A new spin on protein dynamics

Site-directed spin labeling is a general method for investigating structure and conformational switching in soluble and membrane proteins. It will also be an important tool for exploring protein backbone dynamics. A semi-empirical analysis of nitroxide sidechain dynamics in spin-labeled proteins reveals contributions from fluctuations in backbone dihedral angles and rigid-body (collective) motions of alpha helices. Quantitative analysis of sidechain dynamics is sometimes possible, and contributions from backbone modes can be expressed in terms of relative order parameters and rates. Dynamic sequences identified by site-directed spin labeling correlate with functional domains, and so nitroxide scanning could provide an efficient strategy for identifying such domains in high-molecular weight proteins, supramolecular complexes and membrane proteins.     

Structural origins of nitroxide side chain dynamics on membrane protein α-helical sites

Understanding the structure and dynamics of membrane proteins in their native, hydrophobic environment is important to understanding how these proteins function. EPR spectroscopy in combination with site-directed spin labeling (SDSL) can measure dynamics and structure of membrane proteins in their native lipid environment; however, until now the dynamics measured have been qualitative due to limited knowledge of the nitroxide spin label's intramolecular motion in the hydrophobic environment. Although several studies have elucidated the structural origins of EPR line shapes of water-soluble proteins, EPR spectra of nitroxide spin-labeled proteins in detergents or lipids have characteristic differences from their water-soluble counterparts, suggesting significant differences in the underlying molecular motion of the spin label between the two environments. To elucidate these differences, membrane-exposed α-helical sites of the leucine transporter, LeuT, from Aquifex aeolicus, 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, the nitroxide ring of a disulfide-linked spin label side chain (R1) is resolved and makes contacts with hydrophobic residues on the protein surface. The spin label at site I204 on LeuT makes a nontraditional hydrogen bond with the ortho-hydrogen on its nearest neighbor F208, whereas the spin label at site F177 makes multiple van der Waals contacts with a hydrophobic pocket formed with an adjacent helix. These results coupled with the spectral effect of mutating the i ± 3, 4 residues suggest that the spin label has a greater affinity for its local protein environment in the low dielectric than on a water-soluble protein surface. The simulations of the EPR spectra presented here suggest the spin label oscillates about the terminal bond nearest the ring while maintaining weak contact with the protein surface. Combined, the results provide a starting point for determining a motional model for R1 on membrane proteins, allowing quantification of nitroxide dynamics in the aliphatic environment of detergent and lipids. In addition, initial contributions to a rotamer library of R1 on membrane proteins are provided, which will assist in reliably modeling the R1 conformational space for pulsed dipolar EPR and NMR paramagnetic relaxation enhancement distance determination.     

Toward the fourth dimension of membrane protein structure: insight into dynamics from spin-labeling EPR spectroscopy

Trapping membrane proteins in the confines of a crystal lattice obscures dynamic modes essential for interconversion between multiple conformations in the functional cycle. Moreover, lattice forces could conspire with detergent solubilization to stabilize a minor conformer in an ensemble thus confounding mechanistic interpretation. Spin labeling in conjunction with electron paramagnetic resonance (EPR) spectroscopy offers an exquisite window into membrane protein dynamics in the native-like environment of a lipid bilayer. Systematic application of spin labeling and EPR identifies sequence-specific secondary structures, defines their topology and their packing in the tertiary fold. Long range distance measurements (60 ?-80 ?) between pairs of spin labels enable quantitative analysis of equilibrium dynamics and triggered conformational changes. This review highlights the contribution of spin labeling to bridging structure and mechanism. Efforts to develop methods for determining structures from EPR restraints and to increase sensitivity and throughput promise to expand spin labeling applications in membrane protein structural biology.     

