Navigating Membrane Protein Structure, Dynamics, and Energy Landscapes Using Spin Labeling and EPR Spectroscopy

Abstract

A detailed understanding of the functional mechanism of a protein entails the characterization of its energy landscape. Achieving this ambitious goal requires the integration of multiple approaches including determination of high-resolution crystal structures, uncovering conformational sampling under distinct biochemical conditions, characterizing the kinetics and thermodynamics of transitions between functional intermediates using spectroscopic techniques, and interpreting and harmonizing the data into novel computational models. With increasing sophistication in solution-based and ensemble-oriented biophysical approaches such as electron paramagnetic resonance (EPR) spectroscopy, atomic resolution structural information can be directly linked to conformational sampling in solution. Here, we detail how recent methodological and technological advances in EPR spectroscopy have contributed to the elucidation of membrane protein mechanisms. Furthermore, we aim to assist investigators interested in pursuing EPR studies by providing an introduction to the technique, a primer on experimental design, and a description of the practical considerations of the method toward generating high quality data.

Keywords: DEER; Dynamics; EPR; Membrane proteins; Site-directed spin labeling; Structure.

Figure 1. Biophysical methods to study protein structure and dynamics

Whereas x-ray crystallography is the most robust method to determine high resolution structures of small and large proteins, cryoelectron microscopy is best suited for large proteins and protein complexes. Despite its utility to investigate dynamic properties, current molecular size constraints limit the applicability of liquid state NMR to <50,000 MW. In contrast, EPR and fluorescence spectroscopies can interrogate dynamic processes regardless of size or complexity. The application of these probe-based methods to proteins of known structure amplifies the interpretation of data toward understanding mechanism.

Figure 2. EPR methods report on the ensemble of conformations in solution

In a DEER experiment, each molecule in solution reports a characteristic inter-probe distance consistent with its conformation. The distance distribution reports these distances as a function of their frequency within the ensemble. Thus, discrete conformations undergoing equilibrium fluctuations in solution (inward-facing and outward-facing) at ambient temperatures are represented as distinct distance populations in the DEER distance distribution (yellow and green) in the solid state. Therefore, individual conformations can be described using distance parameters generated from ensemble-based measurements.

Figure 3. Site-directed spin labeling and correlation of the EPR spectrum with local structure

(A) Targeted cysteine mutagenesis introduces a sulfhydryl moiety for the attachment of a nitroxide spin label, such as MTSSL. Rotational isomerization of MTSSL predominantly around the bonds highlighted in gray is reflected in the EPR spectral lineshape. (B) The degree of rotational freedom of the label is determined by the local packing environment. Fast rotational correlation times (~1ns) correspond to spin labels attached to surface-exposed sites. Tertiary contact interactions or buried sites that restrict spin label motion reduce the rate and amplitude of isomerization leading to broadening of the lineshape. The dashed line emphasizes the progressive appearance of a slow motion component associated with restricted rotation.

Figure 4. EPR spectroscopy at a glance

Summary of the methods in EPR highlighting structural interpretation and caveats for each method.

Figure 5. Spin label solvent accessibility and the correlation with local environment

(A) The differential solubility of fast-relaxing PRAs (NiEDDA and O₂) allows the determination of spin label environment. Nitroxide scanning of an α-helix that is asymmetrically solvated between aqueous and hydrocarbon milieu will report the gradient of oxygen accessibility toward the center of the bilayer in accordance with helix periodicity. The dotted line highlights the site of expected maximum in O₂ accessibility. The NiEDDA accessibility profile, which probes water exposure, is 180° out-of-phase with the O₂ profile. (B) Two spin labeled sites are shown on a model of LeuT (PDB 2A65), which are used to probe the membrane-water interface. An approximate position for this interface is outlined by an orange box. (C) Power saturation experiments showing the reduction in signal intensity as a function of microwave power. (D) The high NiEDDA accessibility at site 480 in LeuT relative to O₂ and N₂ determined from power saturation curves (inset) suggests a water exposed position of the spin label. (D) In contrast, the high O₂ accessibility at site 488 indicates that the spin label samples the lipid bilayer.

