Spin-labeled Side Chains Research Articles

Time-Resolved EPR Spectroscopy of Channelrhodopsin-2 Helix B Movements

The light-gated dimeric cation channel channelrhodopsin-2 (ChR2) is one of the most important optogenetic tools. Upon light activation ChR2 undergoes conformational changes, the most prominent ones include a movement of transmembrane helix B. In the present work, we apply time resolved continuous wave EPR spectroscopy to follow spectral changes of a spin label bound to position C79 located in helix B. We observed an increase of the motional freedom of the spin label side chain in illuminated ChR2. The recovery of the underlying light-induced conformational change in the dark is correlated with the recovery of the P480 state of ChR2. The observed conformational changes might be thus key elements responsible for desensitizing the channel for cation conduction.

Use of Site-Directed Spin Labeling EPR Spectroscopy to Study Protein-LPS Interactions.

Site-directed spin labeling EPR (electron paramagnetic resonance) spectroscopy is a technique used to identify the local conformational changes at a specific residue of interest within a purified protein in response to a ligand. Here, we describe the site-directed spin labeling EPR spectroscopy methodology to monitor changes in the side-chain motion in soluble lipopolysaccharide transport proteins upon the addition of lipopolysaccharide (LPS). A comparison of the spectral overlays of the spin-labeled protein in the absence and presence of LPS provides a qualitative visualization of how LPS binding affects the motion of each spin-labeled site tested within the protein. No change in the spectral lineshapes of a spin-labeled protein in the absence and presence of LPS indicates that the site is not affected by LPS binding, while differences in the spectral lineshapes indicate that LPS does affect the mobility of the spin label side chain within the protein structure. This is a powerful readout of conformational changes at specific residues of interest that can be used to identify a specific site as a reporter of changes induced by ligand binding and to map out the effects of ligand binding through an array of reporter sites within a protein. With the use of AquaStar tubing, protein concentrations as low as 2μM allow for up to a 100-fold excess of LPS. This methodology may also be applied to other protein-ligand or protein-protein interactions with minor adaptations.

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.

Dynamics and Environmental Characteristics of Spin Labels in a KvAP Voltage Sensor by Molecular Dynamics Simulations.

The nitroxide spin label is the most widely used probe for electron paramagnetic resonance (EPR) spectroscopy studies of the structure and function of biomolecules. However, the role of surrounding environments in determining the dynamics of nitroxide spin labels in biological complex systems remains to be clarified. This study aims to characterize the dynamics and environmental structure of spin labels in the voltage-sensing domain (VSD) of a KvAP potassium channel by means of molecular dynamics (MD) studies. MD simulations for unlabeled and 132 spin-labeled KvAP-VSD models (spin labels introduced at positions 20-151) were carried out in a phospholipid bilayer to evaluate conformational dynamics of nitroxide spin-label side chains in the VSD. Structural flexibility, conformational freedom, and orientation of the spin-label side chains were investigated in relation to their dynamics in different microenvironments. The analysis of MD data showed that the attached spin-label probe did not severely perturb the protein dynamics. The conformational freedoms of the nitroxide side chain vary with the physical structure of the surrounding environments. The two terminal dihedral angles of the nitroxide side chain tend to cluster and adopt several preferred rotameric states. From the nearest-neighbor analysis, the spin label can be exposed to either a homogeneous or heterogeneous environment with various exposure scenarios. The dynamical movement of KvAP-VSD is high at a water-exposed site, moderate in the membrane, and low in the protein core. Understanding the structure and dynamics behaviors of spin labels helps to manage the experimental uncertainty and avoid misleading interpretation in relation to the protein structure.

Structure and dynamics of spin label side chains in KvAP voltage-sensor domain: an all-atom MD simulation study

Site-directed spin labeling (SDSL) combined with continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy has become a useful approach to investigate the structural and dynamical properties of biomolecules, especially membrane protein. In this method, spin label side chains due to their motion highly depending on the local environment were employed as external molecules to probe the structure and dynamics of the membrane protein. The close relationships between the dynamics of the spin label side chain and protein structure have been studied for T4 lysozyme. Besides, molecular dynamics (MD) simulations were widely applied as a powerful tool to study the microscopic behavior of the proteins, completing experimental approaches. Here, we presented an all-atom MD simulation study of the nitroxide spin label (MTSSL) in the X-ray structure of a potassium channel voltage-sensor domain (VSD) from Aeropyrum Pernix (KvAP).

