DEER Spectroscopy Research Articles

Lipid-Dependent Conformational Transitions in KvAP are Driven by Voltage Sensing Domain

Recent electrophysiological studies have shown that the mechanism of voltage-driven gating in Kv channels is exquisitely sensitive to the composition of the lipid membrane. For instance, non-phosphate lipids can dramatically right-shift the voltage dependence (G-V curve) of KvAP (1,2) in a way that promotes the stabilization of the “down” conformation of the voltage sensor. We evaluated the conformations of KvAP's isolated VSD by means of site directed spin labeling CW EPR and DEER spectroscopy through reconstitution in lipids with (POPC:POPG) or without (DOTAP) phosphate groups. Our data suggested a novel Tilt-Shift model for the mechanism of voltage sensing with a ∼3 A upward tilt and simultaneous ∼2 A axial shift of S4 (3). We have extended these measurements to evaluate the effects on the KvAP pore domain and quantify the DOTAP molar fraction dependence on the conformation of the inner gate. While in the full-length channel DOTAP reconstitution triggers conformational rearrangements in both the S4 segment and the S6 inner bundle gate, in the absence of voltage sensing domain, DOTAP is unable to generate significant rearrangements in S6. This result is consistent with the idea that S4 movements are allosterically transmitted to the inner bundle gate. Although the mechanistic correlation between voltage-dependent and lipid-dependent gating in voltage sensing domains remains to be fully characterized, it is clear that just as with electric fields, the interacting lipids play a determinant role in defining the equilibrium between the activated and resting states of voltage sensing domain.1. D. Schmidt, Q. X. Jiang, R. MacKinnon, Nature 444, 775 (2006)2. H. Zheng, W. Liu, L. Y. Anderson, Q. X. Jiang, Nat Commun 2, 250 (2011)3. Q. Li, S. Wanderling, P. Sompornpisut, E. Perozo, Nat Mol Struct Bio l21, 244 (2014)

Evaluating DEER Distance Profiles in Terms of Protein Conformational Ensembles

Distance profiles obtained from DEER spectroscopy provide a wealth of information regarding distances between spin labels in macromolecular systems. We have been evaluating the utility of DEER distance profiles to provide information regarding changes in conformational sampling ensembles in HIV-1 protease. In this workshop we will present our approaches and developments related to assessing errors in the data and how the data are related back to protein conformational sampling states. We will also show that sample freezing methods do not alter HIV distance ensembles within errors of the measurements.

Actin Straightens the Myosin Relay Helix during the Powerstroke

We have used transient time-resolved FRET, (TR)2FRET, to resolve the structural kinetics of the myosin relay helix during the actin-activated power stroke. The relay helix plays a critical role in force generation in all myosin isoforms coupling structural changes in the ATPase site with rotation of the light chain binding domain and force generation. Previous work demonstrated that the relay helix of the myosin II motor domain from Dictyostelium Discoideum exists in dynamic equilibrium between a bent pre-powerstroke state stabilized by ATP, and a straight post-powerstroke state which predominates in the presence of ADP or when the nucleotide binding site is empty. We predict that actin binding to the myosin.ADP.Pi complex will reverse helix bending. This transition should parallel the myosin power-stroke. To test this hypothesis, a Cys-lite Dictyostelium myosin motor domain was labeled at two introduced Cys residues shown to detect relay helix bending using DEER, TR-FRET and (TR)2FRET spectroscopy. We preformed a (TR)2FRET experiment measuring the structural distribution of the relay helix every millisecond after rapid mixing of myosin.ADP/Pi with actin. This experiment showed that actin binding straightens the bent helix. We characterized the actin dependence of helix straightening and then correlated this dependence with the kinetics of actin binding and phosphate dissociation from the myosin active site. Numerical simulations were used to model a system containing pre and post-powerstroke structural states, actin binding, hydrolysis and phosphate dissociation. We globally fit this model to the (TR)2FRET, actin binding, and phosphate dissociation transients. From this analysis, we are able to determine the timing of helix straightening with respect to actin binding and the critical step of phosphate dissociation which acts as the thermodynamic driving force for myosin's force generating powerstroke.

Characterization of the E506Q and H537A dysfunctional mutants in the E. coli ABC transporter MsbA.

MsbA is a member of the ABC transporter superfamily that is specifically found in Gram-negative bacteria and is homologous to proteins involved in both bacterial and human drug resistance. The E506Q and H537A mutations have been introduced and used for crystallization of other members of the ABC transporter protein family, including BmrA and the ATPase domains MalK, HlyB-NBD, and MJ0796, but have not been previously studied in detail or investigated in the MsbA lipid A exporter. We utilized an array of biochemical and EPR spectroscopy approaches to characterize the local and global effects of these nucleotide binding domain mutations on the E. coli MsbA homodimer. The lack of cell viability in an in vivo growth assay confirms that the presence of the E506Q or H537A mutations within MsbA creates a dysfunctional protein. To further investigate the mode of dysfunction, a fluorescent ATP binding assay was used and showed that both mutant proteins maintain their ability to bind ATP, but ATPase assays indicate hydrolysis is severely inhibited by each mutation. EPR spectroscopy data using previously identified and characterized reporter sites within the nucleotide binding domain along with ATP detection assays show that hydrolysis does occur over time in both mutants, though more readily in the H537A protein. DEER spectroscopy demonstrates that both proteins studied are purified in a closed dimer conformation, indicating that events within the cell can induce a stable, closed conformation of the MsbA homodimer that does not reopen even in the absence of nucleotide.

