Methanethiosulfonate Spin Label Research Articles

Spin Labeling of Surface Cysteines Using a Bromoacrylaldehyde Spin Label

Structural investigations of proteins and their biological complexes are now frequently complemented by distance constraints between spin labeled cysteines generated using double electron–electron resonance (DEER) spectroscopy, via site directed spin labeling (SDSL). Methanethiosulfonate spin label (MTSSL), has become ubiquitous in the SDSL of proteins, however, has limitations owing to its high number of rotamers, and reducibility. In this article we introduce the use of bromoacrylaldehyde spin label (BASL) as a cysteine spin label, demonstrating an advantage over MTSSL due to its increased selectivity for surface cysteines, eliminating the need to ‘knock out’ superfluous cysteine residues. Applied to the multidomain protein, His domain protein tyrosine phosphatase (HD-PTP), we show that BASL can be easily added in excess with selective labeling, whereas MTSSL causes protein precipitation. Furthermore, using DEER, we were able to measure a single cysteine pair distance in a three cysteine domain within HD-PTP. The label has a further advantage of comprising a sulfide in a three-bond tether, making it a candidate for protein binding and in-cell studies.

Site-directed spin labeling-electron paramagnetic resonance spectroscopy in biocatalysis: Enzyme orientation and dynamics in nanoscale confinement

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy probes the otherwise inaccessible structural information in complex biological systems. We recently extended SDSL-EPR to reveal the relative orientation and backbone dynamics of enzymes upon encapsulation in mesoporous nanostructures, which set the structural basis underlying the observed biocatalytic activity. Our strategy had generated interest in the biocatalysis community, and thus in this resource article, we contribute an introduction to the principles and experimental procedure that generalize SDSL-EPR to heterogeneous biocatalysis. We will focus on enzymes in mesoporous materials with examples demonstrating the methods and cautions of potential pitfalls. The ultimate goal is to provide the biocatalysis community with a powerful resource to fill in a long-standing knowledge gap in heterogeneous biocatalysis and the structure-function relationship of enzymes at the interface of enzyme-mesoporous materials and utilize the structural insights to guide the rational design of porous platforms for enzyme immobilization. • Enzyme orientation is determined by using EPR to probe its contact with nanoscale pores • Enzyme dynamics in nanoscale pores and associated catalytic activities are revealed • Protocols and procedures are given for experimental data acquisition, analysis, and interpretation • Various EPR techniques and spin labels can be extended to heterogeneous biocatalysis Enzyme immobilization in nanoscale confinement offers enhanced enzyme protection and reusability, yet the structure-function relationship of the entrapped enzymes remains elusive. Site-directed spin labeling in combination with electron paramagnetic resonance (EPR) spectroscopy has primarily been applied in determining protein structural information in complex biological systems. In this resource article, we introduce the extension of the X-band continuous-wave EPR with a methanethiosulfonate spin label to reveal enzyme structural basis upon immobilization into nanoscale confinement. Using our recent works as examples, we demonstrate the method for determining enzyme orientation and backbone dynamics upon immobilization with a special focus on generalizing the method to heterogeneous biocatalysis and cautions in each step. Furthermore, we summarize the recent findings in other EPR techniques and spin labels so that the whole EPR toolbox can be extended to heterogeneous biocatalysis. In this resource article, Yang and colleagues introduce a general procedure that can reveal the orientation and backbone dynamics of enzymes entrapped in mesoporous materials based on metal-organic materials (MOMs) by using X-band SDSL-EPR. They demonstrate the method by using recently published works focusing on enzyme-MOM interfaces, as well as highlight cautions and potential solutions at each step. They then summarize a variety of other EPR techniques and spin labels in order to pinpoint their potential applications in heterogeneous biocatalysis in the near future.

In Situ Labeling and Distance Measurements of Membrane Proteins in E. coli Using Finland and OX063 Trityl Labels.