Electrostatics at Peptide-Lipid Interface in Nano-Bio Hybrid Systems by Spin-Labeling EPR

The ionization states of individual amino acid residues of membrane proteins are difficult to decipher or assign directly in the lipid membrane environment. The effective pK(a) values of protein groups are determined by a complex interplay between local polarity, Coulomb interactions, and a structural reorganization. The analysis is further complicated by the dearth of information about gradients in polarity, electric potentials, and hydration at the protein-membrane interface. The goal of our work is to develop spin-labeling EPR methods for assessing effects of membrane surface potential, local environment at the protein-membrane interface, and water penetration along this interface on effective pK(a) of membrane-burred ionisable groups. In this work we report on developing pH-sensitive ionizable EPR labels and related methods to 1) profile a heterogeneous dielectric environment along the α-helix of a WALP peptide integrated in a lipid bilayer; 2) asses the effect of solid support, specifically silica nanoparticles, on effective pK(a) of membrane-burred ionisable sidechains; and 3) use DEER to assess the location of spin-labeled sidechains. pH-sensitive EPR labels were attached to cysteine residues positioned at various depth within lipid bilayers at the peptide-lipid interface. We have shown that effective pK(a) of membrane-burred sidechain can be significantly shifted by varying the membrane surface charge density. The silica support exerted pronounced effects on WALP dynamics and the effective pK(a) of the ionizable probe. It was demonstrated that the silica nanoparticles shift the effective pK(a) of the ionizable nitroxide probe in a membrane depth-dependent manner. Upon protonation of the membrane-burred model ionisable sidechain the silica support caused significant changes in the membrane association of WALP peptide that is not observed when WALP is integrated into unilamellar phospholipid vesicles of similar curvature.Supported by NSF 1508607 to TIS.

Mapping out the Degree of Freedom of Hosted Enzymes in Confined Spatial Environments

Summary The integration of enzymes with solid materials is crucial for promoting their industrialization. Understanding the enzyme behavior upon association within a confined space, though of fundamental importance for biocomposite development, remains a persistent, unresolved challenge. Here, we present a comprehensive elucidation of the spatial environment’s impact on hosted enzymes’ degree of freedom and, consequently, their accompanying reactivity. Site-directed spin labeling in combination with electron paramagnetic resonance spectroscopy allows the direct detection of host-guest interactions at atomic resolution, while the tailorable synthesis of covalent organic frameworks (COFs) enables an evaluation of factors affecting such interactions. Specifically, lysozyme is found to be more constrained and less active along with increasing hydrophilicity of the COFs. These results support the establishment of a connection between the hydrophilicity of the spatial environment and the resulting biocomposites’ reactivity, enabling the prediction of the performance of unknown biocomposites. This study provides a unique insight into the mechanistic pathways underpinning biocatalysis.

EPR Techniques to Probe Insertion and Conformation of Spin-Labeled Proteins in Lipid Bilayers.

Electron paramagnetic resonance (EPR) spectroscopy of spin-labeled membrane proteins is a valuable biophysical technique to study structural details and conformational transitions of proteins close to their physiological environment, for example, in liposomes, membrane bilayers, and nanodiscs. Unlike in nuclear magnetic resonance (NMR) spectroscopy, having only one or few specific side chains labeled at a time with paramagnetic probes makes the size of the object under investigation irrelevant in terms of technique sensitivity. As a drawback, extensive site-directed mutagenesis is required in order to analyze the properties of the protein under investigation. EPR can provide detailed information on side chain dynamics of large membrane proteins or protein complexes embedded in membranes with an exquisite sensitivity for flexible regions and on water accessibility profiles across the membrane bilayer. Moreover, distances between the two spin-labeled side chains in membrane proteins can be detected with high precision at cryogenic temperatures. The application of EPR to membrane proteins still presents some challenges in terms of sample preparation, sensitivity and data interpretation, thus it is difficult to give ready-to-go methodological recipes. However, new technological developments (arbitrary waveform generators) and new spin labels spectroscopically orthogonal to nitroxides increased the range of applicability from in vitro toward in-cell EPR experiments. This chapter is an updated version of the one published in the first edition of the book and describes the state of the art in the application of nitroxide-based site-directed spin labeling EPR to membrane proteins, addressing new tools such as arbitrary waveform generators and spectroscopically orthogonal labels, such as Gd(III)-based labels. We will present challenges in sample preparation and data analysis for functional and structural membrane protein studies using site-directed spin labeling techniques and give experimental details on EPR techniques providing information on side chain dynamics and water accessibility using nitroxide probes. An updated optimal Q-band DEER setup for nitroxide probes will be described, and its extension to gadolinium-containing samples will be addressed.

