Jeschke, Gunnar
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Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules
Distance distribution information obtained by pulsed dipolar EPR spectroscopy provides an important contribution to many studies in structural biology. Increasingly, such information is used in integrative structural modeling, where it delivers unique restraints on the width of conformational ensembles. In order to ensure reliability of the structural models and of biological conclusions, we herein define quality standards for sample preparation and characterization, for measurements of distributed dipole-dipole couplings between paramagnetic labels, for conversion of the primary time-domain data into distance distributions, for interpreting these distributions, and for reporting results. These guidelines are substantiated by a multi-laboratory benchmark study and by analysis of data sets with known distance distribution ground truth. The study and the guidelines focus on proteins labeled with nitroxides and on double electron-electron resonance (DEER aka PELDOR) measurements and provide suggestions on how to proceed analogously in other cases.
DEER distance measurements on proteins
Distance distributions between paramagnetic centers in the range of 1.8 to 6 nm in membrane proteins and up to 10 nm in deuterated soluble proteins can be measured by the DEER technique. The number of paramagnetic centers and their relative orientation can be characterized. DEER does not require crystallization and is not limited with respect to the size of the protein or protein complex. Diamagnetic proteins are accessible by site-directed spin labeling. To characterize structure or structural changes, experimental protocols were optimized and techniques for artifact suppression were introduced. Data analysis programs were developed, and it was realized that interpretation of the distance distributions must take into account the conformational distribution of spin labels. First methods have appeared for deriving structural models from a small number of distance constraints. The present scope and limitations of the technique are illustrated.
Relaxation-based distance measurements between a nitroxide and a lanthanide spin label
Distance measurements by electron paramagnetic resonance techniques between labels attached to biomacromolecules provide structural information on systems that cannot be crystallized or are too large to be characterized by NMR methods. However, existing techniques are limited in their distance range and sensitivity. It is anticipated by theoretical considerations that these limits could be extended by measuring the enhancement of longitudinal relaxation of a nitroxide label due to a lanthanide complex label at cryogenic temperatures. The relaxivity of the dysprosium complex with the macrocyclic ligand DOTA can be determined without direct measurements of longitudinal relaxation rates of the lanthanide and without recourse to model compounds with well defined distance by analyzing the dependence of relaxation enhancement on either temperature or concentration in homogeneous glassy frozen solutions. Relaxivities determined by the two calibration techniques are in satisfying agreement with each other. Error sources for both techniques are examined. A distance of about 2.7 nm is measured in a model compound of the type nitroxide–spacer–lanthanide complex and is found in good agreement with the distance in a modeled structure. Theoretical considerations suggest that an increase of the upper distance limit requires measurements at lower fields and temperatures.
Photo-CIDNP MAS NMR beyond the T1 limit by fast cycles of polarization extinction and polarization generation
In nanosecond-laser flash photo-CIDNP MAS NMR, polarization generation (PG) proceeds much faster than longitudinal spin relaxation. With a nanosecond-laser setup linked to the NMR console the repetition time of the experiment is then limited by the minimum recycle delay of the NMR spectrometer and the maximum repetition rate of laser flashes. These limits can only be reached if polarization left after the NMR experiment is completely canceled before the next laser flash. We introduce a presaturation pulse sequence, based on three (π/2) 13C pulses and optimized timing and phase cycling that allows for such efficient polarization extinction (PE). The technique is demonstrated on selectively isotope labeled bacterial reaction centers (RCs) of Rhodobacter (Rb.) sphaeroides wildtype (WT). High-quality 13C photo-CIDNP MAS NMR spectra are obtained using cycle rates up to 4 Hz. The PE–PG strategy proposed here provides a general experimental scheme for reduction of measurement time in magnetic resonance experiments based on fast PG.
Laser-Induced Magnetic Dipole Spectroscopy
Pulse electron paramagnetic resonance measurements of nanometer scale distance distributions have proven highly effective in structural studies. They exploit the magnetic dipole-dipole coupling between spin labels site-specifically attached to macromolecules. The most commonly applied technique is double electron-electron resonance (DEER, also called pulsed electron double resonance (PELDOR)). Here we present the new technique of laser-induced magnetic dipole (LaserIMD) spectroscopy based on optical switching of the dipole-dipole coupling. In a proof of concept experiment on a model peptide, we find, already at a low quantum yield of triplet excitation, the same sensitivity for measuring the distance between a porphyrin and a nitroxide label as in a DEER measurement between two nitroxide labels. On the heme protein cytochrome C, we demonstrate that LaserIMD allows for distance measurements between a heme prosthetic group and a nitroxide label, although the heme triplet state is not directly observable by an electron spin echo.