In situ investigation of membrane proteins is a challenging task. Previously we demonstrated that nitroxide labels combined with pulsed ESR spectroscopy is a promising tool for this purpose. However, the nitroxide labels suffer from poor stability, high background labeling, and low sensitivity. Here we show that Finland (FTAM) and OX063 based labels enable labeling of the cobalamin transporter BtuB and BamA, the central component of the β‐barrel assembly machinery (BAM) complex, in E coli. Compared to the methanethiosulfonate spin label (MTSL), trityl labels eliminated the background signals and enabled specific in situ labeling of the proteins with high efficiency. The OX063 labels show a long phase memory time (TM) of ≈5 μs. All the trityls enabled distance measurements between BtuB and an orthogonally labeled substrate with high selectivity and sensitivity down to a few μm concentration. Our data corroborate the BtuB and BamA conformations in the cellular environment of E. coli.

In situ observation of conformational dynamics and protein ligand-substrate interactions in outer-membrane proteins with DEER/PELDOR spectroscopy.

Observing structure and conformational dynamics of membrane proteins at high-resolution in their native environments is challenging because of the lack of suitable techniques. We have developed an approach for high-precision distance measurements in the nanometer range for outer membrane proteins (OMPs) in intact E. coli and native membranes. OMPs in Gram-negative bacteria rarely have reactive cysteines. This enables in-situ labeling of engineered cysteines with a methanethiosulfonate functionalized nitroxide spin label (MTSL) with minimal background signals. Following overexpression of the target protein, spin labeling is performed with E. coli or isolated outer membranes (OM) under selective conditions. The interspin distances are measured in-situ using pulsed electron-electron double resonance spectroscopy (PELDOR or DEER). The residual background signals, which are problematic for in-situ structural biology, contributes specifically to the intermolecular part of the signal and can be selectively removed to extract the desired interspin distance distribution. The initial cloning stage can take 5–7 d and the subsequent protein expression, OM isolation, spin labeling, PELDOR experiment, and the data analysis typically take 4–5 d. The described protocol provides a general strategy for observing protein-ligand/substrate interactions, oligomerization, and conformational dynamics of OMPs in the native OM and intact E. coli.

An Approach for Exploring Novel Conformational States and Membrane Organization of BtuB in Whole Cells using EPR Spectroscopy

TonB dependent transporters (TBDTs) are a family of outer membrane proteins (OMPs) in Gram-negative bacteria that facilitate the active transport of nutrients across the outer membrane (OM). The energy for active transport is derived from inner membrane (IM) proton motive force (pmf) through a reversible coupling to IM protein TonB. Our current understanding of the molecular function of TBDTs is mainly based on high-resolution structural and biophysical characterizations in detergent purified and reconstituted lipid vesicles. Unfortunately, TBDT has never been reconstituted and structural measurements have never been made on an active transport system. Recent work has shown that it is possible to spin label the OMP, BtuB in Gram-negative bacteria, thereby enabling the study of TBDT systems using EPR spectroscopy.1 In our present work, we altered the labeling strategy to allow attachment of methanethiosulfonate spin label in cells that are optimized for their metabolic activity. In these preparations we find that specific labeling of single cysteine residues on the extracellular surface of BtuB can be achieved under conditions where rapid vitamin B12 transport is taking place. Distance measurements using pulse EPR require spin label pairs, and to our surprise many cysteine pairs that are expressed in this system fail to label even though the corresponding single labels can be efficiently labeled. This problem is due to the redox homeostasis in the periplasm and it can be overcome by the selection of appropriate mutant strains and labeling strategy. We have used this strategy to explore the supramolecular organization of OMPs and to identify conformational states of BtuB that have not been seen in purified protein.1. Joseph et al. (2015) Angew. Chem. Int. Ed. 54, 1-5.This work was supported by NIGMS GM-035215.

Free Energy Landscape of the Complete Transport Cycle in a Key Bacterial Transporter.