Toward Precise Interpretation of DEER-Based Distance Distributions: Insights from Structural Characterization of V1 Spin-Labeled Side Chains.

Pulsed electron paramagnetic resonance experiments can measure individual distances between two spin-labeled side chains in proteins in the range of ∼1.5-8 nm. However, the flexibility of traditional spin-labeled side chains leads to diffuse spin density loci and thus distance distributions with relatively broad peaks, thereby complicating the interpretation of protein conformational states. Here we analyzed the spin-labeled V1 side chain, which is internally anchored and hence less flexible. Crystal structures of V1-labeled T4 lysozyme constructs carrying the V1 side chain on α-helical segments suggest that V1 side chains adopt only a few discrete rotamers. In most cases, only one rotamer is observed at a given site, explaining the frequently observed narrow distance distribution for doubly V1-labeled proteins. We used the present data to derive guidelines that may allow distance interpretation of other V1-labeled proteins for higher-precision structural modeling.

Determination of nitroxide spin label conformations via PELDOR and X-ray crystallography.

Pulsed electron-electron double resonance (PELDOR or DEER) in combination with site-directed spin labelling has emerged as an important method for measuring nanometer distance constraints that are used to obtain coarse-grained structures of biomolecules or to follow their conformational changes. Translating measured spin-spin distances between spin labels into structural information requires taking the conformational flexibility of spin label side chains into account. Here, we present an analysis of orientation selective PELDOR data recorded on six singly MTSSL-labelled azurin mutants. The analysis yielded conformational MTSSL ensembles, which are considerably narrower than those predicted using in silico spin labeling methods but match well with spin label conformations found in the corresponding crystal structures. The possible reasons and consequences for predicting spin label conformers in the fold of biomolecules are discussed.

Probing Structural Dynamics and Topology of the KCNE1 Membrane Protein in Lipid Bilayers via Site-Directed Spin Labeling and Electron Paramagnetic Resonance Spectroscopy.

KCNE1 is a single transmembrane protein that modulates the function of voltage-gated potassium channels, including KCNQ1. Hereditary mutations in the genes encoding either protein can result in diseases such as congenital deafness, long QT syndrome, ventricular tachyarrhythmia, syncope, and sudden cardiac death. Despite the biological significance of KCNE1, the structure and dynamic properties of its physiologically relevant native membrane-bound state are not fully understood. In this study, the structural dynamics and topology of KCNE1 in bilayered lipid vesicles was investigated using site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy. A 53-residue nitroxide EPR scan of the KCNE1 protein sequence including all 27 residues of the transmembrane domain (45-71) and 26 residues of the N- and C-termini of KCNE1 in lipid bilayered vesicles was analyzed in terms of nitroxide side-chain motion. Continuous wave-EPR spectral line shape analysis indicated the nitroxide spin label side-chains located in the KCNE1 TMD are less mobile when compared to the extracellular region of KCNE1. The EPR data also revealed that the C-terminus of KCNE1 is more mobile when compared to the N-terminus. EPR power saturation experiments were performed on 41 sites including 18 residues previously proposed to reside in the transmembrane domain (TMD) and 23 residues of the N- and C-termini to determine the topology of KCNE1 with respect to the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) lipid bilayers. The results indicated that the transmembrane domain is indeed buried within the membrane, spanning the width of the lipid bilayer. Power saturation data also revealed that the extracellular region of KCNE1 is solvent-exposed with some of the portions partially or weakly interacting with the membrane surface. These results are consistent with the previously published solution NMR structure of KCNE1 in micelles.

