Nanoscale Distance Measurements Research Articles

The use of single-molecule FRET for the characterization of Holliday junctions containing human telomeric DNA: a methodological approach for nanoscale distance, mobility, and stability measurements

The use of single-molecule FRET for the characterization of Holliday junctions containing human telomeric DNA: a methodological approach for nanoscale distance, mobility, and stability measurements

Mechanism of Electron Spin Decoherence in a Partially Deuterated Glassy Matrix.

Long electron spin coherence lifetimes are essential for applications in quantum information science and electron paramagnetic resonance, for instance, for nanoscale distance measurements in biomolecular systems using double electron-electron resonance. We experimentally investigate the decoherence dynamics under the Hahn echo sequence of the organic radical d18-TEMPO in a variably deuterated frozen water:glycerol matrix. The coherence time (phase memory time) TM scales with proton concentration as [1H]-0.65. For selectively deuterated matrices, decoherence is accelerated in the presence of proton clustering, that is, substantial short-range density in the proton-proton radial distribution functions (<3 Å). Simulations using molecular dynamics and many-body spin quantum dynamics show excellent agreement with experiment and show that geminal proton pairs such as CH2 and OH2 groups are major decoherence drivers. This provides a predictive tool for designing molecular systems with long electron spin coherence times.

Identification of Patients with Pancreatic Cancer by Electron Paramagnetic Resonance Spectroscopy of Fatty Acid Binding to Human Serum Albumin.

An effective biological marker for pancreatic adenocarcinoma (PAC) is not available so far. Here, we investigate how electron paramagnetic resonance (EPR) spectroscopy of spin-labeled fatty acid (FA) molecules binding to human serum albumin (HSA) in human serum is a suitable method for the identification of patients with PAC through detection of PAC-induced changes of FA binding to albumin. The functionality of HSA to bind FA is investigated in serum samples of 35 patients with PAC, 26 patients with benign pancreatic tumors (BPD), and 24 healthy individuals by continuous wave (CW) EPR spectroscopy by simply dissolving 16-DOXYL stearic acid as spin-labeled FA. It is found that FA binding to HSA in PAC is significantly modified when compared with healthy and BPD individuals. The PAC group could best be discriminated from the healthy group based on EPR characteristics at the loading ratio of 1:4 (HSA:FA), while patients with PAC and BPD are distinguishable at a loading ratio of 1:6. Using nanoscale distance measurements through double electron-electron resonance (DEER), it is found that the distribution of FAs in the HSA of one PAC patient is similar to that of FAs in healthy individuals. Combining all EPR spectroscopic data, this leads to a tentative molecular interpretation of only small changes in hydration at the protein's surface as origin of the detectable characteristics for PAC patients. Thus, EPR of FA/HSA binding is a simple and promising tool for clinical detection of patients with PAC and needs to be tested with larger ensembles of different patient groups.

Triplet Fullerenes as Prospective Spin Labels for Nanoscale Distance Measurements by Pulsed Dipolar EPR Spectroscopy

AbstractPrecise nanoscale distance measurements by pulsed electron paramagnetic resonance (EPR) spectroscopy play a crucial role in structural studies of biomolecules. The properties of the spin labels used in this approach determine the sensitivity limits, attainable distances, and proximity to biological conditions. Herein, we propose and validate the use of photoexcited fullerenes as spin labels for pulsed dipolar (PD) EPR distance measurements. Hyperpolarization and the narrower spectrum of fullerenes compared to other triplets (e.g., porphyrins) boost the sensitivity, and superior relaxation properties allow PD EPR measurements up to a near‐room temperature. This approach is demonstrated using fullerene–nitroxide and fullerene–triarylmethyl pairs, as well as a supramolecular complex of fullerene with nitroxide‐labeled protein. Photoexcited triplet fullerenes can be considered as new spin labels with outstanding spectroscopic properties for future structural studies of biomolecules.

Triplet Fullerenes as Prospective Spin Labels for Nanoscale Distance Measurements by Pulsed Dipolar EPR Spectroscopy.

Precise nanoscale distance measurements by pulsed electron paramagnetic resonance (EPR) spectroscopy play a crucial role in structural studies of biomolecules. The properties of the spin labels used in this approach determine the sensitivity limits, attainable distances, and proximity to biological conditions. Herein, we propose and validate the use of photoexcited fullerenes as spin labels for pulsed dipolar (PD) EPR distance measurements. Hyperpolarization and the narrower spectrum of fullerenes compared to other triplets (e.g., porphyrins) boost the sensitivity, and superior relaxation properties allow PD EPR measurements up to a near-room temperature. This approach is demonstrated using fullerene-nitroxide and fullerene-triarylmethyl pairs, as well as a supramolecular complex of fullerene with nitroxide-labeled protein. Photoexcited triplet fullerenes can be considered as new spin labels with outstanding spectroscopic properties for future structural studies of biomolecules.

