A pressure-jump EPR system to monitor millisecond conformational exchange rates of spin-labeled proteins.

Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) using nitroxide spin labels is a well-established technology for mapping site-specific secondary and tertiary structure and for monitoring conformational changes in proteins of any degree of complexity, including membrane proteins, with high sensitivity. SDSL-EPR also provides information on protein dynamics in the time scale of ps-µs using continuous wave lineshape analysis and spin lattice relaxation time methods. However, the functionally important time domain of µs-ms, corresponding to large-scale protein motions, is inaccessible to those methods. To extend SDSL-EPR to the longer time domain, the perturbation method of pressure-jump relaxation is implemented. Here, we describe a complete high-pressure EPR system at Q-band for both static pressure and millisecond-timescale pressure-jump measurements on spin-labeled proteins. The instrument enables pressure jumps both up and down from any holding pressure, ranging from atmospheric pressure to the maximum pressure capacity of the system components (~3500 bar). To demonstrate the utility of the system, we characterize a local folding-unfolding equilibrium of T4 lysozyme. The results illustrate the ability of the system to measure thermodynamic and kinetic parameters of protein conformational exchange on the millisecond timescale.

Viewing rare conformations of the ß2 adrenergic receptor with pressure-resolved DEER spectroscopy.

The ß2 adrenergic receptor (ß2AR) is an archetypal G protein coupled receptor (GPCR). One structural signature of GPCR activation is a large-scale movement (ca. 6 to 14 Å) of transmembrane helix 6 (TM6) to a conformation which binds and activates a cognate G protein. The ß2AR exhibits a low level of agonist-independent G protein activation. The structural origin of this basal activity and its suppression by inverse agonists is unknown but could involve a unique receptor conformation that promotes G protein activation. Alternatively, a conformational selection model proposes that a minor population of the canonical active receptor conformation exists in equilibrium with inactive forms, thus giving rise to basal activity of the ligand-free receptor. Previous spin-labeling and fluorescence resonance energy transfer experiments designed to monitor the positional distribution of TM6 did not detect the presence of the active conformation of ligand-free ß2AR. Here we employ spin-labeling and pressure-resolved double electron-electron resonance spectroscopy to reveal the presence of a minor population of unliganded receptor, with the signature outward TM6 displacement, in equilibrium with inactive conformations. Binding of inverse agonists suppresses this population. These results provide direct structural evidence in favor of a conformational selection model for basal activity in ß2AR and provide a mechanism for inverse agonism. In addition, they emphasize 1) the importance of minor populations in GPCR catalytic function; 2) the use of spin-labeling and variable-pressure electron paramagnetic resonance to reveal them in a membrane protein; and 3) the quantitative evaluation of their thermodynamic properties relative to the inactive forms, including free energy, partial molar volume, and compressibility.

Angiotensin Analogs with Divergent Bias Stabilize Distinct Receptor Conformations.

"Biased" G protein-coupled receptor (GPCR) agonists preferentially activate pathways mediated by G proteins or ß-arrestins. Here, we use double electron-electron resonance spectroscopy to probe the changes that ligands induce in the conformational distribution of the angiotensin II type I receptor. Monitoring distances between 10 pairs of nitroxide labels distributed across the intracellular regions enabled mapping of four underlying sets of conformations. Ligands from different functional classes have distinct, characteristic effects on the conformational heterogeneity of the receptor. Compared to angiotensin II, the endogenous agonist, agonists with enhanced Gq coupling more strongly stabilize an "open" conformation with an accessible transducer-binding site. ß-arrestin-biased agonists deficient in Gq coupling do not stabilize this open conformation but instead favor two more occluded conformations. These data suggest a structural mechanism for biased ligand action at the angiotensin receptor that can be exploited to rationally design GPCR-targeting drugs with greater specificity of action.

DEER Spectroscopy Measurements Reveal Multiple Conformations of HIV-1 SOSIP Envelopes that Show Similarities with Envelopes on Native Virions.

HIV-1 Envelope (Env) mediates viral-host membrane fusion after binding host-receptor CD4 and coreceptor. Soluble envelopes (SOSIPs), designed to mimic prefusion conformational states of virion-bound envelopes, are proposed immunogens for eliciting neutralizing antibodies, yet only static structures are available. To evaluate conformational landscapes of ligand-free, CD4-bound, inhibitor-bound, and antibody-bound SOSIPs, we measured inter-subunit distances throughout spin-labeled SOSIPs using double electron-electron resonance (DEER) spectroscopy and compared results to soluble and virion-bound Env structures, and single-molecule fluorescence resonance energy transfer (smFRET)-derived dynamics of virion-bound Envs. Unliganded SOSIP measurements were consistent with closed, neutralizing antibody-bound structures and shielding of non-neutralizing epitopes, demonstrating homogeneity at Env apex, increased flexibility near Env base, and no evidence for the intra-subunit flexibility near Env apex suggested by smFRET. CD4 binding increased inter-subunit distances and heterogeneity, consistent with rearrangements required for coreceptor binding. Results suggest similarities between SOSIPs and virion-bound Envs and demonstrate DEER's relevance for immunogen design.

