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.     

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.     

Recent advances and applications of site-directed spin labeling

Site-directed spin labeling has become a popular biophysical tool for the characterization of protein structure, dynamics and conformational change. This method is well suited and widely used to study small soluble proteins, membrane proteins and large protein complexes. Recent advances in site-directed spin labeling methodology have occurred in two areas. The first involves an understanding of the conformations and local dynamics of the spin-labeled sidechain, including the features of proteins that influence electron paramagnetic resonance lineshape. The second advance is the application of pulse techniques to determine long-range distances and distance distributions in proteins. During the past two years, these technical developments have been used to address several important problems concerning the molecular function of proteins.     

Modeling a spin-labeled fusion peptide in a membrane: implications for the interpretation of EPR experiments

Site-directed spin-labeling and electron paramagnetic resonance are powerful tools for studying structure and conformational dynamics of proteins, especially in membranes. The position of the spin label is used as an indicator of the position of the site to which it is attached. The interpretation of these experiments is based on the assumptions that the spin label does not affect the peptide configuration and that it has a fixed orientation and distance with respect to the protein backbone. Here, the validity of these assumptions is examined through implicit membrane molecular dynamics simulations of the influenza hemagglutinin fusion peptide that has been labeled with methanethiosulfonate spin label. We find that the methanethiosulfonate spin label can occasionally induce peptide orientations that differ from those adopted by the wild-type peptide. Furthermore, the spin-label resides, on average, several Angstroms deeper in the membrane than the corresponding backbone C(alpha)-atom even at sites pointing toward the solvent. The nitroxide spin label exhibits flexibility and adopts various configurations depending on the surrounding residues.     

Mapping backbone dynamics in solution with site-directed spin labeling: GCN4-58 bZip free and bound to DNA

In site-directed spin labeling, a nitroxide-containing side chain is introduced at selected sites in a protein. The EPR spectrum of the labeled protein encodes information about the motion of the nitroxide on the nanosecond time scale, which has contributions from the rotary diffusion of the protein, from internal motions in the side chain, and from backbone fluctuations. In the simplest model for the motion of noninteracting (surface) side chains, the contribution from the internal motion is sequence independent, as is that from protein rotary diffusion. Hence, differences in backbone motions should be revealed by comparing the sequence-dependent motions of nitroxides at structurally homologous sites. To examine this model, nitroxide side chains were introduced, one at a time, along the GCN4-58 bZip sequence, for which NMR (15)N relaxation experiments have identified a striking gradient of backbone mobility along the DNA-binding region [Bracken et al. (1999) J. Mol. Biol. 285, 2133]. Spectral simulation techniques and a simple line width measure were used to extract dynamical parameters from the EPR spectra, and the results reveal a mobility gradient similar to that observed in NMR relaxation, indicating that side chain motions mirror backbone motions. In addition, the sequence-dependent side chain dynamics were analyzed in the DNA/protein complex, which has not been previously investigated by NMR relaxation methods. As anticipated, the backbone motions are damped in the DNA-bound state, although a gradient of motion persists with residues at the DNA-binding site being the most highly ordered, similar to those of helices on globular proteins.     

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.     

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.     

Molecular motion of spin labeled side chains in the C-terminal domain of RGL2 protein: a SDSL-EPR and MD study

Five singly spin labeled side chains at surface sites in the C-terminal domain of RGL2 protein have been analyzed to investigate the general relationship between nitroxide side chain mobility and protein structure. At these sites, the structural perturbation produced by replacement of a native residue with a nitroxide side chain appears to be very slight at the level of the backbone fold. The primary determinants of the nitroxide side chain mobility are backbone dynamics and tertiary interactions. On the exposed surfaces of alpha-helices, the side chain mobility is not restricted by tertiary interactions but appears to be determined by backbone dynamics, while in loop sites, the side chain mobility is even higher. For a better understanding of the changes in the EPR spectral line shape, molecular dynamics simulations were performed and found in agreement with EPR spectral data.     