The structural flexibility of the human copper chaperone Atox1: Insights from combined pulsed EPR studies and computations.

Metallochaperones are responsible for shuttling metal ions to target proteins. Thus, a metallochaperone's structure must be sufficiently flexible both to hold onto its ion while traversing the cytoplasm and to transfer the ion to or from a partner protein. Here, we sought to shed light on the structure of Atox1, a metallochaperone involved in the human copper regulation system. Atox1 shuttles copper ions from the main copper transporter, Ctr1, to the ATP7b transporter in the Golgi apparatus. Conventional biophysical tools such as X-ray or NMR cannot always target the various conformational states of metallochaperones, owing to a requirement for crystallography or low sensitivity and resolution. Electron paramagnetic resonance (EPR) spectroscopy has recently emerged as a powerful tool for resolving biological reactions and mechanisms in solution. When coupled with computational methods, EPR with site-directed spin labeling and nanoscale distance measurements can provide structural information on a protein or protein complex in solution. We use these methods to show that Atox1 can accommodate at least four different conformations in the apo state (unbound to copper), and two different conformations in the holo state (bound to copper). We also demonstrate that the structure of Atox1 in the holo form is more compact than in the apo form. Our data provide insight regarding the structural mechanisms through which Atox1 can fulfill its dual role of copper binding and transfer.

Complementary-addressed site-directed spin labeling of long natural RNAs.

Nanoscale distance measurements by pulse dipolar Electron paramagnetic resonance (EPR) spectroscopy allow new insights into the structure and dynamics of complex biopolymers. EPR detection requires site directed spin labeling (SDSL) of biomolecule(s), which remained challenging for long RNAs up-to-date. Here, we demonstrate that novel complementary-addressed SDSL approach allows efficient spin labeling and following structural EPR studies of long RNAs. We succeeded to spin-label Hepatitis C Virus RNA internal ribosome entry site consisting of ≈330 nucleotides and having a complicated spatial structure. Application of pulsed double electron–electron resonance provided spin–spin distance distribution, which agrees well with the results of molecular dynamics (MD) calculations. Thus, novel SDSL approach in conjunction with EPR and MD allows structural studies of long natural RNAs with nanometer resolution and can be applied to systems of biological and biomedical significance.

Interaction of triarylmethyl radicals with DNA termini revealed by orientation-selective W-band double electron-electron resonance spectroscopy.

Spin labels selectively attached to biomolecules allow high-accuracy nanoscale distance measurements using pulsed electron paramagnetic resonance (EPR), in many cases providing the only access to the structure of complex biosystems. Triarylmethyl (TAM) radicals have recently emerged as a new class of spin labels expanding the applicability of the method to physiological temperatures. Along with other factors, the accuracy of the obtained distances crucially relies on the understanding of interactions between biomolecules and spin labels. In this work, we consider such crucial interactions and their impact on pulsed EPR distance measurements in TAM-labeled DNAs. Using orientation-selective high-frequency (94 GHz) double electron-electron resonance (DEER) we demonstrate strong specific interactions between DNA termini and TAM labels, leading to a significant restriction of their conformational mobility. An understanding of such interactions guides the way to select optimum TAM-labeling strategies, thus refining nanoscale EPR distance measurements in nucleic acids and their complexes under physiological conditions.

Triarylmethyl Labels: Toward Improving the Accuracy of EPR Nanoscale Distance Measurements in DNAs.

Triarylmethyl (trityl, TAM) based spin labels represent a promising alternative to nitroxides for EPR distance measurements in biomolecules. Herewith, we report synthesis and comparative study of series of model DNA duplexes, 5'-spin-labeled with TAMs and nitroxides. We have found that the accuracy (width) of distance distributions obtained by double electron-electron resonance (DEER/PELDOR) strongly depends on the type of radical. Replacement of both nitroxides by TAMs in the same spin-labeled duplex allows narrowing of the distance distributions by a factor of 3. Replacement of one nitroxide by TAM (orthogonal labeling) leads to a less pronounced narrowing but at the same time gains sensitivity in DEER experiment due to efficient pumping on the narrow EPR line of TAM. Distance distributions in nitroxide/nitroxide pairs are influenced by the structure of the linker: the use of a short amine-based linker improves the accuracy by a factor of 2. At the same time, a negligible dependence on the linker length is found for the distribution width in TAM/TAM pairs. Molecular dynamics calculations indicate greater conformational disorder of nitroxide labels compared to TAM ones, thus rationalizing the experimentally observed trends. Thereby, we conclude that double spin-labeling using TAMs allows obtaining narrower spin-spin distance distributions and potentially more precise distances between labeling sites compared to traditional nitroxides.