Mapping Membrane Protein Backbone Dynamics: A Comparison of Site-Directed Spin Labeling with NMR N-Relaxation Measurements

The ability to detect nanosecond backbone dynamics with site-directed spin labeling (SDSL) in soluble proteins has been well established. However, for membrane proteins, the nitroxide appears to have more interactions with the protein surface, potentially hindering the sensitivity to backbone motions. To determine whether membrane protein backbone dynamics could be mapped with SDSL, a nitroxide was introduced at 55 independent sites in a model polytopic membrane protein, TM0026. Electron paramagnetic resonance spectral parameters were compared with NMR ¹⁵N-relaxation data. Sequential scans revealed backbone dynamics with the same trends observed for the R₁ relaxation rate, suggesting that nitroxide dynamics remain coupled to the backbone on membrane proteins.     

Mapping Membrane Protein Backbone Dynamics: A Comparison of Site-Directed Spin Labeling with NMR 15N-Relaxation Measurements

The ability to detect nanosecond backbone dynamics with site-directed spin labeling (SDSL) in soluble proteins has been well established. However, for membrane proteins, the nitroxide appears to have more interactions with the protein surface, potentially hindering the sensitivity to backbone motions. To determine whether membrane protein backbone dynamics could be mapped with SDSL, a nitroxide was introduced at 55 independent sites in a model polytopic membrane protein, TM0026. Electron paramagnetic resonance spectral parameters were compared with NMR ¹⁵N-relaxation data. Sequential scans revealed backbone dynamics with the same trends observed for the R₁ relaxation rate, suggesting that nitroxide dynamics remain coupled to the backbone on membrane proteins.     

Simulation of nitroxide electron paramagnetic resonance spectra from brownian trajectories and molecular dynamics simulations

A simulated continuous wave electron paramagnetic resonance spectrum of a nitroxide spin label can be obtained from the Fourier transform of a free induction decay. It has been previously shown that the free induction decay can be calculated by solving the time-dependent stochastic Liouville equation for a set of Brownian trajectories defining the rotational dynamics of the label. In this work, a quaternion-based Monte Carlo algorithm has been developed to generate Brownian trajectories describing the global rotational diffusion of a spin-labeled protein. Also, molecular dynamics simulations of two spin-labeled mutants of T4 lysozyme, T4L F153R1, and T4L K65R1 have been used to generate trajectories describing the internal dynamics of the protein and the local dynamics of the spin-label side chain. Trajectories from the molecular dynamics simulations combined with trajectories describing the global rotational diffusion of the protein are used to account for all of the dynamics of a spin-labeled protein. Spectra calculated from these combined trajectories correspond well to the experimental spectra for the buried site T4L F153R1 and the helix surface site T4L K65R1. This work provides a framework to further explore the modeling of the dynamics of the spin-label side chain in the wide variety of labeling environments encountered in site-directed spin labeling studies.     

Identifying conformational changes with site-directed spin labeling

Site-direct spin labeling combined with electron paramagnetic resonance (EPR) spectroscopy is a powerful tool for detecting structural changes in proteins. This review provides examples that illustrate strategies for interpreting the data in terms of specific rearrangements in secondary and tertiary structure. The changes in the mobility and solvent accessibility of the spin label side chains, and in the distances between spin labels, report (i) rigid body motions of alpha-helices and beta-strands (ii) relative movements of domains and (iii) changes in secondary structure. Such events can be monitored in the millisecond time-scale, making it possible to follow structural changes during function. There is no upper limit to the size of proteins that can be investigated, and only 50-100 picomoles of protein are required. These features make site-directed spin labeling an attractive approach for the study of structure and dynamics in a wide range of systems.     