Folding and Pigment Binding of Light-Harvesting Chlorophyll a/b Protein (LHCIIb)
The major light-harvesting chlorophyll a/b protein (LHCIIb) is one of the most abundant proteins of the chloroplast in green plants. It contains roughly half of the chlorophylls involved in photosynthesis, and exhibits an unusual ability to self-organize in vitro. Simply mixing the apoprotein, native or recombinant, with its pigments, chlorophyll a, chlorophyll b, and xanthophylls, in detergent solution, suffices to trigger protein folding and the assembly of about 18 pigments in their correct binding sites. A study of the mechanism of this self-organization seems worthwhile since (1) our knowledge about membrane protein folding is scarce compared to what we know about the folding of water-soluble proteins, (2) the mechanism of LHCIIb formation in vitro may give useful clues about the so-far unknown pathway of its assembly in the chloroplast, and (3) a thorough understanding of the process may facilitate the application of recombinant LHCIIb in hybrid constructs such as photovoltaic devices or the construction of potentially useful proteins or other polymers that spontaneously bind other dyes at a similarly high density.
A Comparative Electron Paramagnetic Resonance Study of the Nucleotide-Binding Domains’ Catalytic Cycle in the Assembled Maltose ATP-Binding Cassette Importer
We present a quantitative analysis of conformational changes of the nucleotide-binding subunits, MalK2, of the maltose ATP-binding cassette importer MalFGK2 during the transport cycle. Distance changes occurring between selected residues were monitored in the full transporter by site-directed spin-labeling electron paramagnetic resonance spectroscopy and site-directed chemical cross-linking. We considered S83C and A85C from the conserved Q-loop and V117C located on the outer surface of MalK. Additionally, two native cysteines (C350, C360) were included in the study. On ATP binding, small rearrangements between the native sites, and no distance changes between positions 117 were detected. In contrast, positions 85 come closer together in the ATP-bound state and in the vanadate-trapped intermediate and move back toward the apo-state after ATP hydrolysis. The distance between positions 83 is shown to slightly decrease on ATP binding, and to further decrease after ATP hydrolysis. Results from cross-linking experiments are in agreement with these findings. The data are compared with in silico spin-labeled x-ray structures from both isolated MalK2 and the MalFGK2-E complex. Our results are consistent with a slightly modified “tweezers-like” model of closure and reopening of MalK2 during the catalytic cycle, and show an unforeseen potential interaction between MalK and the transmembrane subunit MalG.
Gd(III)-PyMTA Label Is Suitable for In-Cell EPR
Distance measurement in the nanometer range by electron paramagnetic resonance spectroscopy (EPR) in combination with site-directed spin labeling is a very powerful tool to monitor the structure and dynamics of biomacromolecules in their natural environment. However, in-cell application is hampered by the short lifetime of the commonly used nitroxide spin labels in the reducing milieu inside a cell. Here, we demonstrate that the Gd(III) based spin label Gd-PyMTA is suitable for in-cell EPR. Gd-PyMTA turned out to be cell compatible and was proven to be inert in in-cell extracts of Xenopus laevis oocytes at 18 °C for more than 24 h. The proline rich peptide H-AP10CP10CP10-NH2 was site-directedly spin labeled with Gd-PyMTA at both cysteine moieties. The resulting peptide, H-AP10C(Gd-PyMTA)P10C(Gd-PyMTA)P10-NH2, as well as the model compound Gd-spacer-Gd, which consists of a spacer of well-known stiffness, were microinjected into Xenopus laevis oocytes, and the Gd(III)–Gd(III) distances were determined by double electron–electron resonance (DEER) spectroscopy. To analyze the intracellular peptide conformation, a rotamer library was set up to take the conformational flexibility of the tether between the Gd(III) ion and the Cα of the cysteine moiety into account. The results suggest that the spin labeled peptide H-AP10C(Gd-PyMTA)P10C(Gd-PyMTA)P10-NH2 is inserted into cell membranes, coinciding with a conformational change of the oligoproline from a PPII into a PPI helix.
Backbone Structure of Transmembrane Domain IX of the Na+/Proline Transporter PutP of Escherichia coli
The backbone structure is determined by site-directed spin labeling, double electron electron resonance measurements of distances, and modeling in terms of a helix-loop-helix construct for a transmembrane domain that is supposed to line the translocation pathway in the 54.3 kDa Na+/proline symporter PutP of Escherichia coli. The conformational distribution of the spin labels is accounted for by a rotamer library. An ensemble of backbone models with a root mean-square deviation of less than 2 Å is obtained. These models exhibit a pronounced kink near residue T341, which is involved in substrate binding. The kink may be associated with a hinge that allows the protein to open and close an inwardly oriented cavity.
Characterization of the Primary Radical Pair in Reaction Centers of Heliobacillus mobilis by 13C Photo-CIDNP MAS NMR
Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in membrane fragments of heliobacterium Heliobacillus mobilis without further isolation by 13C magic-angle spinning (MAS) solid-state NMR under continuous illumination with white light. In the 13C photo-CIDNP MAS NMR spectra of heliobacterial membrane fragments, two sets of signals are observed, allowing characterization of the primary radical pair. One set, showing enhanced absorptive (positive) signals, arises from the BChl g donor, while the set of emissive (negative) signals is assigned to the 81-hydroxy Chl a acceptor. Hence, under these sample conditions, both donor and acceptor sides are either monomeric or composed of identical cofactors. The occurrence of the differential relaxation (DR) mechanism suggests a donor triplet lifetime in the microsecond range. It appears that the occurrence of the solid-state photo-CIDNP effect is a general feature of primary radical pairs in natural photosynthesis.