Site-directed Spin Labeling EPR Spectroscopy Research Articles

Structural Flexibility of Tau in Its Interaction with Microtubules as Viewed by Site-Directed Spin Labeling EPR Spectroscopy.

Tau is a microtubule-associated protein that belongs to the Intrinsically Disordered Proteins (IDPs) family. IDPs or Intrinsically Disordered Regions (IDRs) play key roles in protein interaction networks and their dysfunctions are often related to severe diseases. Defined by their lack of stable secondary and tertiary structures in physiological conditions while being functional, these proteins use their inherent structural flexibility to adapt to and interact with various binding partners. Knowledges on the structural dynamics of IDPs and their different conformers are crucial to finely decipher fundamental biological processes controlled by mechanisms such as conformational adaptations or switches, induced fit, or conformational selection events. Different mechanisms of binding have been proposed: among them, the so-called folding-upon-binding in which the IDP adopts a certain conformation upon interacting with a partner protein, or the formation of a "fuzzy" complex in which the IDP partly keeps its dynamical character at the surface of its partner. The dynamical nature and physicochemical properties of unbound as well as bound IDPs make this class of proteins particularly difficult to characterize by classical bio-structural techniques and require specific approaches for the fine description of their inherent dynamics.Among other techniques, Site-Directed Spin Labeling combined with Electron Paramagnetic Resonance (SDSL-EPR) spectroscopy has gained much interest in this last decade for the study of IDPs. SDSL-EPR consists in grafting a paramagnetic label (mainly a nitroxide radical) at selected site(s) of the macromolecule under interest followed by its observation using and/or combining different EPR strategies. These nitroxide spin labels detected by continuous wave (cw) EPR spectroscopy are used as perfect reporters or "spy spins" of their local environment, being able to reveal structural transitions, folding/unfolding events, etc. Another approach is based on the measurement of inter-label distance distributions in the 1.5-8.0nm range using pulsed dipolar EPR experiments, such as Double Electron-Electron Resonance (DEER) spectroscopy. The technique is then particularly well suited to study the behavior of Tau in its interaction with its physiological partner: microtubules (MTs). In this chapter we provide a detailed experimental protocol for the labeling of Tau protein and its EPR study while interacting with preformed (Paclitaxel-stabilized) MTs, or using Tau as MT inducer. We show how the choice of nitroxide label can be crucial to obtain functional information on Tau/tubulin complexes.

Electron paramagnetic resonance spectroscopic characterization of the human KCNE3 protein in lipodisq nanoparticles for structural dynamics of membrane proteins

One of the major challenges in solubilization of membrane proteins is to find the optimal physiological environment for their biophysical studies. EPR spectroscopy is a powerful biophysical technique for studying the structural and dynamic properties of macromolecules. However, the challenges in the membrane protein sample preparation and flexible motion of the spin label limit the utilization of EPR spectroscopy to a majority of membrane protein systems in a physiological membrane-bound state. Recently, lipodisq nanoparticles or styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) have emerged as a membrane mimetic system for investigating the structural studies of membrane proteins. However, its detail characterization for membrane protein studies is still poorly understood. Recently, we characterized the potassium channel membrane protein KCNQ1 voltage sensing domain (KCNQ1-VSD) and KCNE1 reconstituted into lipodisq nanoparticles using EPR spectroscopy. In this study, the potassium channel accessory protein KCNE3 containing flexible N- and C-termini was encapsulated into proteoliposomes and lipodisq nanoparticles and characterized for studying its structural and dynamic properties using nitroxide based site-directed spin labeling EPR spectroscopy. CW-EPR lineshape analysis data indicated an increase in spectral line broadenings with the addition of the styrene-maleic acid (SMA) polymer which approaches close to the rigid limit providing a homogeneous stabilization of the protein-lipid complex. Similarly, EPR DEER measurements indicated an enhanced quality of distance measurements with an increase in the phase memory time (Tm) values upon incorporation of the sample into lipodisq nanoparticles, when compared to proteoliposomes. These results agree with the solution NMR structural structure of the KCNE3 and EPR studies of other membrane proteins in lipodisq nanoparticles. This study along with our earlier studies will provide the reference characterization data that will provide benefit to the membrane protein researchers for studying structural dynamics of challenging membrane proteins.