Conformational Flexibility and Structure in High-Pressure Excited States of Apomyoglobin Revealed by SDSL-EPR

Proteins exist in an ensemble of conformations at equilibrium, and while higher energy (“excited”) states may play important functional roles, under normal conditions they are too sparsely populated to detect. The application of high hydrostatic pressure may be used to populate excited states, but characterization of the high-pressure conformational ensemble is complicated by the presence of multiple conformations exchanging on the μs-ms time scale. The method of site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) is ideally suited to explore such excited states. The intrinsic timescale of EPR (ns) is fast compared to that of conformational exchange, so the EPR spectrum captures a snapshot of conformational equilibrium frozen in time and can reveal the existence of multiple conformational substates. For each resolved substate, the spectral lineshape encodes information on the local backbone dynamics and tertiary fold. In the present work, a set of spin-labeled mutants of both holo- and apomyoglobin were studied in the pressure range 0 - 2 kbar. Many EPR spectra in the well-ordered holomyoglobin native state are essentially pressure independent, demonstrating that the internal motion of the spin label side chain is pressure insensitive. Thus, pressure-dependent spectral changes observed in more flexible systems can be directly interpreted in terms of protein compressibility rather than side chain effects. Pressurization of apomyoglobin up to 2 kbar populates a low-lying excited state that has been designated a “high-pressure molten globule,” although the work presented here shows it to be distinct from the conventional low pH molten globule that is an intermediate along the folding pathway. Interestingly, the EPR spectra of apomyoglobin at 2 kbar suggest the existence of a conformation with native-like dynamics within the ensemble of conformations that comprise the high-pressure molten globule.

Structural Basis for Allosteric Coupling at the Membrane-Protein Interface in Gloeobacter violaceus Ligand-gated Ion Channel (GLIC)

Ligand binding at the extracellular domain of pentameric ligand-gated ion channels initiates a relay of conformational changes that culminates at the gate within the transmembrane domain. The interface between the two domains is a key structural entity that governs gating. Molecular events in signal transduction at the interface are poorly defined because of its intrinsically dynamic nature combined with functional modulation by membrane lipid and water vestibules. Here we used electron paramagnetic resonance spectroscopy to delineate protein motions underlying Gloeobacter violaceus ligand-gated ion channel gating in a membrane environment and report the interface conformation in the closed and the desensitized states. Extensive intrasubunit interactions were observed in the closed state that are weakened upon desensitization and replaced by newer intersubunit contacts. Gating involves major rearrangements of the interfacial loops, accompanied by reorganization of the protein-lipid-water interface. These structural changes may serve as targets for modulation of gating by lipids, alcohols, and amphipathic drug molecules.

The Attachment Affinity of Hemoglobin toward Silver-Containing Bioactive Glass Functionalized with Glutaraldehyde

Bioactive glasses belonging to the 56SiO2·(40 - x)CaO·4P2O5·xAg2O system, with x = 0, 2, and 8 mol %, were surface functionalized with the protein coupling agent glutaraldehyde (GA) and further evaluated in terms of hemoglobin affinity. The bare and GA-functionalized samples were investigated before and after protein attachment, by electron paramagnetic resonance (EPR) spectroscopy combined with spin-labeling procedure. Methanethiosulfonate spin label was used to explore the local environment of β-93 cysteine in horse hemoglobin, in terms of spin label side chain mobility. The EPR simulation methods were employed to quantify the rotational correlational times and fraction of the immobilized spin labels. The EPR absorption spectrum was further exploited to estimate the amount of hemoglobin loaded on the substrates. The surface elemental composition obtained by X-ray photoelectron spectroscopy revealed similar tendency in terms of surface coverage. Changes in surface architecture, that is, changes in surface morphology after protein coverage, were observed by scanning electron microscopy. It was concluded that GA improves the stability of protein attachment and induces polymerization of hemoglobin molecules.