The backrub motion: how protein backbone shrugs when a sidechain dances

Surprisingly, the frozen structures from ultra-high-resolution protein crystallography reveal a prevalent, but subtle, mode of local backbone motion coupled to much larger, two-state changes of sidechain conformation. This "backrub" motion provides an influential and common type of local plasticity in protein backbone. Concerted reorientation of two adjacent peptides swings the central sidechain perpendicular to the chain direction, changing accessible sidechain conformations while leaving flanking structure undisturbed. Alternate conformations in sub-1 angstroms crystal structures show backrub motions for two-thirds of the significant Cbeta shifts and 3% of the total residues in these proteins (126/3882), accompanied by two-state changes in sidechain rotamer. The Backrub modeling tool is effective in crystallographic rebuilding. For homology modeling or protein redesign, backrubs can provide realistic, small perturbations to rigid backbones. For large sidechain changes in protein dynamics or for single mutations, backrubs allow backbone accommodation while maintaining H bonds and ideal geometry.     

Probing the helical tilt and dynamic properties of membrane-bound phospholamban in magnetically aligned bicelles using electron paramagnetic resonance spectroscopy

Wild-type phospholamban (WT-PLB), a Ca(2+)-ATPase (SERCA) regulator in the sarcoplasmic reticulum membrane, was studied using TOAC nitroxide spin labeling, magnetically aligned bicelles, and electron paramagnetic resonance (EPR) spectroscopy to ascertain structural and dynamic information. Different structural domains of PLB (transmembrane segment: positions 42 and 45, loop region: position 20, and cytoplasmic domain: position 10) were probed with rigid TOAC spin labels to extract the transmembrane helical tilt and structural dynamic information, which is crucial for understanding the regulatory function of PLB in modulating Ca(2+)-ATPase activity. Aligned experiments indicate that the transmembrane domain of wild-type PLB has a helical tilt of 13°±4° in DMPC/DHPC bicelles. TOAC spin labels placed on the WT-PLB transmembrane domain showed highly restricted motion with more than 100ns rotational correlation time (τ(c)); whereas the loop, and the cytoplasmic regions each consists of two distinct motional dynamics: one fast component in the sub-nanosecond scale and the other component is slower dynamics in the nanosecond range.     

Differential backbone dynamics of companion helices in the extended helical coiled‐coil domain of a bacterial chemoreceptor

Cytoplasmic domains of transmembrane bacterial chemoreceptors are largely extended four‐helix coiled coils. Previous observations suggested the domain was structurally dynamic. We probed directly backbone dynamics of this domain of the transmembrane chemoreceptor Tar from Escherichia coli using site‐directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. Spin labels were positioned on solvent‐exposed helical faces because EPR spectra for such positions reflect primarily polypeptide backbone movements. We acquired spectra for spin‐labeled, intact receptor homodimers solubilized in detergent or inserted into native E. coli lipid bilayers in Nanodiscs, characterizing 16 positions distributed throughout the cytoplasmic domain and on both helices of its helical hairpins, one amino terminal to the membrane‐distal tight turn (N‐helix), and the other carboxyl terminal (C‐helix). Detergent solubilization increased backbone dynamics for much of the domain, suggesting that loss of receptor activities upon solubilization reflects wide‐spread destabilization. For receptors in either condition, we observed an unanticipated difference between the N‐ and C‐helices. For bilayer‐inserted receptors, EPR spectra from sites in the membrane‐distal protein‐interaction region and throughout the C‐helix were typical of well‐structured helices. In contrast, for approximately two‐thirds of the N‐helix, from its origin as the AS‐2 helix of the membrane‐proximal HAMP domain to the beginning of the membrane‐distal protein‐interaction region, spectra had a significantly mobile component, estimated by spectral deconvolution to average approximately 15%. Differential helical dynamics suggests a four‐helix bundle organization with a pair of core scaffold helices and two more dynamic partner helices. This newly observed feature of chemoreceptor structure could be involved in receptor function.     