Osmolyte perturbation reveals conformational equilibria in spin‐labeled proteins

Recent evidence suggests that proteins at equilibrium can exist in a manifold of conformational substates, and that these substates play important roles in protein function. Therefore, there is great interest in identifying regions in proteins that are in conformational exchange. Electron paramagnetic resonance spectra of spin‐labeled proteins containing the nitroxide side chain (R1) often consist of two (or more) components that may arise from slow exchange between conformational substates (lifetimes > 100 ns). However, crystal structures of proteins containing R1 have shown that multicomponent spectra can also arise from equilibria between rotamers of the side chain itself. In this report, it is shown that these scenarios can be distinguished by the response of the system to solvent perturbation with stabilizing osmolytes such as sucrose. Thus, site‐directed spin labeling (SDSL) emerges as a new tool to explore slow conformational exchange in proteins of arbitrary size, including membrane proteins in a native‐like environment. Moreover, equilibrium between substates with even modest differences in conformation is revealed, and the simplicity of the method makes it suitable for facile screening of multiple proteins. Together with previously developed strategies for monitoring picosecond to millisecond backbone dynamics, the results presented here expand the timescale over which SDSL can be used to explore protein flexibility.     

Selective spin-labeling of the ribosomal proteins of 70S ribosomes from Escherichia coli.

We have used a series of N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl) maleimide spin labels of different length to label, covalently and selectively, the most reactive sulfhydryl groups of 70S ribosomal proteins of Escherichia coli. Under short periods of labeling (1--2 min), less than two spin labels per ribosome are incorporated and were shown to be distributed mainly on five ribosomal proteins in the following order: S18 greater than S21, L27 greater than S17, and S12. With a long period of labeling (3 h) up to 13 spin labels are attached to the ribosome, and protein S1 is the most labeled. The shape of the electron paramagnetic resonance (epr) signal shows two components with a predominance for the strongly immobilized orientation, and the percentage of these components in each spectra has been evaluated. When the distance between the nitroxide group and the maleimide-attaching group exceeds 6 A (1 A = 0.1 nm) the strongly immobilized orientation disappears. The effect of magnesium ions on these selectively spinlabeled ribosomes shows that the dissociation into subunits does not affect the epr signal, but more spin labels are incorporated into the subunits if labeling is performed under conditions of dissociation.     

Protein structural dynamics revealed by site-directed spin labeling and multifrequency EPR

Multifrequency electron paramagnetic resonance (EPR), combined with site-directed spin labeling, is a powerful spectroscopic tool to characterize protein dynamics. The lineshape of an EPR spectrum reflects combined rotational dynamics of the spin probe's local motion within a protein, reorientations of protein domains, and overall protein tumbling. All these motions can be restricted and anisotropic, and separation of these motions is important for thorough characterization of protein dynamics. Multifrequency EPR distinguishes between different motions of a spin-labeled protein, due to the frequency dependence of EPR resolution to fast and slow motion of a spin probe. This gives multifrequency EPR its unique capability to characterize protein dynamics in great detail. In this review, we analyze what makes multifrequency EPR sensitive to different rates of spin probe motion and discuss several examples of its usage to separate spin probe dynamics and overall protein dynamics, to characterize protein backbone dynamics, and to resolve protein conformational states.     

Orientation of SecA and SecB in complex, derived from disulfide cross-linking

SecA is the ATPase that acts as the motor for protein export in the general secretory, or Sec, system of Escherichia coli. The tetrameric cytoplasmic chaperone SecB binds to precursors of exported proteins before they can become stably folded and delivers them to SecA. During this delivery step, SecB binds to SecA. The complex between SecA and SecB that is maximally active in translocation contains two protomers of SecA bound to a tetramer of SecB. The aminoacyl residues on each protein that are involved in binding the other have previously been identified by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy; however, that study provided no information concerning the relative orientation of the proteins within the complex. Here we used our extensive collection of single-cysteine variants of the two proteins and subjected pairwise combinations of SecA and SecB to brief oxidation to identify residues in close proximity. These data were used to generate a model for the orientation of the two proteins within the complex.     