Figure 6. Monitoring global conformational changes with DEER spectroscopy

(A) The four-pulse protocol for DEER spectroscopy is designed to interrogate distance-dependent dipolar interactions between spin A and spin B. The inversion Π pulse on spin B modulates the echo decay of spin A as a function of time t, and the frequency of the resulting oscillation is inversely proportional to the average distance. The decay rate of the spin echo modulation is informed by the distribution of distances in the sample. (B) A simulated conformational change between two states of discrete energies as shown in the spin echo decay (top panels) is manifested by distinct r_av and σ in the unimodal distance distribution. The middle panels illustrate an equilibrium between two states, which is the sum of contributions from each conformation. For simplicity, each conformation is equally populated. In the bottom panels, a shift in the equilibrium (induced by ligand binding, for instance) toward an increase in population of the short distance component is visualized in the spin echo decay. The dotted lines in P(r) show the position of r_av.

Figure 7. Correlation of global structural rearrangements with local helix packing in MsbA

(A) Model of the MsbA homodimer in the open, Apo (PDB 3B5W) and the closed, AMP-PNP-bound (PDB 3B60) states showing symmetry-related sites for spin label incorporation. Individual monomers are identified by the color scheme. (B) EPR spectra of spin labels at these positions and the corresponding distance distributions (C) in the Apo and ADP-Vi-bound states (trapped post-hydrolysis). Labels at 561 and 162 show opposite distance changes between states, consistent with rigid body movement of helices in an alternating access mechanism. Although separated by ~50Å in the Apo state (C), spin labels at site 121 are within 20Å in the ADP-Vi-bound state as indicated by broadening of the EPR lineshape (arrow in B). (D) Formation of a closed conformation on the intracellular side according to distance analysis is consistent with changes in the NiEDDA accessibility profile of transmembrane helix 3 induced by ADP-Vi.

Figure 8. EPR distance measurements and structure elucidation

Analysis of protein structure using EPR distance measurements requires triangulation of spin label positions. (A) Triangles represent the least dense labeling strategy that can identify whether a motif is undergoing conformational reorganization. (B) More dense strategies, like quadranges, narrow the possible space that spin label can occupy thereby providing a more detailed description of protein structure and more informative restraints for modeling. (C) To effectively identify positions in three dimensions a pyramid scheme is required.

Figure 9. EPR reveals equilibria that can be used to describe energy landscapes and mechanism

LeuT is a Na+-coupled amino acid transporter. We conducted DEER experiments that monitored the conformational transitions on the extracellular and intracellular sides, shown here for helix 6/intracellular loop 3 (orange). We observed conformational equilibria between inward-facing, outward-facing and occluded conformations associated with apo (ligand-free, black), Na+-bound (blue) and Na+/Leu-bound (red) conditions. These were used along with structural characterization of intermediate states (numerically identified) and a biochemical description of transport to produce a novel description of alternating access in LeuT.

Figure 10. Impact of protein aggregates on the spin echo decay and the resulting distance distribution

(A) Preparative size exclusion chromatography demonstrating different levels of protein aggregation for the same mutant as indicated by the leading shoulder. The traces were normalized by area. The peak fractions pooled for DEER analysis are highlighted by a gray rectangle. Changes in the intermolecular background of the DEER experiment tracked with the presence of aggregated species (B), introducing a long distance artifact in the distance distributions (C).

Figure 11. Detergent and lipid environments and the consequence on the DEER signal background

Solvation of membrane protein in (A) detergent micelles, (B) liposomes and (C) Nanodiscs. Liposome reconstitution often introduces more than one protein copy per liposome. As a result, the higher effective spin concentration increases the contribution of the background decay in the DEER signal relative to proteomicelles and nanodiscs (D).