Dynamics of the HP1 Hinge Region with DNA Measured by Site-Directed Spin Labeling-EPR Spectroscopy

Dynamics of the HP1 Hinge Region with DNA Measured by Site-Directed Spin Labeling-EPR Spectroscopy

Structural dynamics of the chromo-shadow domain and chromodomain of HP1 bound to histone H3K9 methylated peptide, as measured by site-directed spin-labeling EPR spectroscopy

The structural dynamics of the chromo-shadow domain (CSD) and chromodomain (CD) of human HP1 proteins essential for heterochromatin formation were investigated at the nanosecond and nanometer scales by site-directed spin labeling electron paramagnetic resonance and pulsed double resonance spectroscopy. Distance measurements showed that the spin-labeled CSD of human HP1α and HP1γ tightly dimerizes. Unlike CD-CD interaction observed in fission yeast HP1 in an inactivated state (Canzio et al., 2013), the two CDs of HP1α and HP1γ were spatially separated from each other, dynamically mobile, and ready for a Brownian search for H3K9-tri-methyl(me3) on histones. Complex formation of the CD with H3K9me3 slowed dynamics of the domain due to a decreased diffusion constant. CSD mobility was significantly (∼1.3-fold) lower in full-length HP1α than in HP1γ, suggesting that the immobilized conformation of human HP1α shows an auto-inactivated state. Differential properties of HP1α and HP1γ to form the inactive conformation could be relevant to its physiological role in the heterochromatin formation in a cell.

Investigation of Structural Dynamics of the KCNE3 in a Lipid Bilayer Membrane using Site-Directed Spin Labeling EPR Spectroscopy

KCNE3, a single pass membrane protein, is responsible for controlling the function of multiple potassium channels, one of which is the KCNQ1 channel. The binding of KCNE3 with KCNQ1 is vital in transepithelial potassium ion recycling which has been associated with disorders such as cystic fibrosis. Although KCNE3 has great biological significance, its structural and dynamic information in a native like environment is poorly understood. In this study, we have utilized site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) spectroscopic technique to investigate structural dynamics of KCNE3 in lipid bilayers. KCNE3 was overexpressed and purified in LMPG detergent micelles. MTSL spin label was introduced followed by the incorporation of the protein into POPC/POPG lipid bilayer. Continuous wave (CW)-EPR spectral lineshape analysis was performed on several spin labeling sites including transmembrane domain and extracellular domain of KCNE3 in LMPG detergent micelles and POPC/POPG lipid bilayes to obtain the spin label sidechain mobility and the rotational correlation times. Our EPR data indicated that the nitroxide spin label sidechains in liposomes have slower motion for the sites in the transmembrane domain of KCNE3 when compared to that in the extracellular region of KCNE3. Our results are consistent with the previously published solution NMR structure of KCNE3.

Conformational Dynamics of Sensory Rhodopsin II in Nanolipoprotein and Styrene-Maleic Acid Lipid Particles.