PepTSo is a proton-coupled bacterial symporter, from the major facilitator superfamily (MFS), which transports di-/tripeptide molecules. The recently obtained crystal structure of PepTSo provides an unprecedented opportunity to gain an understanding of functional insights of the substrate transport mechanism. Binding of the proton and peptide molecule induces conformational changes into occluded (OC) and outward-facing (OF) states, which we are able to characterize using molecular dynamics (MD) simulations. The structural knowledge of the OC and OF state is important to fully understand the major energy barrier associated with the transport cycle. In order to gain functional insight into the interstate dynamics, we performed extensive all atom MD simulations. The Markov state model was constructed to identify the free energy barriers between the states, and kinetic information on intermediate pathways was obtained using the transition pathway theory (TPT). TPT shows that the OF state is obtained by the movement of TM1 and TM7 at the extracellular side approximately 12–16 Å away from each other, and the inward movement of TM4 and TM10 at the intracellular halves to 3–4 Å characterizes the OC state. Helix distance distributions obtained from MD simulations were compared with experimental double electron–electron resonance spectroscopy and were found to be in excellent agreement with previous studies. We also predicted the optimal positions for placement of methane thiosulfonate spin label probes to capture the slowest protein dynamics. Our finding sheds light on the conformational cycle of this key membrane transporter and the functional relationships between the multiple intermediate states.

IKMTSL-PTE, a Phospholipid-Based EPR Probe for Surface Electrostatic Potential of Biological Interfaces at Neutral pH: Effects of Temperature and Effective Dielectric Constant of the Solvent.

The synthesis and characterization of a lipidlike electrostatic spin probe, (S)-2,3-bis(palmitoyloxy)propyl 2-((4-(4-(dimethylamino)-2-ethyl-1-oxyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-2-yl)benzyl)disulfanyl)ethyl phosphate (IKMTSL-PTE), are being reported. The intrinsic pKa0 of IKMTSL-PTE was determined by X-band (9.5 GHz) electron paramagnetic resonance (EPR) titration of a water-soluble model compound, 4-(dimethylamino)-2-ethyl-2-(4-(((2-hydroxyethyl)disulfanyl)methyl)phenyl)-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl (IKMTSL-ME), an adduct of methanethiosulfonate spin label IKMTSL and 2-mercaptoethanol. The pKa0 of IKMTSL-ME in bulk aqueous solutions was found to be significantly higher than that of 4-(((2-hydroxyethyl)disulfanyl)methyl)-2,2,3,5,5-pentamethylimidazolidin-1-oxyl (IMTSL-ME), an adduct of the corresponding methanethiosulfonate spin label IMTSL and 2-mercaptoethanol (17 °C, pKa0 = 6.16 ± 0.03 vs 20 °C, pKa0 = 3.33 ± 0.03, respectively). A series of EPR titration experiments with IKMTSL-ME in aqueous solutions containing 0-60% v/v isopropanol have been carried out at 17 and 48 °C to determine the effects of temperature and bulk dielectric permittivity constant, ε, on the probe pKa. A linear relationship between the probe pKa and ε has been established and found to be essentially the same at 17 and 48 °C. The polarity term contributing to the pKa of IKMTSL-PTE at an uncharged lipidlike interface was determined by incorporating the probe into electrically neutral micelles formed from nonionic detergent Triton X-100, and it was found, similar to IMTSL-PTE, to be negative. In negatively charged DMPG lipid bilayers, IKMTSL-PTE exhibits ionization transitions with significantly higher pKa values than those previously reported for IMTSL-PTE (e.g., at 17 °C, pKai = 7.80 ± 0.03 vs pKa0 = 5.70 ± 0.05). The surface electrostatic potentials of DMPG lipid bilayers calculated using IKMTSL-PTE titration data were found to be somewhat lower than those calculated using IMTSL-PTE. The lower values measured by IKMTSL-PTE are the likely consequences of the structure of the linker that positions the reporter nitroxide further away from the bilayer plane into aqueous phase. Overall, the ionization transitions of IKMTSL-PTE with pKa values close to the neutral pH range make this lipidlike molecule a valuable spectroscopic EPR probe for studying the electrostatic phenomena at biological interfaces, including lipid bilayer/membrane protein systems, that could be unstable in the acidic pH range accessible by the previously available probes.