Rotamer Library of Spin Labeled Cysteines Attached to T4 Lysozyme Deduced from Molecular Dynamics Simulations Constrained by Double Electron-Electron Resonance (Deer) Experiments

DEER (Double Electron Electron Resonance) is a powerful pulsed EPR technique allowing the determination of spin-spin distance histograms between side-directed nitroxide label sites on a protein in their native environment. In this study, a novel molecular dynamics simulation method, the restrained ensemble (RE) method, has been used for the refinement of spin labels inserted in T4 Lysozyme (T4L) structures. In the RE method, the spin-spin distance distribution histograms calculated from a multiple-copy molecular dynamics simulations are forced, via a special ensemble-based energy restraint, to match those extracted from 51 EPR/DEER experimental spin-spin pairs. To examine the effect of the restraint on the rotameric state of the spin label at the 37 different sites in T4L, conventional long molecular dynamics (MD) and locally enhanced sampling (LES) simulations were also performed. As expected, the distance histograms obtained from the RE simulations are very similar to those obtained from experiment; however, the distance histograms from conventional long MD and LES simulations deviate from experiment. From the analysis of these simulations, a general trend is found in rotamer population distribution along five dihedral angles connecting the nitroxide ring to the protein backbone. Overall, the rotamer population obtained from the RE simulation agrees with those obtained from x-ray structure. From the analysis of the dynamics of nitroxide atoms in spin label side chains, a force field for a pseudo nitroxide dummy atom has been parameterized. This pseudo nitroxide was used in an RE simulation coupled with the EPR/DEER experimental distance distribution data to refine distorted structures of T4L. Thus, experimental restraints from DEER experiments in conjunction with RE simulations can be used to refine structural propertied of biological systems.

High-Resolution Structure of a Protein Spin-Label in a Solvent-Exposed β-Sheet and Comparison with DEER Spectroscopy

X-ray crystallography has been a useful tool in the development of site-directed spin labeling by resolving rotamers of the nitroxide spin-label side chain in a variety of α-helical environments. In this work, the crystal structure of a doubly spin-labeled N8C/K28C mutant of the B1 immunoglobulin-binding domain of protein G (GB1) was solved. The double mutant formed a domain-swapped dimer under crystallization conditions. Two rotameric states of the spin-label were resolved at the solvent-exposed α-helical site, at residue 28; these are in good agreement with rotamers previously reported for helical structures. The second site, at residue 8 on an interior β-strand, shows the presence of three distinct solvent-exposed side-chain rotamers. One of these rotamers is rarely observed within crystal structures of R1 sites and suggests that the H(α) and S(δ) hydrogen bond that is common to α-helical sites is absent at this interior β-strand residue. Variable temperature continuous wave (CW) experiments of the β-strand site showed two distinct components that were correlated to the rotameric states observed in crystallography. Interestingly, the CW data at room temperature could be fit without the use of an order parameter, which is consistent with the lack of the H(α) and S(δ) interaction. Additionally, double electron electron resonance (DEER) spectroscopy was performed on the GB1 double mutant in its monomeric form and yielded a most probable interspin distance of 25 ± 1 Å. In order to evaluate the accuracy of the measured DEER distance, the rotamers observed in the crystal structure of the domain-swapped GB1 dimer were modeled into a high-resolution structure of the wild type monomeric GB1. The distances generated in the resulting GB1 structural models match the most probable DEER distance within ~2 Å. The results are interesting as they indicate by direct experimental measurement that the rotameric states of R1 found in this crystal provide a very close match to the most probable distance measured by DEER.

Simulating the dynamics and orientations of spin-labeled side chains in a protein-DNA complex.

Site-directed spin labeling, wherein a nitroxide side chain is introduced into a protein at a selected mutant site, is increasingly employed to investigate biological systems by electron spin resonance (ESR) spectroscopy. An understanding of the packing and dynamics of the spin label is needed to extract the biologically relevant information about the macromolecule from ESR measurements. In this work, molecular dynamics (MD) simulations were performed on the spin-labeled restriction endonuclease, EcoRI in complex with DNA. Mutants of this homodimeric enzyme were previously constructed, and distance measurements were performed using the double electron electron resonance experiment. These correlated distance constraints have been leveraged with MD simulations to learn about side chain packing and preferred conformers of the spin label on sites in an α-helix and a β-strand. We found three dihedral angles of the spin label side chain to be most sensitive to the secondary structure where the spin label was located. Conformers sampled by the spin label differed between secondary structures as well. C(α)-C(α) distance distributions were constructed and used to extract details about the protein backbone mobility at the two spin labeled sites. These simulation studies enhance our understanding of the behavior of spin labels in proteins and thus expand the ability of ESR spectroscopy to contribute to knowledge of protein structure and dynamics.

