as compared to fluorescence lifetimes (Figure 3). Indeed, no REES has been observed for the denatured proteins such as HSA [39,40], cytoskeletal protein tubulin [21], pH (low) insertion peptide (pHLIP) derived from bacteriorhodopsin [41], oligomeric lectin protein concanavalin A [42], and the homodimeric E. coli toxin, controller of cell death B (CCdB) at pH 4 [43].

Figure 3. REES for native and unfolded states of proteins.

Shown are the schematic representation of a protein in native and unfolded states along with their representative REES data. The unfolded/denatured state is shown with both the presence and absence of any residual structure. Typically, upon excitation at the excitation maximum, the emission maximum of the native protein exhibits blue-shifted fluorescence emission maximum compared to the unfolded states. The bound/restricted and free/bulk water molecules are denoted as purple and blue spheres, respectively. λ_ex and λ_em correspond to excitation and emission wavelengths, respectively. See text for details.

Interestingly, several proteins have been shown to exhibit significant REES in denatured proteins, which demonstrates that the Trp residue(s) or the covalently labelled extrinsic probes are partially shielded from bulk water suggesting the presence of residual structure even in denatured state (see Figure 3). The slow solvent relaxation in the denatured protein monitored by REES has been first demonstrated for erythroid spectrin [44]. Since then, several proteins have been shown to exhibit REES in their denatured states, for instance, in Ca²⁺-binding milk protein, α-lactalbumin [45], brain spectrin [46], nuclear factor (NF)-kB essential modulator (NEMO) [47], bovine and horse seminal plasma proteins PDC-109 [48] and HSP1/2 [49], respectively. The uniqueness of REES approach is such that it is sensitive to detect the presence of residual structures in denatured proteins which is not possible using conventional circular dichroism (CD) measurements.

In protein folding, the molten globule (MG) state is considered to be an important intermediate that can remain in equilibrium with the tightly packed native (N) state and has important cellular implications [50–53]. REES has been utilized to explore the organization and dipolar relaxation dynamics in MG states of soluble proteins such as ferredoxin [54], α-lactalbumin [45,55], ovalbumin [56], HSA [39,40,57], and CCdB at low pH [43]. However, the observed REES effects in the MG states are system-dependent. For instance, the magnitude of REES (7 nm) remains the same in N and MG states in ovalbumin. Likewise, α-lactalbumin shows similar magnitude of REES in N and MG-I states, however, the REES magnitude in MG-II state of α-lactalbumin is identical to denatured state. Interestingly, in case of HSA and ferredoxin, the MG state has significantly increased REES (~6-11 nm) compared to the modest REES observed in N state (~2-3 nm). Based on the above considerations, although REES is a valuable approach to probe loosely packed MG states, which remain in equilibrium with N state, additional experiments are needed to validate the REES results.

Contrary to classical structure-function paradigm, intrinsically disordered proteins (IDP) do not require unique structures to perform their biological functions [58]. In addition, the structure-less IDPs can also undergo disordered-to-ordered transitions upon binding to their respective biological partners. Importantly, REES has been utilized as a sensitive tool to monitor the structural plasticity of IDPs. For example, in the case of amyloidogenic k-casein, the microenvironment of the sole Trp residue [59] and the ordered water molecules within the collapsed globule state [60] have been characterized using REES. In case of α-synuclein, which lacks Trp residue, the interaction with phospholipid membranes has been probed by incorporating non-native Trp residues at various locations [61]. Thus, REES has been helpful to distinguish subtle but important structural alterations in IDPs.