Internal Dynamics of the 3-Pyrroline-N-Oxide Ring in Spin-Labeled Proteins.

Site-directed spin labeling is a versatile tool to study structure as well as dynamics of proteins using EPR spectroscopy. Methanethiosulfonate (MTS) spin labels tethered through a disulfide linkage to an engineered cysteine residue were used in a large number of studies to extract structural as well as dynamic information on the protein from the rotational dynamics of the nitroxide moiety. The ring itself was always considered to be a rigid body. In this contribution, we present a combination of high-resolution X-ray crystallography and EPR spectroscopy of spin-labeled protein single crystals demonstrating that the nitroxide ring inverts fast at ambient temperature while exhibiting nonplanar conformations at low temperature. We have used quantum chemical calculations to explore the potential energy that determines the ring dynamics as well as the impact of the geometry on the magnetic parameters probed by EPR spectroscopy.

Versatile Trityl Spin Labels for Nanometer Distance Measurements on Biomolecules In Vitro and within Cells.

Structure determination of biomacromolecules under in-cell conditions is a relevant yet challenging task. Electron paramagnetic resonance (EPR) distance measurements in combination with site-directed spin labeling (SDSL) are a valuable tool in this endeavor but the usually used nitroxide spin labels are not well-suited for in-cell measurements. In contrast, triarylmethyl (trityl) radicals are highly persistent, exhibit a long relaxation time and a narrow spectral width. Here, the synthesis of a versatile collection of trityl spin labels and their application in in vitro and in-cell trityl-iron distance measurements on a cytochrome P450 protein are described. The trityl labels show similar labeling efficiencies and better signal-to-noise ratios (SNR) as compared to the popular methanethiosulfonate spin label (MTSSL) and enabled a successful in-cell measurement.

Versatile Trityl Spin Labels for Nanometer Distance Measurements on Biomolecules In Vitro and within Cells

AbstractStructure determination of biomacromolecules under in‐cell conditions is a relevant yet challenging task. Electron paramagnetic resonance (EPR) distance measurements in combination with site‐directed spin labeling (SDSL) are a valuable tool in this endeavor but the usually used nitroxide spin labels are not well‐suited for in‐cell measurements. In contrast, triarylmethyl (trityl) radicals are highly persistent, exhibit a long relaxation time and a narrow spectral width. Here, the synthesis of a versatile collection of trityl spin labels and their application in in vitro and in‐cell trityl–iron distance measurements on a cytochrome P450 protein are described. The trityl labels show similar labeling efficiencies and better signal‐to‐noise ratios (SNR) as compared to the popular methanethiosulfonate spin label (MTSSL) and enabled a successful in‐cell measurement.

Density functional theory studies of MTSL nitroxide side chain conformations attached to an activation loop.

A quantum mechanical (QM) method rooted on density functional theory (DFT) has been employed to determine conformations of the methane-thiosulfonate spin label (MTSL) attached to a fragment extracted from the activation loop of Aurora-A kinase. The features of the calculated energy surface revealed low energy barriers between isoenergetic minima, and the system could be described in a population of 76 rotamers that can be also considered for other systems since it was found that the chi_{3} , chi_{4} and chi_{5} do not depend on the previous two dihedral angles. Conformational states obtained were seen to be comparable to those obtained in the α-helix systems studied previously, indicating that the protein backbone does not affect the torsional profiles significantly and suggesting the possibility to use determined conformations for other protein systems for further modelling studies.Electronic supplementary materialThe online version of this article (doi:10.1007/s00214-016-1859-z) contains supplementary material, which is available to authorized users.