RNA-Binding to Archaeal RNA Polymerase Subunits F/E: A DEER and FRET Study

RNA polymerases (RNAP) carry out transcription, the first step in the highly regulated process of gene expression. RNAPs are complex multisubunit enzymes, which undergo extensive structural rearrangements during the transcription cycle (initiation-elongation-termination). They accommodate interactions with the nucleic acid scaffold of transcription complexes (template DNA, DNA/RNA hybrid, and nascent RNA) and interact with a plethora of transcription factors. Here we focused on the RNAP-F/E subcomplex, which forms a stable heterodimer that binds the nascent RNA and thereby stimulates the processivity of elongation complexes. We used the pulsed-EPR method DEER and fluorescence spectroscopy to probe for conformational changes within the F/E dimer. Our results demonstrate that, upon binding of RNA, F/E remains in a stable conformation, which suggests that it serves as a structurally rigid guiding rail for the growing RNA chain during transcription.

Accurate Distance Constraints for RNA Structures Using Deer Spectroscopy

Understanding structure-function relationships in RNA and RNA-protein complexes requires robust methods for obtaining structural information on a variety of length scales. DEER (double electron-electron resonance) is emerging as a powerful method for very accurate (± 2 A over 15-80 A) distance measurements between pairs of nitroxide labels that can be placed using several available conjugation sites in RNA nucleobases or phosphodiester linkages. Here, we show the potential for DEER spectroscopy in monitoring global RNA folding and also small changes in RNA structure within a model system that is based on the Hammerhead ribozyme. This catalytically active RNA, a three-helix junction motif with a buried active site, undergoes cation-dependent folding transitions that are linked to activity. Nitroxide labels placed at strategic positions allow helix-docking and active-site core rearrangements to be monitored by measuring the dipolar coupling between paramagnetic sites. This poster will present the results of DEER measurements obtained at both X-band and Q-band, where the higher-frequency Q-band spectroscopy significantly enhances the sensitivity of this technique. Mg2+-dependent global folding, and evidence for a smaller local structural change with higher added metal concentrations, are both observed in this RNA. Since labels can be placed at targeted sites within both nucleic acids and proteins, and there is no inherent limitation on macromolecular size, DEER spectroscopy has potential for obtaining high-resolution structural information in complex RNAs and in large RNA or DNA-protein complexes.

Accessing the distance range of interest in biomolecules: Site-directed spin labeling and DEER spectroscopy

Investigations on the structure and function of biomolecules often depend on the availability of topological information to build up structural models or to characterize conformational changes during function. Electron paramagnetic resonance (EPR) spectroscopy in combination with site–directed spin labeling (SDSL) allow to determine intra- and intermolecular distances in the range from 4–70 Å, covering the range of interest for biomolecules. The approach does not require crystalline samples and is well suited also for molecules exhibiting intrinsic flexibility. This article is intended to give an overview on pulsed EPR in conjunction with SDSL to study protein interactions as well as conformational changes, exemplified on the tRNA modifying enzyme MnmE.

Accessing the distance range of interest in biomolecules: Site-directed spin labeling and DEER spectroscopy

Investigations on the structure and function of biomolecules often depend on the availability of topological information to build up structural models or to characterize conformational changes during function. Electron paramagnetic resonance (EPR) spectroscopy in combination with site–directed spin labeling (SDSL) allow to determine intra- and intermolecular distances in the range from 4–70 A, covering the range of interest for biomolecules. The approach does not require crystalline samples and is well suited also for molecules exhibiting intrinsic flexibility. This article is intended to give an overview on pulsed EPR in conjunction with SDSL to study protein interactions as well as conformational changes, exemplified on the tRNA modifying enzyme MnmE.

Characterization of the L511P and D512G Mutations in the MsbA Lipid Flippase

MsbA is a 65kDa lipid flippase found in the inner membrane of Gram-negative bacteria such as E. coli and S. typhimurium. As a member of the ABC transporter superfamily, MsbA contains two nucleotide binding domains and two transmembrane domains, one from each of its two monomers. ABC transporters transport a diverse group of substrates from lipids to antibiotics and their dysfunction contributes to a number of human pathologies including cystic fibrosis. As an essential protein in E. coli, the deletion or dysfunction of MsbA results in the toxic accumulation of lipid A in the inner membrane resulting in membrane instability and cell death. The L511P and D512G mutations have been previously identified through mutational analysis as dysfunctional nucleotide binding domain mutations specific to MsbA and were suggested to have a lower affinity for ATP. To further understand the cause of dysfunction in these point mutations, in vivo growth assays, in vitro ATPase activity assays, DEER and CW EPR spectroscopy studies throughout the ATP hydrolysis cycle were conducted. L511P and D512G were each paired with nine different reporter residues, each in or near an important conserved nucleotide binding domain motif and compared to the reporter residues alone. To identify the stage in the ATP hydrolysis cycle in which the L511P and D512G mutations are dysfunctional, the local tertiary interactions before, during, and after ATP hydrolysis were monitored by EPR spectroscopy at each stage of the ATP hydrolysis cycle.