Styrene-maleic acid lipid particles (SMALPs) provide stable water-soluble nanocontainers for lipid-encased membrane proteins. Possible effects of the SMA-stabilized lipid environment on the interaction dynamics between functionally coupled membrane proteins remain to be elucidated. The photoreceptor sensory rhodopsin II, NpSRII and its cognate transducer, NpHtrII, of Natronomonas pharaonis form a transmembrane complex, NpSRII2 /NpHtrII2 that plays a key role in negative phototaxis and provides a unique model system to study the light-induced transfer of a conformational signal between two integral membrane proteins. Photon absorption induces transient structural changes in NpSRII comprising an outward movement of helix F that cause further conformational alterations in NpHtrII. We applied site-directed spin labeling and time-resolved optical and EPR spectroscopy to compare the conformational dynamics of NpSRII2 /NpHtrII2 reconstituted in SMALPs with that of nanolipoprotein particle and liposome preparations. NpSRII and NpSRII2 /NpHtrII2 show similar photocycles in liposomes and nanolipoprotein particles. An accelerated decay of the M photointermediate found for SMALPs can be explained by a high local proton concentration provided by the carboxylic groups of the SMA polymer. Light-induced large-scale conformational changes of NpSRII2 /NpHtrII2 observed in liposomes and nanolipoprotein particles are affected in SMALPs, indicating restrictions of the protein's conformational freedom.

Characterization of KCNE1 inside Lipodisq Nanoparticles for EPR Spectroscopic Studies of Membrane Proteins.

EPR spectroscopic studies of membrane proteins in a physiologically relevant native membrane-bound state are extremely challenging due to the complexity observed in inhomogeneity sample preparation and dynamic motion of the spin-label. Traditionally, detergent micelles are the most widely used membrane mimetics for membrane proteins due to their smaller size and homogeneity, providing high-resolution structure analysis by solution NMR spectroscopy. However, it is often difficult to examine whether the protein structure in a micelle environment is the same as that of the respective membrane-bound state. Recently, lipodisq nanoparticles have been introduced as a potentially good membrane mimetic system for structural studies of membrane proteins. However, a detailed characterization of a spin-labeled membrane protein incorporated into lipodisq nanoparticles is still lacking. In this work, lipodisq nanoparticles were used as a membrane mimic system for probing the structural and dynamic properties of the integral membrane protein KCNE1 using site-directed spin labeling EPR spectroscopy. The characterization of spin-labeled KCNE1 incorporated into lipodisq nanoparticles was carried out using CW-EPR titration experiments for the EPR spectral line shape analysis and pulsed EPR titration experiment for the phase memory time (Tm) measurements. The CW-EPR titration experiment indicated an increase in spectral line broadening with the addition of the SMA polymer which approaches close to the rigid limit at a lipid to polymer weight ratio of 1:1, providing a clear solubilization of the protein-lipid complex. Similarly, the Tm titration experiment indicated an increase in Tm values with the addition of SMA polymer and approaches ∼2 μs at a lipid to polymer weight ratio of 1:2. Additionally, CW-EPR spectral line shape analysis was performed on six inside and six outside the membrane spin-label probes of KCNE1 in lipodisq nanoparticles. The results indicated significant differences in EPR spectral line broadening and a corresponding inverse central line width between spin-labeled KCNE1 residues located inside and outside of the membrane for lipodisq nanoparticle samples when compared to lipid vesicle samples. These results are consistent with the solution NMR structure of KCNE1. This study will be beneficial for researchers working on studying the structural and dynamic properties of membrane proteins.

Site-Directed Spin Labeling EPR Spectroscopy of the Cytoplasmic Tail of Influenza a M2

The M2 protein is a 97 residue homotetrameric, multifunctional ion channel that plays critical roles during the influenza infection cycle. While a variety of high-resolution biophysical techniques have been used to characterize the transmembrane domain (residues 22-46) and the juxtamembrane C-terminal region (46-62), less is known about the conformation and dynamics of the remaining residues of the C-terminal cytoplasmic tail. Here, we use site-directed spin labeling electron paramagnetic spectroscopy (SDSL-EPR) experiments to probe the secondary structure and membrane topology of cytoplasmic tail residues 60-80 when the protein is reconstituted into lipid bilayers. Cholesterol is essential for the role the C-terminal domain of the M2 protein plays in viral budding. SDSL-EPR data is collected in both in the presence and absence of cholesterol.