A bifunctional spin label reports the structural topology of phospholamban in magnetically-aligned bicelles

We have applied a bifunctional spin label and EPR spectroscopy to determine membrane protein structural topology in magnetically-aligned bicelles, using monomeric phospholamban (PLB) as a model system. Bicelles are a powerful tool for studying membrane proteins by NMR and EPR spectroscopies, where magnetic alignment yields topological constraints by resolving the anisotropic spectral properties of nuclear and electron spins. However, EPR bicelle studies are often hindered by the rotational mobility of monofunctional Cys-linked spin labels, which obscures their orientation relative to the protein backbone. The rigid and stereospecific TOAC label provides high orientational sensitivity but must be introduced via solid-phase peptide synthesis, precluding its use in large proteins. Here we show that a bifunctional methanethiosulfonate spin label attaches rigidly and stereospecifically to Cys residues at i and i+4 positions along PLB’s transmembrane helix, thus providing orientational resolution similar to that of TOAC, while being applicable to larger membrane proteins for which synthesis is impractical. Computational modeling and comparison with NMR data shows that these EPR experiments provide accurate information about helix tilt relative to the membrane normal, thus establishing a robust method for determining structural topology in large membrane proteins with a substantial advantage in sensitivity over NMR.

EPR Distance Measurements in Native Proteins with Genetically Encoded Spin Labels.

The genetic encoding of nitroxide amino acids in combination with electron paramagnetic resonance (EPR) distance measurements enables precise structural studies of native proteins, i.e. without the need for mutations to create unique reactive sites for chemical labeling and thus with minimal structural perturbation. We here report on in vitro DEER measurements in native E. coli thioredoxin (TRX) that establish the nitroxide amino acid SLK-1 as a spectroscopic probe that reports distances and conformational flexibilities in the enzyme with nonmutated catalytic centers that are not accessible by the use of the traditional methanethiosulfonate spin label (MTSSL). We generated a rotamer library for SLK-1 that in combination with molecular dynamics (MD) simulation enables predictions of distance distributions between two SLK-1 labels incorporated into a target protein. Toward a routine use of SLK-1 for EPR distance measurements in proteins and the advancement of the approach to intracellular environments, we study the stability of SLK-1 in E. coli cultures and lysates and establish guidelines for protein expression and purification that offer maximal nitroxide stability. These advancements and insights provide new perspectives for facile structural studies of native, endogenous proteins by EPR distance measurements.

Proton Transverse Relaxation as a Sensitive Probe for Structure Determination in Solid Proteins.

Solid-state nuclear magnetic resonance (NMR) spectroscopy has been successfully applied to elucidate the atomic-resolution structures of insoluble proteins. The major bottleneck is the difficulty to obtain valuable long-distance structural information. Here, we propose the use of distance restraints as long as 32 Å, obtained from the quantification of transverse proton relaxation induced by a methanethiosulfonate spin label (MTSL). Combined with dipolar proton-proton distance restraints, this method allows us to obtain protein structures with excellent precision from single spin-labeled 1 mg protein samples using fast magic angle spinning.

ATP-dependent Conformational Changes Trigger Substrate Capture and Release by an ECF-type Biotin Transporter

Energy-coupling factor (ECF) transporters for vitamins and metal ions in prokaryotes consist of two ATP-binding cassette-type ATPases, a substrate-specific transmembrane protein (S component) and a transmembrane protein (T component) that physically interacts with the ATPases and the S component. The mechanism of ECF transporters was analyzed upon reconstitution of a bacterial biotin transporter into phospholipid bilayer nanodiscs. ATPase activity was not stimulated by biotin and was only moderately reduced by vanadate. A non-hydrolyzable ATP analog was a competitive inhibitor. As evidenced by cross-linking of monocysteine variants and by site-specific spin labeling of the Q-helix followed by EPR-based interspin distance analyses, closure and reopening of the ATPase dimer (BioM2) was a consequence of ATP binding and hydrolysis, respectively. A previously suggested role of a stretch of small hydrophobic amino acid residues within the first transmembrane segment of the S units for S unit/T unit interactions was structurally and functionally confirmed for the biotin transporter. Cross-linking of this segment in BioY (S) using homobifunctional thiol-reactive reagents to a coupling helix of BioN (T) indicated a reorientation rather than a disruption of the BioY/BioN interface during catalysis. Fluorescence emission of BioY labeled with an environmentally sensitive fluorophore was compatible with an ATP-induced reorientation and consistent with a hypothesized toppling mechanism. As demonstrated by [(3)H]biotin capture assays, ATP binding stimulated substrate capture by the transporter, and subsequent ATP hydrolysis led to substrate release. Our study represents the first experimental insight into the individual steps during the catalytic cycle of an ECF transporter in a lipid environment.