Monitoring RNA base structure and dynamics using site-directed spin labeling

Site-directed spin labeling utilizes site-specific attachment of a stable nitroxide radical to probe the structure and dynamics of macromolecules. In the present study, a 4-thiouridine base is introduced at each of six different positions in a 23-nucleotide RNA molecule. The 4-thiouridine derivatives were subsequently modified with one of three methanethiosulfonate nitroxide reagents to introduce a spin label at specific sites. The electron paramagnetic resonance spectra of the labeled RNAs were analyzed in terms of nitroxide motion and the RNA solution structure. At a base-paired site in the RNA helix, where the nitroxide has weak or no local interactions, motion of the nitroxide is apparently dominated by rotation about bonds within the probe. The motion is similar to that found for a structurally related probe on helical sites in proteins, suggesting a similar mode of motion. At other sites that are hydrogen bonded and stacked within the helix, local interactions within the RNA molecule modulate the nitroxide motion in a manner consistent with expectations based on the known structure. For a base that is not structurally constrained, the mobility is higher than at any other site, presumably due to motion of the base itself. These results demonstrate the general utility of the 4-thiouridine/methanethiosulfonate coupling method to introduce nitroxide spin labels into RNA and the ability of the resulting label to probe local structure and dynamics.     

Dap-SL: a new site-directed nitroxide spin labeling approach for determining structure and motions in synthesized peptides and proteins

A new approach for site-directed placement of nitroxide spin labels in chemically synthesized peptides and proteins is described. The scheme takes advantage of a novel diaminopropionic acid scaffold to independently control backbone and side chain elongation. The result is a spin-labeled side chain, referred to as Dap-SL, in which an amide bond forms a linker between the nitroxide and the peptide backbone. The method was demonstrated in a series of helical peptides. Circular dichroism and nuclear magnetic resonance showed that Dap-SL introduces only a minor perturbation in the helical structure. The electron paramagnetic resonance spectrum of the singly labeled species allowed for determination of the spin label rotational correlation time and suggests that the Dap-SL side chain is more flexible than the modified Cys side chain frequently used in site-directed spin label studies. Spectra of the doubly labeled peptides indicate a mixture of 3(10)-helix and alpha-helix, which parallels findings from previous studies. The scheme demonstrated here offers a fundamentally new approach for introducing spin labels into proteins and promises to significantly extend biophysical investigations of large proteins and receptors. In addition, the technique is readily modified for incorporation of any biophysical probe.     

Transmembrane organization of yeast syntaxin-analogue Sso1p

Membrane fusion in secretory pathways is thought to be mediated by SNAREs. It is proposed that membrane fusion transits through hemifusion, a condition in which the outer leaflets of the bilayers are mixed, but the inner leaflets are not. Hemifusion then proceeds to the fusion pore that connects the two internal contents. It is believed that the transmembrane domains (TMDs) of the fusion proteins play an essential role in the transition from hemifusion to the fusion pore. In this work, the structure, dynamics, and membrane topology of the TMD of Sso1p, a target membrane (t-) SNARE involved in the trafficking from Golgi to plasma membrane in yeast, was investigated using site-directed spin labeling and EPR spectroscopy. The EPR analysis of spin-labeled mutants showed that the TMD of Sso1p is a well-defined membrane spanning alpha-helix. The results also indicate that there is an equilibrium between the monomers and the oligomers. The oligomerization is mainly mediated through the interaction at the N-terminal half of the TMD, whereas the C-terminal half is free of the tertiary interaction. Additionally, the isotropic hyperfine splitting values were examined for nitroxide-scanning mutants, and it was found that the hyperfine splitting values show a V-shaped profile across the bilayer. Thus, hyperfine splitting may be used as an additional parameter to measure bilayer immersion depths of nitroxide.     

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.     

Thermodynamic insights into proteins from NMR spin relaxation studies.

NMR spin relaxation measurements of picosecond to nanosecond timescale backbone and sidechain fluctuations of protein molecules, and subsequent entropic interpretation yield interesting, but sometimes counterintuitive, insights into proteins. The stabilities of proteins and protein interactions are achieved through enthalpy-entropy compensation, which is partitioned between the backbone and sidechains depending on the nature of the system.