Elucidating the design principles of photosynthetic electron-transfer proteins by site-directed spin labeling EPR spectroscopy

Site-directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy is a powerful tool to determine solvent accessibility, side-chain dynamics, and inter-spin distances at specific sites in biological macromolecules. This information provides important insights into the structure and dynamics of both natural and designed proteins and protein complexes. Here, we discuss the application of SDSL EPR spectroscopy in probing the charge-transfer cofactors in photosynthetic reaction centers (RC) such as photosystem I (PSI) and the bacterial reaction center (bRC). Photosynthetic RCs are large multi-subunit proteins (molecular weight≥300kDa) that perform light-driven charge transfer reactions in photosynthesis. These reactions are carried out by cofactors that are paramagnetic in one of their oxidation states. This renders the RCs unsuitable for conventional nuclear magnetic resonance spectroscopy investigations. However, the presence of native paramagnetic centers and the ability to covalently attach site-directed spin labels in RCs makes them ideally suited for the application of SDSL EPR spectroscopy. The paramagnetic centers serve as probes of conformational changes, dynamics of subunit assembly, and the relative motion of cofactors and peptide subunits. In this review, we describe novel applications of SDSL EPR spectroscopy for elucidating the effects of local structure and dynamics on the electron-transfer cofactors of photosynthetic RCs. Because SDSL EPR Spectroscopy is uniquely suited to provide dynamic information on protein motion, it is a particularly useful method in the engineering and analysis of designed electron transfer proteins and protein networks. This article is part of a Special Issue entitled Biodesign for Bioenergetics — the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

The guanine nucleotide exchange factor Ric-8A induces domain separation and Ras domain plasticity in Gαi1

Heterotrimeric G proteins are activated by exchange of GDP for GTP at the G protein alpha subunit (Gα), most notably by G protein-coupled transmembrane receptors. Ric-8A is a soluble cytoplasmic protein essential for embryonic development that acts as both a guanine nucleotide exchange factor (GEF) and a chaperone for Gα subunits of the i, q, and 12/13 classes. Previous studies demonstrated that Ric-8A stabilizes a dynamically disordered state of nucleotide-free Gα as the catalytic intermediate for nucleotide exchange, but no information was obtained on the structures involved or the magnitude of the structural fluctuations. In the present study, site-directed spin labeling (SDSL) together with double electron-electron resonance (DEER) spectroscopy is used to provide global distance constraints that identify discrete members of a conformational ensemble in the Gαi1:Ric-8A complex and the magnitude of structural differences between them. In the complex, the helical and Ras-like nucleotide-binding domains of Gαi1 pivot apart to occupy multiple resolved states with displacements as large as 25 Å. The domain displacement appears to be distinct from that observed in Gαs upon binding of Gs to the β2 adrenergic receptor. Moreover, the Ras-like domain exhibits structural plasticity within and around the nucleotide-binding cavity, and the switch I and switch II regions, which are known to adopt different conformations in the GDP- and GTP-bound states of Gα, undergo structural rearrangements. Collectively, the data show that Ric-8A induces a conformationally heterogeneous state of Gαi and provide insight into the mechanism of action of a nonreceptor Gα GEF.

Spin Labeling Studies of Transmembrane Signaling and Transport: Applications to Phototaxis, ABC Transporters and Symporters.

Membrane proteins still represent a major challenge for structural biologists. This chapter will focus on the application of continuous wave and pulsed EPR spectroscopy on spin-labeled membrane proteins. Site-directed spin labeling EPR spectroscopy has evolved as a powerful tool to study the structure and dynamics of proteins, especially membrane proteins, as this method works largely independently of the size and complexity of the biological system under investigation. This chapter describes applications of this technique to three different systems: the archaeal photoreceptor/-transducer complex SRII/HtrII as an example for transmembrane signaling and two transport systems, the histidine ATP-binding cassette transporter HisQMP, and the sodium-proline symporter PutP.