Abstract 111: HDL-apolipoprotein A-I Exchange Is Impaired in the Metabolic Syndrome

Objective: Metabolic syndrome (MetSyn) is associated with a host of cardiovascular risk factors including increased waist circumference, triglycerides, blood pressure, fasting glucose, and reduced high density lipoprotein cholesterol (HDL-C). Changes in HDL function, especially the ability to participate in reverse cholesterol transport, are more indicative of the atheroprotective properties of HDL compared to HDL-C levels alone. In this study, we determine the association of HDL-apolipoproteinA-I (apoA-I) exchange, a measure of HDL remodeling/apoA-I exchange, with MetSyn and its individual components. Methods and Results: We analyzed HDL-apoA-I exchange (HAE) in the plasma of 60 well-characterized subjects with MetSyn and 14 healthy control subjects. HAE measurements were obtained using electron paramagnetic resonance (EPR) following incubation of plasma with lipid-freeapoA-I containing a methanethiosulfonate spin label. HAE positively correlated with plasma HDL-C and apoA-I levels, and inversely correlated with fasting blood glucose levels, blood pressure, BMI, and triglycerides. Multiple linear regression showed that HAE is significantly correlated with MetSyn (HAE: r2 = 0.57, P<0.0001), though MetSyn subjects on statins exhibited significantly higher HAE compared to subjects not on statins. Conclusions: MetSyn has a significant negative impact on HDL remodelin/apoA-I exchange, as measured by HAE. HAE is a strong identifier of MetSyn status even after adjusting for individual components of MetSyn.

Using Molecular Dynamics Simulations to Interpret Fluorescence and EPR Data on Labeled SERCA and Phospholamban

We have performed molecular dynamics simulations of the sarcoplasmic reticulum Ca2+-ATPase (SERCA) and phospholamban (PLB) labeled with spectroscopic probes in order to establish a foundation for analyzing fluorescence and EPR data from this system. Site-specific labeling of a protein can provide insight into local structural dynamics, based on fluorescence quenching, anisotropy, or EPR measurements. Labelling can also provide structural information like distances to another label as measured with FRET or DEER and orientation in aligned bicelles as measured with EPR. To interpret the experimental results in a structural context, we have undertaken an approach involving X-ray crystallography and computational simulations. To perform these simulations, we developed CHARMM force-field parameters for the probes used. SERCA was labeled at position C674 with the fluorescent probe IAEDANS. A crystal structure of IAEDANS-labeled SERCA was used as a starting point for molecular dynamics simulations. The transition dipole autocorrelation functions were calculated and were shown to agree with experimental measurements of fluorescence anisotropy. Interprobe distance R and orientation factor κ2, determined from the simulations were used to predict lifetime changes due to FRET. PLB was labeled with the bifunctional methanethiosulfonate spin-label (BSL) at position F32C/A36C, and EPR data was acquired on magnetically aligned bicelles, which allows determining the orientation of spin labels with respect to the applied magnetic field. By combining EPR studies and molecular dynamics simulations, we have shown that this approach is capable of directly reporting the structural topology of monomeric PLB. These results show that we have established reliable frameworks for integrating spectroscopic experiments and molecular simulations in the SERCA-PLB complex. Spectroscopic studies were performed at Minnesota Biophysical Spectroscopy Center and computational work at the Minnesota Supercomputing Institute. This work was supported by NIH (GM27906, AR007612).