Lipid and Membrane Mimetic Environments Modulate Spin Label Side Chain Configuration in the Outer Membrane Protein A

In the present work, the factors that determine EPR line shapes from spin labels at the protein-hydrocarbon interface of a β-barrel membrane protein are examined. The EPR spectra from hydrocarbon facing sites in the outer membrane protein A (OmpA) are highly dependent upon the detergent or lipid into which OmpA is reconstituted. In general, line shapes at these sites are correlated with the solvent accessibility in the supporting amphiphile. A notable exception is CHAPS, which yields rigid limit EPR line shapes for labels at every position along a transmembrane β-strand in OmpA. EPR line shapes from the surface of OmpA are not strongly influenced by steric interference with neighboring side chains, but are modulated by solutes that should interact with hydrophobic surfaces. These results suggest that differences in EPR spectra in different supporting environments are not the result of differences in protein dynamics but are a result of different configurations or rotameric states that are assumed by the label. This conclusion is supported by distance measurements across the OmpA β-barrel, which indicate that labels yielding more motionally restricted line shapes interact more closely with the protein surface. These results have implications for the use of spin-label-derived distance constraints in protein structure determination and demonstrate that spin labels on membrane proteins provide a highly sensitive probe for the environment surrounding a membrane protein.

Nitroxide Spin Label Side Chain Dynamics of Solvent Exposed Sites on Membrane Proteins

Understanding the structure and functional dynamics of membrane proteins in their native, hydrophobic environment is key to understanding how proteins function. EPR spectroscopy in combination with site directed spin labeling (SDSL) has the potential to quantify structure and dynamics of proteins of arbitrary weight in their native lipid environment. Several studies have elucidated the structural origins of CW EPR lineshapes of water-soluble proteins; however, CW EPR spectra of nitroxide spin labeled proteins in a detergent/lipid environment have characteristic differences from their water-soluble counterparts. Membrane protein spectra are generally broader and frequently contain multiple spectral components. In this study, membrane exposed sites on model α-helical and β-barrel proteins LeuT and OmpA, respectively, were investigated using X-ray crystallography, mutational analysis, nitroxide side chain derivatives, and spectral simulations in order to obtain a motional model of the nitroxide. For each crystal structure of spin labeled LeuT, the nitroxide ring of the spin label is resolved and makes contacts with hydrophobic residues on the protein surface. In addition to the crystal structures, the spectral effect of nearest neighbor mutations and the spectral simulations suggest the spin label has a greater affinity for its local protein environment compared to water-soluble proteins. These results for spin labeled sites on LeuT and OmpA begin to explain the observed trends in membrane protein CW EPR spectra.

Understanding the Membrane Docking Geometry of GRP1 PH Domain Using Site-Directed Spin-Labeling and EPR

PH domains are membrane targeting motifs that typically recruit signaling proteins to specific membrane surfaces in response to the appearance of lipid second messengers. Despite their functional significance, little is known about how PH domains dock to the bilayer surface on a structural level. Here we focus on the representative PH domain of the General Receptor for Phosphoinositides 1 (GRP1 PH), which specifically binds the signaling lipid phosphatidylinositol (3,4,5)-trisphosphate (PIP3). To elucidate the membrane docking geometry of this PH domain, site-directed spin-labeling and EPR power saturation method are being employed. Single cysteine residues are introduced at positions scattered over the membrane binding face and subsequently labeled with a thio-reactive spin-label. The EPR power saturation method is utilized to measure the bilayer depth of each spin-labeled side chain when the PH domain is docked to a PIP3-containing synthetic lipid bilayer. The resulting membrane depths, in conjunction with the known crystal structure, will be used to model the penetration depth and angle of the PH domain relative to the bilayer surface. The latest findings will be presented at the meeting.