Light-induced switching of HAMP domain conformation and dynamics revealed by time-resolved EPR spectroscopy

HAMP domains are widely abundant signaling modules. The putative mechanism of their function comprises switching between two distinct states. To unravel these conformational transitions, we apply site-directed spin labeling and time-resolved EPR spectroscopy to the phototactic receptor/transducer complex NpSRII/NpHtrII. We characterize the kinetic coupling of NpHtrII to NpSRII along with the activation period of the transducer and follow the transient conformational signal. The observed transient shift towards a more compact state of the HAMP domain upon light-activation agrees with structure-based calculations. It thereby validates the two modeled signaling states and integrates the domain’s dynamics into the current model.

Assessing induced folding within the intrinsically disordered C-terminal domain of the Henipavirus nucleoproteins by site-directed spin labeling EPR spectroscopy

This work aims at characterizing structural transitions within the intrinsically disordered C-terminal domain of the nucleoprotein (NTAIL) from the Nipah and Hendra viruses, two recently emerged pathogens gathered within the Henipavirus genus. To this end, we used site-directed spin labeling combined with electron paramagnetic resonance spectroscopy to investigate the α-helical-induced folding that Henipavirus NTAIL domains undergo in the presence of the C-terminal X domain of the phosphoprotein (PXD). For each NTAIL protein, six positions located within four previously proposed molecular recognition elements (MoREs) were targeted for spin labeling, with three of these positions (475, 481, and 487) falling within the MoRE responsible for binding to PXD (Box3). A detailed analysis of the impact of the partner protein on the labeled NTAIL variants revealed a dramatic modification in the environment of the spin labels grafted within Box3, with the observed modifications supporting the formation of an induced α-helix within this region. In the free state, the slightly lower mobility of the spin labels grafted within Box3 as compared to the other positions suggests the existence of a transiently populated α-helix, as already reported for measles virus (MeV) NTAIL. Comparison with the well-characterized MeV NTAIL–PXD system, allowed us to validate the structural models of Henipavirus NTAIL–PXD complexes that we previously proposed. In addition, this study highlighted a few notable differences between the Nipah and Hendra viruses. In particular, the observation of composite spectra for the free form of the Nipah virus NTAIL variants spin labeled in Box3 supports conformational heterogeneity of this partly pre-configured α-helix, with the pre-existence of stable α-helical segments. Altogether these results provide insights into the molecular mechanisms of the Henipavirus NTAIL–PXD binding reaction.

Probing structural transitions in both structured and disordered proteins using site‐directed spin‐labeling EPR spectroscopy

EPR spectroscopy is a technique that specifically detects unpaired electrons. EPR-sensitive reporter groups (spin labels or spin probes) can be introduced into biological systems via site-directed spin-labeling (SDSL). The basic strategy of SDSL involves the introduction of a paramagnetic group at a selected protein site. This is usually accomplished by cysteine-substitution mutagenesis, followed by covalent modification of the unique sulfydryl group with a selective reagent bearing a nitroxide radical. In this review we briefly describe the theoretical principles of this well-established approach and illustrate how we successfully applied it to investigate structural transitions in both human pancreatic lipase (HPL), a protein with a well-defined α/β hydrolase fold, and the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N(TAIL) ) upon addition of ligands and/or protein partners. In both cases, SDSL EPR spectroscopy allowed us to document protein conformational changes at the residue level. The studies herein summarized show that this approach is not only particularly well-suited to study IDPs that inherently escape atomistic description by X-ray crystallography but also provides dynamic information on structural transitions occurring within well-characterized structured proteins for which X-ray crystallography can only provide snapshots of the initial and final stages.