Characterizing solution surface loop conformational flexibility of the GM2 activator protein.

GM2AP has a β-cup topology with numerous X-ray structures showing multiple conformations for some of the surface loops, revealing conformational flexibility that may be related to function, where function is defined as either membrane binding associated with ligand binding and extraction or interaction with other proteins. Here, site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy and molecular dynamic (MD) simulations are used to characterize the mobility and conformational flexibility of various structural regions of GM2AP. A series of 10 single cysteine amino acid substitutions were generated, and the constructs were chemically modified with the methanethiosulfonate spin label. Continuous wave (CW) EPR line shapes were obtained and subsequently simulated using the microscopic order macroscopic disorder (MOMD) program. Line shapes for sites that have multiple conformations in the X-ray structures required two spectral components, whereas spectra of the remaining sites were adequately fit with single-component parameters. For spin labeled sites L126C and I66C, spectra were acquired as a function of temperature, and simulations provided for the determination of thermodynamic parameters associated with conformational change. Binding to GM2 ligand did not alter the conformational flexibility of the loops, as evaluated by EPR and NMR spectroscopies. These results confirm that the conformational flexibility observed in the surface loops of GM2AP crystals is present in solution and that the exchange is slow on the EPR time scale (>ns). Furthermore, MD simulation results are presented and agree well with the conformational heterogeneity revealed by SDSL.

Automated structure refinement for a protein heterodimer complex using limited EPR spectroscopic data and a rigid-body docking algorithm: a three-dimensional model for an ankyrin-CDB3 complex.

We report here specialized functions incorporated recently in the rigid-body docking software toolkit TagDock to utilize electron paramagnetic resonance derived (EPR-derived) interresidue distance measurements and spin-label accessibility data. The TagDock package extensions include a custom methanethiosulfonate spin label rotamer library to enable explicit, all-atom spin-label side-chain modeling and scripts to evaluate spin-label surface accessibility. These software enhancements enable us to better utilize the biophysical data routinely available from various spin-labeling experiments. To illustrate the power and utility of these tools, we report the refinement of an ankyrin:CDB3 complex model that exhibits much improved agreement with the EPR distance measurements, compared to model structures published previously.

Structural Studies of the Docking Complex between Cytochrome P450 (CYP101) and Putidaredoxin (PDX) by Double Electron-Electron Resonance (DEER)

P450cam (CYP101) is an archetype of the cytochrome P450 superfamily of heme mono-oxygenases. This enzyme hydroxylates camphor to 5-exo-hydroxy-camphor following two distinct electron transfers from its unique [2Fe-2S] redox partner, Pdx (Putidaredoxin). Other ferredoxin-type proteins, such as adrenodoxin, are only able transfer the first electron and lack the features of the complex of P450:Pdx required for the second electron transfer. This specificity of the protein electron donor suggests that geometry of the protein-protein complex may play a critical role in the electron transfer process. Moreover, we have observed in previous studies that P450cam shifts from open to closed conformation upon binding camphor, and that oxidized but not reduced Pdx induces a shift of substrate-bound P450cam from the closed to the open conformation. The above results lead us to pursue structural information about the solution complex of P450cam and Pdx in different redox and coordination states. Several combinations of site-directed spin labeling (SDSL) sites were made with single methanethiosulfonate (MTSL) spin-labels on P450 and Pdx. The inter-spin label distance constraints in the P450:Pdx complex were measured by Double Electron Electron Resonance (DEER or PELDOR). While these studies show that the solution complex between P450cam and Pdx is consistent with that reported in recent crystallographic studies, the studies also show little difference in the docking conformation of P450cam(oxidized)-Pdx(oxidized) and P450cam(reduced CO)-Pdx(reduced). We will also report DEER studies of the conformational states of P450cam bound to CN- and NO. These results will help clarify the structural factors responsible for the role of Pdx as a structural effector of P450cam function.