Protonation state of glutamate 73 regulates the formation of a specific dimeric association of mVDAC1.

The voltage-dependent anion channel (VDAC) is the most abundant protein in the outer mitochondrial membrane and constitutes the primary pathway for the exchange of ions and metabolites between the cytosol and the mitochondria. There is accumulating evidence supporting VDAC's role in mitochondrial metabolic regulation and apoptosis, where VDAC oligomerization has been implicated with these processes. Herein, we report a specific pH-dependent dimerization of murine VDAC1 (mVDAC1) identified by double electron-electron resonance and native mass spectrometry. Intermolecular distances on four singly spin-labeled mVDAC1 mutants were used to generate a model of the low-pH dimer, establishing the presence of residue E73 at the interface. This dimer arrangement is different from any oligomeric state previously described, and it forms as a steep function of pH with an apparent pKa of 7.4. Moreover, the monomer-dimer equilibrium affinity constant was determined using native MS, revealing a nearly eightfold enhancement in dimerization affinity at low pH. Mutation of E73 to either alanine or glutamine severely reduces oligomerization, demonstrating the role of protonated E73 in enhancing dimer formation. Based on these results, and the known importance of E73 in VDAC physiology, VDAC dimerization likely plays a significant role in mitochondrial metabolic regulation and apoptosis in response to cytosolic acidification during cellular stress.

Saturation Recovery EPR and Nitroxide Spin Labeling for Exploring Structure and Dynamics in Proteins.

Experimental techniques capable of determining the structure and dynamics of proteins are continuously being developed in order to understand protein function. Among existing methods, site-directed spin labeling in combination with saturation recovery (SR) electron paramagnetic resonance spectroscopy contributes uniquely to the determination of secondary and tertiary protein structure under physiological conditions, independent of molecular weight and complexity. In addition, SR of spin labeled proteins was recently demonstrated to be sensitive to conformational exchange events with characteristic lifetimes on the order of µs, a time domain that presents a significant challenge to other spectroscopic techniques. In this chapter, we present the theoretical background necessary to understand the capabilities of SR as applied to spin labeled proteins, the instrumental requirements, and practical experimental considerations necessary to obtain interpretable data, and the use of SR to obtain information on protein: (1) secondary structure via solvent accessibility measurements, (2) tertiary structure using interspin distance measurements, and (3) conformational exchange.

High-Pressure EPR and Site-Directed Spin Labeling for Mapping Molecular Flexibility in Proteins.

High hydrostatic pressure is a powerful probe of protein conformational flexibility. Pressurization reveals regions of elevated compressibility, and thus flexibility, within individual conformational states, but also shifts conformational equilibria such that "invisible" excited states become accessible for spectroscopic characterization. The central aim of this chapter is to describe recently developed instrumentation and methodologies that enable high-pressure site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) experiments on proteins and to demonstrate the information content of these experiments by highlighting specific recent applications. A brief introduction to the thermodynamics of proteins under pressure is presented first, followed by a discussion of the principles underlying SDSL-EPR detection of pressure effects in proteins, and the suitability of SDSL-EPR for this purpose in terms of timescale and ability to characterize conformational heterogeneity. Instrumentation and practical considerations for variable-pressure continuous wave EPR and pressure-resolved double electron-electron resonance (PR DEER) experiments are reviewed, and finally illustrations of data analysis using recent applications are presented. Although high-pressure SDSL-EPR is in its infancy, the recent applications presented highlight the considerable potential of the method to (1) identify compressible (flexible) regions in a folded protein; (2) determine thermodynamic parameters that relate conformational states in equilibrium; (3) populate and characterize excited states of proteins undetected at atmospheric pressure; (4) reveal the structural heterogeneity of conformational ensembles and provide distance constraints on the global structure of pressure-populated states with PR DEER.

SIGNAL TRANSDUCTION. Structural basis for nucleotide exchange in heterotrimeric G proteins.

G protein-coupled receptors (GPCRs) relay diverse extracellular signals into cells by catalyzing nucleotide release from heterotrimeric G proteins, but the mechanism underlying this quintessential molecular signaling event has remained unclear. Here we use atomic-level simulations to elucidate the nucleotide-release mechanism. We find that the G protein α subunit Ras and helical domains-previously observed to separate widely upon receptor binding to expose the nucleotide-binding site-separate spontaneously and frequently even in the absence of a receptor. Domain separation is necessary but not sufficient for rapid nucleotide release. Rather, receptors catalyze nucleotide release by favoring an internal structural rearrangement of the Ras domain that weakens its nucleotide affinity. We use double electron-electron resonance spectroscopy and protein engineering to confirm predictions of our computationally determined mechanism.

Structural Insights into the Dynamic Process of ß2-Adrenergic Receptor Signaling.