Probing the Structural and Dynamic Properties of KCNE1 Using Site-Directed Spin Labeling EPR Spectroscopy

KCNE1 (minK) is a membrane protein known to modulate the function of the KCNQ1 voltage-gated potassium ion channel in the human heart. KCNE1 has been over expressed and purified into SDS detergent and reconstituted into POPC/POPG lipid vesicles. We show that that KCNE1 maintains its ability to associate with and modulate KCNQ1 K+ channel activity when placed into POPC/POPG liposomes. Functional measurements of KCNE1 proteoliposomes were performed in Xenopus oocytes expressing KCNQ1 (KV7.1). Our CD measurements show that KCNE1 undergoes a global change in secondary structure when removed from a micelle environment and placed in a lipid bilayer. CW-EPR and Power Saturation EPR experiments have been used to examine the structural and dynamic properties of KCNE1 in micelles and lipid bilayer vesicles. The results indicate that KCNE1 inserts into the POPC/POPG vesicles, S28C and R32C are solvent-exposed and located outside the membrane, and S64C and F54C are buried inside the POPC/POPG lipid bilayer. The differences of KCNE1 placed into either micelles or lipid bilayers will be discussed.

Structural Rearrangements Underlying Mg2+ Dependent Gating in Cora

The recent structures of CorA from Thermotoga maritima have provided an excellent model for a molecular understanding of Mg2+ transport. So far all crystal structures, obtained at high divalent cation concentrations, appear to be in a “closed” conformation. Macroscopic currents recorded from oocytes macropatches show that Mg2+-binding to the cytoplasmic side may act as a gating factor, defining CorA as a Mg2+-activated, Mg2+-channel. The role of the putative Mg2+ sensor was tested by engineering a salt bridge between residue D253 and D89 at the interface of the each monomer. The presence of a positive charge at either residue was sufficient to lock CorA in a closed conformation independent of the free [Mg2+].Using site-directed spin-labeling EPR spectroscopy, we were able to detect Mg2+-dependent conformational transitions with an apparent KD close to 2 mM, a concentration in the physiological range of free [Mg2+] in cells. The structural rearrangements associated with CorA gating were measured on 106 positions along the stalk helix and TM1 segment, near the fivefold axis of symmetry. Strikingly, a significant increase in the spin-spin dipolar coupling at the tip of the stalk helix was detected from CW-EPR and DEER measurements. Mapping these EPR-determined conformational changes on the CorA crystal structure suggest an explicit structural mechanism for CorA opening, where an umbrella-like closing motion of the stalk helix would lead to an expansion of the pore immediately after the kink. This conformational wave propagate to the pore forming helix TM1 that rotates, ultimately increasing the diameter of the permeation pathway. This mechanism is supported by an increase in both, NieddA accessibility and probe mobility with a concomitant decrease in spin-spin coupling along the permeation pathway. Altogether, these results suggest a plausible molecular mechanism of gating in Magnesium channel related to CorA.

Mapping the Global Conformation of the Phi29 Packaging RNA Dimer Using Deer Distance Constraints

The insertion of bacteriophage phi29 genomic DNA into its preformed procapsid requires the DNA packaging motor, which is the strongest known biological motor. The packaging motor is an intricate ring-shape protein/RNA complex. The RNA component, called the packaging RNA (pRNA), is indispensable for motor function, and may play an essential role in motor ATPase activity. Current structural information on pRNA is limited, which hinders our effort on understanding motor function. Here, we use site-directed spin labeling and pulse EPR spectroscopy to map the global structure of a pRNA dimer that has been shown to be a functional intermediate in assembling the ring-shaped pRNA complex in the packaging motor. In our studies, nitroxide pairs were attached to specific sites of a truncated monomeric pRNA construct, the labeled monomers were then assembled into dimers in the presence of Mg2+, and inter-nitroxide distances were measured using DEER (Double Electron-Electron Resonance) spectroscopy. In parallel, an unbiased pool of models that contains variable pRNA conformations was generated, which treats pRNA as a 3-way junction construct, and a set of corresponding inter-nitroxide distances was predicted for each model. Intra-molecular DEER distances were used to obtain the monomeric structures of pRNA in dimer, which are then used to build the structural pool for pRNA dimers. A very small number of models were selected. We expect that this work will provide much-needed structural information regarding pRNA, as well as establishing a new methodology for analyzing global conformations in complex RNAs.