Hydration dynamics in membrane protein functional states

Since protein dynamics is intrinsically related to hydrating solvent molecules [1,3,8,12,13], hydration dynamics has been shown to play crucial roles in ligand-protein interactions [47,62], lipid-protein interactions [14,32,63], mediating ion channel functional states [9–11] and ion channel selectivity [15]. REES is therefore useful and has been utilized to distinguish between functionally-relevant conformational states of proteins. The magnitude of REES has been used to monitor the channel and non-channel conformations of the multi-Trp containing peptide, gramicidin [23]. However, these two conformations of gramicidin have distinct architecture. Interestingly, REES approach has also been used to monitor the structural dynamics of ion channel proteins in different functional states with identical architecture and topology. For instance, using site-directed fluorescence and REES approach on the NBD-labelled outer vestibule residues, it has been shown that significant differences exist in hydration dynamics when the pH-gated potassium ion channel KcsA [10,11,64,65] shuttles between open/conductive to open/inactivated conformation [10]. At the molecular level, toxins, blockers and metal ions bind to the outer vestibule and modulate the functional behaviour of potassium channels. Particularly, it has been shown experimentally, for the first time, that the C-type inactivated conformation of KcsA is associated with the presence of restricted/bound water molecules in the outer vestibule, whereas the open/conductive state has a relatively fast solvent relaxation [10]. It is interesting to note that the recovery from C-type inactivation is directly controlled by these buried water molecules as has been shown by long (~17 μs) molecular dynamics simulations [9]. The increased hydration dynamics in the open/conductive conformation of KcsA has been found to be linked with the highly dynamic outer vestibule of KcsA (Figure 4). This study supports the role of differential water dynamics in different functional states of KcsA, and the idea that water could act as a structural component in selectivity filter gating mechanisms of potassium ion channels [9,10].

Figure 4. Solvent relaxation dynamics of KcsA probed by REES in different functional states.

REES of NBD-labelled outer vestibule residues of full-length WT (left) and E71A (right) KcsA reconstituted in liposomes. Measurements at pH 7.5 and pH 4 correspond to closed and open states of the channel. The change in the emission maximum (in nm) as a function of changing the excitation wavelength from 465 to 515 nm is shown as REES (top). The difference REES values (ΔREES) are shown in middle, and the difference values were mapped on the crystal structure of KcsA to highlight the solvent relaxation dynamics changes between different functional states of KcsA (bottom) upon gating. Adapted and modified from ref. [10].

Das et al. [32] has recently utilized the site-directed NBD fluorescence of the functionally-important S3b-S4 loop residues of the isolated voltage sensing domain (VSD) of a voltage-gated K⁺ channel KvAP in its activated (‘Up’) conformation. This sensor loop localizes in the chemically heterogeneous environment in membranes in such a way that while some of the residues are partially exposed to aqueous medium, others partition at the membrane interface. The NBD-labelled S3b-S4 loop of the sensor exhibits significant REES in membranes and micelles, which is an indication of the motionally-restricted environments and the presence of restricted/bound water molecules in both these membrane-mimetic systems. Importantly, the magnitude of REES for most of the loop residues in membranes are lesser compared to micelles, indicating that the sensor loop in membranes experiences relatively less motional constraint imposed by the relaxing water molecules and thus the sensor loop adopts a dynamic/relaxed organization in membranes (Figure 5).

Figure 5. Membrane-induced conformational dynamic variability of the voltage sensor loop.

Schematic representation of the key differences in the organization and dynamics of S3b-S4 loop residues of KvAP-VSD in OG micelles (left) and in PC/PG membranes (right) are shown. Only a part of the S3b and S4 helices are shown and the positions of residues 110 and 117 are indicated by spheres (C_α atoms) in helices. The S3b-S4 loop region exhibits pronounced dynamic variability in membranes as depicted by the wiggly nature of the loop (right). In contrast, the micellar environment imposes motional restriction and induces increased number of discrete conformational substates of the loop region as denoted by more copies of the loop region (left). Changes in hydration dynamics are depicted by the increased ratio of bulk/free (empty circles) vs. restricted/bound (filled circles) water molecules. The curved and straight lines denote the arbitrary micellar and membrane boundaries, respectively. Adapted and modified from ref. [31].

Very recently, REES has been utilized to monitor the gating-related structural dynamics of MgtE magnesium channel by employing site-directed Trp fluorescence [27]. Since MgtE is a multi-tryptophan containing ion channel and analysis of ensemble Trp fluorescence is complicated [24], six single-Trp mutants in the functional Trp-less background of MgtE have been engineered to obtain site-specific information on MgtE in membrane-mimetic systems. Interestingly, gating-induced REES changes in membranes are observed only in Trp residues in the transmembrane domain and not in the Mg²⁺-sensing cytoplasmic N-domain of MgtE, which suggests that the core packing of the N-domain is not significantly altered when the channel shuttles between open and closed states in membranes. Interestingly, a novel analysis of REES data has been successfully used to monitor the changes in conformational substates of the KvAP sensor loop in membrane-mimetics and MgtE during gating (see later).