G-protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmic domain of the ß2-adrenergic receptor (ß2AR) using (19)F-fluorine NMR and double electron-electron resonance spectroscopy. These studies show that unliganded and inverse-agonist-bound ß2AR exists predominantly in two inactive conformations that exchange within hundreds of microseconds. Although agonists shift the equilibrium toward a conformation capable of engaging cytoplasmic G proteins, they do so incompletely, resulting in increased conformational heterogeneity and the coexistence of inactive, intermediate, and active states. Complete transition to the active conformation requires subsequent interaction with a G protein or an intracellular G protein mimetic. These studies demonstrate a loose allosteric coupling of the agonist-binding site and G-protein-coupling interface that may generally be responsible for the complex signaling behavior observed for many GPCRs.

Structure-relaxation mechanism for the response of T4 lysozyme cavity mutants to hydrostatic pressure.

Application of hydrostatic pressure shifts protein conformational equilibria in a direction to reduce the volume of the system. A current view is that the volume reduction is dominated by elimination of voids or cavities in the protein interior via cavity hydration, although an alternative mechanism wherein cavities are filled with protein side chains resulting from a structure relaxation has been suggested [López CJ, Yang Z, Altenbach C, Hubbell WL (2013) Proc Natl Acad Sci USA 110(46):E4306-E4315]. In the present study, mechanisms for elimination of cavities under high pressure are investigated in the L99A cavity mutant of T4 lysozyme and derivatives thereof using site-directed spin labeling, pressure-resolved double electron-electron resonance, and high-pressure circular dichroism spectroscopy. In the L99A mutant, the ground state is in equilibrium with an excited state of only ∼ 3% of the population in which the cavity is filled by a protein side chain [Bouvignies et al. (2011) Nature 477(7362):111-114]. The results of the present study show that in L99A the native ground state is the dominant conformation to pressures of 3 kbar, with cavity hydration apparently taking place in the range of 2-3 kbar. However, in the presence of additional mutations that lower the free energy of the excited state, pressure strongly populates the excited state, thereby eliminating the cavity with a native side chain rather than solvent. Thus, both cavity hydration and structure relaxation are mechanisms for cavity elimination under pressure, and which is dominant is determined by details of the energy landscape.

Mapping protein conformational heterogeneity under pressure with site-directed spin labeling and double electron-electron resonance.

The dominance of a single native state for most proteins under ambient conditions belies the functional importance of higher-energy conformational states (excited states), which often are too sparsely populated to allow spectroscopic investigation. Application of high hydrostatic pressure increases the population of excited states for study, but structural characterization is not trivial because of the multiplicity of states in the ensemble and rapid (microsecond to millisecond) exchange between them. Site-directed spin labeling in combination with double electron-electron resonance (DEER) provides long-range (20-80 Å) distance distributions with angstrom-level resolution and thus is ideally suited to resolve conformational heterogeneity in an excited state populated under high pressure. DEER currently is performed at cryogenic temperatures. Therefore, a method was developed for rapidly freezing spin-labeled proteins under pressure to kinetically trap the high-pressure conformational ensemble for subsequent DEER data collection at atmospheric pressure. The methodology was evaluated using seven doubly-labeled mutants of myoglobin designed to monitor selected interhelical distances. For holomyoglobin, the distance distributions are narrow and relatively insensitive to pressure. In apomyoglobin, on the other hand, the distributions reveal a striking conformational heterogeneity involving specific helices in the pressure range of 0-3 kbar, where a molten globule state is formed. The data directly reveal the amplitude of helical fluctuations, information unique to the DEER method that complements previous rate determinations. Comparison of the distance distributions for pressure- and pH-populated molten globules shows them to be remarkably similar despite a lower helical content in the latter.

Circular dichroism and site-directed spin labeling reveal structural and dynamical features of high-pressure states of myoglobin.

Excited states of proteins may play important roles in function, yet are difficult to study spectroscopically because of their sparse population. High hydrostatic pressure increases the equilibrium population of excited states, enabling their characterization [Akasaka K (2003) Biochemistry 42:10875-85]. High-pressure site-directed spin-labeling EPR (SDSL-EPR) was developed recently to map the site-specific structure and dynamics of excited states populated by pressure. To monitor global secondary structure content by circular dichroism (CD) at high pressure, a modified optical cell using a custom MgF2 window with a reduced aperture is introduced. Here, a combination of SDSL-EPR and CD is used to map reversible structural transitions in holomyoglobin and apomyoglobin (apoMb) as a function of applied pressure up to 2 kbar. CD shows that the high-pressure excited state of apoMb at pH 6 has helical content identical to that of native apoMb, but reversible changes reflecting the appearance of a conformational ensemble are observed by SDSL-EPR, suggesting a helical topology that fluctuates slowly on the EPR time scale. Although the high-pressure state of apoMb at pH 6 has been referred to as a molten globule, the data presented here reveal significant differences from the well-characterized pH 4.1 molten globule of apoMb. Pressure-populated states of both holomyoglobin and apoMb at pH 4.1 have significantly less helical structure, and for the latter, that may correspond to a transient folding intermediate.