Lid Opening and Unfolding in Human Pancreatic Lipase at Low pH Revealed by Site-Directed Spin Labeling EPR and FTIR Spectroscopy

The structural changes induced in human pancreatic lipase (HPL) by lowering the pH were investigated using a combined approach involving the use of site-directed spin labeling coupled to electron paramagnetic resonance (SDSL-EPR) and Fourier transform infrared (ATR-FTIR) spectroscopy. The secondary structure of HPL observed with ATR-FTIR spectroscopy was found to be stable in the pH range of 3.0-6.5, where HPL remained active. Using a spin-label introduced into the lid of HPL at position 249, a reversible opening of the lid controlling the access to the active site was observed by EPR spectroscopy in the pH range of 3.0-5.0. In the same pH range, some structural changes were also found to occur outside the lid in a peptide stretch located near catalytic aspartate 176, using a spin-label introduced at position 181. Below pH 3.0, ATR-FTIR measurements indicated that HPL had lost most of its secondary structure. At these pH levels, the loss of enzyme activity was irreversible and the ability of HPL to bind to lipid emulsions was abolished. The EPR spectrum of the spin-label introduced at position 181, which was typical of a spin-label having a high mobility, confirmed the drastic structural change undergone by HPL in this particular region. The EPR spectrum of the spin-label at position 249 indicated, however, that the environment of this residue within the lid was not affected at pH 3.0 in comparison with that observed in the pH range of 3.0-5.0. This finding suggests that the disulfide bridge between the hinges of the lid kept the secondary structure of the lid intact, whereas the HPL was completely unfolded.

Both maltose-binding protein and ATP are required for nucleotide-binding domain closure in the intact maltose ABC transporter

The maltose transporter MalFGK(2) of Escherichia coli is a member of the ATP-binding cassette superfamily. A periplasmic maltose-binding protein (MBP) delivers maltose to MalFGK(2) and stimulates its ATPase activity. Site-directed spin labeling EPR spectroscopy was used to study the opening and closing of the nucleotide-binding interface of MalFGK(2) during the catalytic cycle. In the intact transporter, closure of the interface coincides not just with the binding of ATP, as seen with isolated nucleotide-binding domains, but requires both MBP and ATP, implying that MBP stimulates ATPase activity by promoting the closure of the nucleotide-binding interface. After ATP hydrolysis, with MgADP and MBP bound, the nucleotide-binding interface resides in a semi-open configuration distinct from the fully open configuration seen in the absence of any ligand. We propose that P(i) release coincides with the reorientation of transmembrane helices to an inward-facing conformation and the final step of maltose translocation into the cell.

Mapping α‐helical induced folding within the intrinsically disordered C‐terminal domain of the measles virus nucleoprotein by site‐directed spin‐labeling EPR spectroscopy

Using site-directed spin-labeling EPR spectroscopy, we mapped the region of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N(TAIL)) that undergoes induced folding. In addition to four spin-labeled N(TAIL) variants (S407C, S488C, L496C, and V517C) (Morin et al. (2006), J Phys Chem 110: 20596-20608), 10 new single-site cysteine variants were designed, purified from E. coli, and spin-labeled. These 14 spin-labeled variants enabled us to map in detail the gain of rigidity of N(TAIL) in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol or the C-terminal domain X (XD) of the viral phosphoprotein. Different regions of N(TAIL) were shown to contribute to a different extent to the binding to XD, while the mobility of the spin labels grafted at positions 407 and 460 was unaffected upon addition of XD; that of the spin labels grafted within the 488-502 and the 505-522 regions was severely and moderately reduced, respectively. Furthermore, EPR experiments in the presence of 30% sucrose allowed us to precisely map to residues 488-502, the N(TAIL) region undergoing alpha-helical folding. The mobility of the 488-502 region was found to be restrained even in the absence of the partner, a behavior that could be accounted for by the existence of a transiently populated folded state. Finally, we show that the restrained motion of the 505-522 region upon binding to XD is due to the alpha-helical transition occurring within the 488-502 region and not to a direct interaction with XD.