Dipolar protein matrix as the relaxing ‘solvent’ for REES effects

As mentioned above, the biological applications of REES are mostly focused to study the solvent (water) relaxation dynamics around the excited-state fluorophore to understand the complex solvation environments in systems like proteins and membrane-mimetics. In general, the dielectric constant inside a protein is lower compared to the protein surface [66], and the hydrophobic interior of proteins has varying degrees of conformational heterogeneity in side chain packing. Interestingly, it has been shown that substantial side chain fluctuations could make the protein core behave like a dense fluid [67,68]. In this regard, the dipolar protein matrix could act as the relaxing ‘solvent’ to give rise to REES effects rather than the dynamics of solvent molecules. Only few studies are available recently, which directly deals with the protein matrix as the relaxing ‘solvent’ for REES effects.

It has been demonstrated that, in enhanced green fluorescent protein (EGFP), the observed REES is solely due to slow dipolar relaxation of the rigid protein matrix around its centrally-localized fluorophore, and is independent of the viscosity and hydration of the surrounding medium [23]. In case of HSA, the magnitude of REES has been used as a reporter of heterogeneity and dynamics of the protein core. Using the extrinsic fluorophore 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (IAEDANS) covalently bound inside a protein core that is solvated by the buried polar side chains, it has been shown that the core of HSA is heterogeneously packed and the side chains solvating the buried fluorophore have relatively restricted motions similar to a viscous dense liquid [57]. The magnitude of REES of IAEDANS bound to HSA has been observed to be similar irrespective of the increase in viscosity of the exterior solvent. The authors attribute these results to the constrained solvation environment of the protein matrix contributed by the heterogenous orientation of the side chains and their slower dipolar relaxation, and not the dynamics of the water molecules around the excited-state fluorophore [57]. Similar behavior of the interior of proteins mimicking viscous liquid-like solvent has also recently been shown for the CcdB at pH 4 using the REES of intrinsic Trp residues [43]. In ligand-binding experiments with fibroblast growth factor-10, the possible role of the protein matrix has also been addressed [62]. Although these studies have shed novel information on the protein organization, the precise contribution of whether the relaxing restricted/bound water molecules or the relaxing protein matrix or both are responsible for the observed REES effects needs to be evaluated carefully with proper controls.

Novel REES analysis to reveal protein structural ensembles

Most of the studies using REES approach are mainly restricted to obtain qualitative information related to understanding the structural organization and dynamics of soluble and membrane proteins using the magnitude of REES [10,20,21,23,32]. However, the disadvantage of relying only on the magnitude of REES but not on the curvature of REES data overlooks crucial structural information in proteins. For instance, in different functional conformations of a protein (like closed vs open, active vs inactive and bound vs unbound etc.), the magnitude of REES can be same in both functional states, but the curvature associated with REES data might be significantly different (Figure 6). Recently, a novel analysis of REES data has been used to demonstrate that the REES can be used as a powerful tool to unmask protein structural ensembles and can potentially give information on the ‘ruggedness’ of the free energy landscape (FEL) [47]. This is done by fitting the steady-state REES data to a Gaussian probability distribution function and extracting the magnitude of area, which is sensitive to protein conformational equilibrium. Importantly, the changing curvature of REES data has the potential to give information on the changing number of discrete conformational substates despite the magnitude of REES being the same, for example, in two functional forms of proteins. Therefore, the magnitude of REES data along with its associated curvature can effectively be used to monitor the changes in protein FEL and the equilibrium of protein conformational states (Figure 6). This novel analysis of REES data has been validated in a single Trp-containing protein NEMO, which is the key regulator in the NF-kB signalling pathway [69]. Novel information regarding the interaction of NEMO with poly-ubiquitin has been obtained using REES, and this is also the first report of the pressure dependence of REES effect [47]. Further, this approach has been used to link the changes in protein conformational substates to enzyme catalysis as in the case of a model sugar cleaving enzyme oligo-1,6-glucosidase 1 [70].

Figure 6. Information obtained from REES data to distinguish protein functional states.

Schematic representation of REES data for two functional states (i.e., conformers A and B) of proteins is shown depicting differences in magnitude of REES (left) and same magnitude of REES with changing curvature (right). Interpretation from magnitude of REES and from the novel analysis of REES data to calculate area using Gaussian probability distribution function (see text and ref. [46] for details) is also shown. λ_ex and λ_em correspond to excitation and emission wavelengths, respectively. See text for details.