Protein-induced changes in DNA structure and dynamics observed with noncovalent site-directed spin labeling and PELDOR

Site-directed spin labeling and pulsed electron–electron double resonance (PELDOR or DEER) have previously been applied successfully to study the structure and dynamics of nucleic acids. Spin labeling nucleic acids at specific sites requires the covalent attachment of spin labels, which involves rather complicated and laborious chemical synthesis. Here, we use a noncovalent label strategy that bypasses the covalent labeling chemistry and show that the binding specificity and efficiency are large enough to enable PELDOR or DEER measurements in DNA duplexes and a DNA duplex bound to the Lac repressor protein. In addition, the rigidity of the label not only allows resolution of the structure and dynamics of oligonucleotides but also the determination of label orientation and protein-induced conformational changes. The results prove that this labeling strategy in combination with PELDOR has a great potential for studying both structure and dynamics of oligonucleotides and their complexes with various ligands.     

Exploring protein conformations in vitro and in cell with EPR distance measurements

The electron–electron double resonance (DEER) method, which provides distance distributions between two spin labels, attached site specifically to biomolecules (proteins and nucleic acids), is currently a well-recognized biophysical tool in structural biology. The most commonly used spin labels are based on nitroxide stable radicals, conjugated to the proteins primarily via native or engineered cysteine residues. However, in recent years, new spin labels, along with different labeling chemistries, have been introduced, driven in part by the desire to study structural and dynamical properties of biomolecules in their native environment, the cell. This mini-review focuses on these new spin labels, which allow for DEER on orthogonal spin labels, and on the state of the art methods for in-cell DEER distance measurements.     

Protein rotational mobility in thylakoid membranes of different polypeptide composition in the wild type and mutant strains of Chlamydomonas reinhardtii

The rotational mobility of thylakoid membrane proteins labeled with a paramagnetic analog of N-ethylmaleimide was investigated by saturation transfer electron spin resonance. In the wild type strain of Chlamydomonas reinhardtii two polypeptides are prominently labeled. They correspond to the 19-kDa subunit of the reaction center I protein and to the 30-kDa subunit of the light harvesting complex. Several polypeptides, most of which are either trypsin or alkaline sensitive, are also labeled. In order to circumvent the lack of specificity during the labeling, we have compared the rotational mobilities of labeled proteins in thylakoid membranes from several mutant strains which lack in photosystem I., ATPase or light harvesting complexes. Comparison of the saturation transfer electron spin resonance spectra obtained with these mutant membranes as well as with trypsin- and alkaline-treated membranes allowed us to characterize the rotational contribution of some of the labeled proteins to the overall protein dynamics observed in the wild type strain. The reaction center I protein undergoes slow rotation as compared to the other labeled proteins. The rotational characteristics of the labeled light harvesting complexes are those of a peptide fragment in the complex which is in rapid motion in unstacked membranes. Stacking of the thylakoid membranes upon Mg²⁺ addition is accompanied by a marked change in shape of the saturation transfer spectra, and corresponds to the appearance of highly immobilized nitroxides. We interpret these changes as arising mainly from the hindrance upon membrane appression, of the labeled fragment of the light harvesting complexes which protrude at the thylakoid outer surface.     

Spin labeling of calcium-dependent phospholipid-binding proteins

Bovine lung annexins p32 and p34 were spin labeled with an iodoacetamidoproxyl spin label, a reagent that reportedly couples with protein methionine residues. Labeling conditions and stoichiometry were studied with the radiolabeled analogue [1-14C]iodoacetamide. As judged by this method, carboxamidomethylation of both p32 and p34 occurred up to a 0.7 mol ratio after 60 h of reaction at 37 degrees C and at pH 4. The two proteins retained Ca2(+)-dependent phospholipid-binding ability both in radiolabeled and in spin-labeled forms. Electron resonance spectra of spin-labeled p32 and p34 showed the features of a partially immobilized spin probe, with rotational correlation time values of 1.15 and 1.25 ns, respectively, which definitely indicate successful spin labeling. Quantitation of ESR spectra by computer double integration indicated 70% spin labeling of both proteins, as anticipated by radiolabeling. The use of spin-labeled p32 and p34 in the study of Ca2(+)-dependent interaction of annexins with biomembranes is proposed.     