Dynamic and Strucutral Effects of Ligand and Coregulator Binding on Estrogen Receptor Ligand Binding Domain Measured by Electron Paramagnetic Resonance

The estrogen receptor (ER) is an important therapeutic target for the treatment and prevention of estrogen responsive forms of breast cancer. Despite the availability of several crystal structures for ER bound to either agonist or antagonist ligands, its molecular mechanism of action still remains unclear. The major structural difference between agonist and antagonist forms can be observed in the position of helix-12 (H12) C-terminus region. Here, we present the results of site directed spin labeling on the H12 region (543) and on the H11-H12 hinge region (530) to monitor the effect of ligands with different biological activity on the solution dynamic and structure of H12. We found that the hinge region is directly affected by allosteric binding of coregulators peptides in a ligand dependent fashion. We characterized the structural changes resulting from ligand/coregulator binding using DEER spectroscopy. Additionally the effects of ligand binding on H12 were directly observable with our 543 labeled ER. When taken together, these results substantially complete our current understanding of the interplay between ligand/coregulator binding and dynamic/structural changes that regulate ER's biological activity.

Characterization of HIV-1 Protease-Inhibitor Interaction by Interflap Distance Measurement, NMR Spectroscopy, and Solution Kinetics

HIV-1 protease (HIV-1 PR) is an important drug target for the treatment of HIV/AIDS. Currently, there are several commercially available protease inhibitors (PIs) that improve the lives of patients. However, viral mutation often renders the PIs less effective after continuous use. In this study, we compare the effects of several PIs such as Indinavir, Atazanavir, Lopinavir, Saquinavir and Nelfinavir on the activity of the wild-type (PMPR), clinical isolate V6 and MDR769 HIV-1 proteases. We also use 2D HSQC NMR of uniformly 15N labeled samples and DEER spectroscopy with K55MTSL as the reporter site to study the conformational change in HIV-1 PR as a function of various inhibitors. Preliminary solution kinetics data show strong inhibition of PMPR by various inhibitors, but not for V6 and MDR769. The NMR spectra of unbound PMPR, V6, and MDR769 differ markedly from one another, and significant changes in the protein chemical shifts of PMPR and V6 are seen in the presence of inhibitors. The DEER distance distribution profiles reveal altered flap conformations in the uninhibited state of V6 and MDR769 compared to PMPR. Finally, in the presence of inhibitors such as Indinavir, the flap conformations in V6 and MDR769 show a minor change, whereas data for PMPR reflects a closing of the flaps to a conformation consistent with X-ray crystallographic structures.

A Model for the Solution Structure of the Rod Arrestin Tetramer

Visual rod arrestin has the ability to self-associate at physiological concentrations. We previously demonstrated that only monomeric arrestin can bind the receptor and that the arrestin tetramer in solution differs from that in the crystal. We employed the Rosetta docking software to generate molecular models of the physiologically relevant solution tetramer based on the monomeric arrestin crystal structure. The resulting models were filtered using the Rosetta energy function, experimental intersubunit distances measured with DEER spectroscopy, and intersubunit contact sites identified by mutagenesis and site-directed spin labeling. This resulted in a unique model for subsequent evaluation. The validity of the model is strongly supported by model-directed crosslinking and targeted mutagenesis that yields arrestin variants deficient in self-association. The structure of the solution tetramer explains its inability to bind rhodopsin and paves the way for experimental studies of the physiological role of rod arrestin self-association.

A site‐directed spin labeling study of arrestin conformation in solution and bound to activated rhodopsin

Visual rod arrestin binds to light-activated phosphorylated rhodopsin (P-Rh*) to terminate receptor signaling. Previous studies have shown that arrestin forms tetramers at physiological concentrations, but only the monomeric form binds to P-Rh*. The arrestin crystal structure reveals a tetramer in which the monomers have different conformations in flexible regions of the molecule (eg, the “finger loop” involved in P-Rh* binding). Interestingly, the structure of the crystal and solution tetramers is different. To explore the monomer structure in the solution tetramer, spin labels were introduced pairwise in the molecule. Distances between the pairs were determined by DEER spectroscopy to provide global structural constraints. The set from which pairs were selected are shown in the figure, and include sites in both the N and C terminal domains. Distance data obtained so far are consistent with the D chain conformation seen in the crystal structure tetramer in which the finger loop is in a bent conformation, rather than extended. Pairs involving residue 344 show broad distance distributions indicating plasticity of the host loop. Upon binding to P-Rh*, the pattern of distance changes observed suggests that the internal structure of the N and C domains remains essentially unchanged, but that specific rearrangements of the finger loop and an adjacent loop containing residue 139 take place.