The first membrane protein for which this novel analysis of REES data has been used to probe the molecular heterogeneity is the isolated voltage sensing domain (VSD) of a voltage-gated K⁺ channel KvAP [32]. The magnitude of REES and the area derived from the REES data using the Gaussian probability distribution function has recently been utilized to decipher the structural dynamics and the conformational heterogeneity of the functionally-important S3b-S4 loop of the voltage sensor in micelles and membranes (see Figure 5). Importantly, it appears that the environmental heterogeneity is strongly correlated with the conformational heterogeneity of the sensor loop in phospholipid membranes, which has lesser number of discrete equilibrium conformational states compared to its organization in detergent micelles [32]. In case of MgtE magnesium channel, this novel REES analysis shows that MgtE-Trp residues might have altered conformational states in membranes compared to micelles. Further, REES results show that not only the dynamics of hydration is significantly different between membrane-mimetic systems, but also indicate the change in conformational states of MgtE during gating [27].

It should be mentioned that the 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 (intrinsic or extrinsic) does not exhibit REES or when the magnitude of REES is negligible. In this regard, the lifetime distribution analysis by maximum entropy method (MEM) is a direct read-out of structural heterogeneity irrespective of the solvent relaxation dynamics around the fluorophore since fluorescence lifetime distribution gives an ultrafast snapshot of the protein population distribution [71]. The combination of novel REES analysis and MEM-lifetime distribution has recently been exploited to support the notion of ligand-induced ‘conformational wave’ from the Mg²⁺-sensing N-domain to transmembrane domain of MgtE, and that the structural integrity of N-domain is preserved during the gating process [27].

Can REES be a potential tool in imaging protein organization in cells?

As discussed above, all studies that utilize REES have been carried out in vitro using the magnitude of REES (i.e., red-shifted emission maximum as a function of increasing excitation wavelength). It should be noted that excitation-energy dependent blue-shifted emission maximum has also been rarely observed although the physical phenomenon of red edge effects remains the same [72]. Importantly, it has recently been demonstrated that, using a confocal microscope set-up, REES could be used as a novel fluorescence-based approach to detect and image fluorescein-labelled ‘subvisible’ toxic amyloid β (Aβ) -protein aggregates in mouse cortical neuronal cells [72]. In this case, the blue-shifted emission maximum upon increasing excitation wavelength has been observed for amyloid constructs (Aβ_15-21 or Aβ_1-40) in solution. Interestingly, in contrast to Aβ_15-21, Aβ_1-40 shows a conventional REES effect in neuronal cells, which has been attributed to unique combination of Aβ_1-40 and the cellular microenvironment. Whether ‘REES microscopy’ can be a potential tool to monitor protein organization in cells needs to be explored further.

Conclusions and future perspectives

It has been ~40 years since the first report of REES in proteins and still REES is continuing to be a powerful approach to monitor the structural organization and dynamics of proteins and membrane-mimetic systems. We have provided a review of recent literature on the applications of REES to get information on dynamic insights into protein structure. Further, we have provided examples of studies using a novel analysis of REES data to probe the conformational heterogeneity by linking the solvent relaxation dynamics around the fluorophore in soluble and membrane proteins. As mentioned before, REES has been done mainly utilizing intrinsic Trp fluorescence and extrinsic fluorophores strategically labelled in a protein. By utilizing the REES approach, it is possible to obtain the dynamic hydration map of proteins in different functional states. However, REES studies probing the hydration dynamics of an entire protein is still lacking and few recent studies have combined site-directed fluorescence labelling and REES approach in consecutive amino acid residues in functionally-important regions of proteins to probe the environmental dynamics and restriction [10,32]. Importantly, REES combined with the MEM lifetime distribution results can be used as a powerful approach to monitor protein structural heterogeneity [65]. Considering the plethora of existing extrinsic fluorophore tool box and the continued development of novel and photostable fluorophores, the suitability of REES in cellular context [72] holds promise of using this approach in imaging various proteins in the near future.

Summary.

• Hydration dynamics is intrinsically related to the structure and function of proteins.

• REES is a well-established wavelength-selective fluorescence approach that arises due to slow rates of solvent (water) relaxation and polar side chain rearrangements in a protein core.

• REES provides qualitative information on the presence of restricted/bound water molecules in proteins and also to monitor the dynamics and packing of side chains in a protein matrix.

• REES is an effective fluorescence approach to investigate diverse physiologically-important phenomena that includes folding and aggregation of soluble and membrane proteins, lipid-protein interactions, protein-protein interactions, membrane dynamics etc.

• Novel quantitative analysis of REES data is useful to get information on the structural changes and the equilibrium of conformational states, i.e., conformational heterogeneity in proteins.