Assessment of the partitioning capacity of high abundant proteins in human cerebrospinal fluid using affinity and immunoaffinity subtraction spin columns

The performance of three different affinity and immunoaffinity subtraction spin columns was investigated for the removal of the most abundant proteins in human cerebrospinal fluid (CSF). A pool of human CSF was processed with the spin columns and both the bound and flow through fractions were compared with each other and with intact CSF using 1D gel electrophoresis and nanoLC–MALDI-TOF/TOF-MS analysis. MASCOT MS/MS ionscores were compared before and after processing with the columns. The non-specific co-removal of proteins bound to the high abundant proteins, so called “sponge effect” was also examined for each spin column. The reproducibility of one of the spin columns, ProteomeLab IgY-12 proteome partitioning spin column, was further investigated by isobaric tags for relative and absolute quantification (iTRAQ) labeling and MS/MS analysis. Overall, 173 unique proteins were identified on a 95% MudPIT confidence scoring level. For all three spin columns, the number of proteins identified and their MASCOT scores were increased up to 10 times. The largest degree of non-specific protein removal was observed for a purely affinity based albumin removal column, where 28 other proteins also were present. The ProteomeLab IgY-12 proteome partitioning spin column showed very high reproducibility when combined with iTRAQ labeling and MS/MS analysis. The combined relative standard deviation (R.S.D.) for the high abundant protein removal, iTRAQ labeling and nanoLC–MALDI-TOF/TOF-MS analysis was less than 17.5%.     

Effects of phytohaemagglutinin, wheat-germ agglutinin, and concanavalin-A on the physical state of sialic acid and membrane proteins in human erythrocyte ghosts: a spin label study

Parallel experiments employing sialic acid- and protein specific spin labels have been performed to monitor the effects on the physical state of this carbohydrate and membrane proteins of human erythrocytes induced by the binding of three lectins, $\text{Phaseolus vulgaris}$ phytohaemagglutinin (PHA), wheat germ agglutinin (WGA), and Concanavalin-A (Con-A). PHA and WGA, both of which are known to bind at different sites on the principal sialoglycoprotein of human erythrocytes, glycophorin, had markedly different effects: compared to control values, PHA decreased the apparent correlation time of the sialic acid specific spin probe by 10% while this parameter was decreased by 33% by WGA. The protein specific spin label also monitored differential effects of these lectins: the relevant electron spin resonance parameter (the W/S ratio) was reduced 33% by PHA and increased by WGA over 17% from that of control values. Con-A, which is known to bind to the principal transmembrane protein, Band 3, had no effect on sialic acid or membrane proteins as assessed by the two spin labels employed. These results suggest that (1) the effects of binding of these different lectins, two of which bind to the same cell surface receptor, can be discriminated by use of spin labeling methods; (2) binding events occuring at the cell surface have distinct and pronounced effects on the physical state of proteins within the membrane; (3) the different results with PHA and WGA both of which bind to glycophorin are indicative of multiple and complex interactions of this glycoprotein with the membrane proteins in the erythrocyte; and (4) that the spin labelling technique has the potential to investigate the relationships between cell-surface binding events to membrane structural-functional interactions.     

New limits of sensitivity of site-directed spin labeling electron paramagnetic resonance for membrane proteins

Site-directed spin labeling electron paramagnetic resonance is a biophysical technique based on the specific introduction of spin labels to one or more sites in diamagnetic proteins, which allows monitoring dynamics and water accessibility of the spin-labeled side chains, as well as nanometer distances between two (or more) labels. Key advantages of this technique to study membrane proteins are addressed, with focus on the recent developments which will expand the range of applicability. Comparison with other biophysical methods is provided to highlight the strength of EPR as complementary tool for structural biology. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.