Cooperatively Folded Conformational Sub-States in Intrinsically Disordered Proteins as Revealed by EPR Spectroscopy

The discovery of intrinsically disordered proteins (IDPs) has started revolutionizing structural biology in recent years. Despite the, from a traditional point of view, lack of well-folded structures IDPs fulfill many vital functions and hence seem to contradict or at least amend the classic structure = function paradigm. The structural and functional characterization of IDPs remains challenging; only a few experimental techniques are suitable and appropriate theoretical concepts are rare. Here, we show that nanoscale distance measurements (double electron-electron resonance, DEER) based on electron paramagnetic resonance (EPR) spectroscopy can provide unique insights into the physical character of IDPs. EPR spectroscopy is used to provide quantitative information about the thermodynamic behavior and populations of cooperatively folded sub-states. The methodology was applied to the IDP Osteopontin (OPN), a cytokine involved in metastasis and tumor progression. We demonstrate that the solution structure of OPN is best described as a heterogeneous structural ensemble in which the individual, transiently formed conformational sub-states are largely different, ranging from extended coils devoid of stabilizing interactions to cooperatively folded compact structures with accentuated side-chain interactions and binding sites preformed to accommodate authentic binding partners. Thus, OPN is best understood as a polymer, whose large conformational space is governed by a subtle interplay of electrostatic and hydrophobic forces and significantly enriched with robust tertiary conformational sub-states. The unprecedented finding that IDPs are able to sample conformations reminiscent of globular stably folded proteins under native conditions, goes far beyond the classical view of intrinsically unstructured proteins and calls for a reassessment of the binary description scheme (ordered vs. disordered) proposed for this enormously important protein class.

Evidence for Water-Tuned Structural Differences in Proteins: An Approach Emphasizing Variations in Local Hydrophilicity

We present experimental evidence for the significant effect that water can have on the functional structure of proteins in solution. Human (HSA) and Bovine Serum Albumin (BSA) have an amino acid sequence identity of 75.52% and are chosen as model proteins. We employ EPR-based nanoscale distance measurements using double electron-electron resonance (DEER) spectroscopy and both albumins loaded with long chain fatty acids (FAs) in solution to globally (yet indirectly) characterize the tertiary protein structures from the bound ligands’ points of view. The complete primary structures and crystal structures of HSA and as of recently also BSA are available. We complement the picture as we have recently determined the DEER-derived solution structure of HSA and here present the corresponding BSA solution structure. The characteristic asymmetric FA distribution in the crystal structure of HSA can surprisingly be observed by DEER in BSA in solution. This indicates that the BSA conformational ensemble in solution seems to be narrow and close to the crystal structure of HSA. In contrast, for HSA in solution a much more symmetric FA distribution was found. Thus, conformational adaptability and flexibility dominate in the HSA solution structure while BSA seems to lack these properties. We further show that differences in amino acid hydropathies of specific structural regions in both proteins can be used to correlate the observed difference in the global (tertiary) solution structures with the differences on the primary structure level.

Host–guest interactions in polycationic human serum albumin bioconjugates

Polycationic human serum albumin, cHSA, as well as cHSA conjugates with multiple polyethylene(oxide) chains of two different lengths were synthesized, and the uptake and release of spin-labeled fatty acid (FA) ligands were quantitatively analyzed by continuous wave (CW) electron paramagnetic resonance (EPR) spectroscopy and nanoscale distance measurements with double electron–electron resonance (DEER) spectroscopy. It is found that the seven FA binding pockets of native HSA are less well accessible by FAs in cHSA and its PEO-conjugates. A large number (at least 25) of FAs can diffusely and electrostatically be bound to the surface of all highly cationic serum albumin variants. These bound FAs show a remarkable resilience against release from both cHSA and PEO conjugates. Conjugation of PEO chains to cHSA had only minute effects on all EPR data, indicating that PEO grafts can be used without reduction of effectiveness in the ligand binding.

Conformational changes of the chaperone SecB upon binding to a model substrate – bovine pancreatic trypsin inhibitor (BPTI)

SecB is a homotetrameric cytosolic chaperone that forms part of the protein translocation machinery in E. coli. Due to SecB, nascent polypeptides are maintained in an unfolded translocation-competent state devoid of tertiary structure and thus are guided to the translocon. In vitro SecB rapidly binds to a variety of ligands in a non-native state. We have previously investigated the bound state conformation of the model substrate bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin labeling and pyrene fluorescence methods. It was shown that SecB undergoes a conformational change during the process of substrate binding. Here, we generated SecB mutants containing but a single cysteine per subunit or an exposed highly reactive new cysteine after removal of the nearby intrinsic cysteines. Quantitative spin labeling was achieved with the methanethiosulfonate spin label (MTS) at positions C97 or E90C, respectively. Highfield (W-band) electron paramagnetic resonance (EPR) measurements revealed that with BPTI present the spin labels are exposed to a more polar/hydrophilic environment. Nanoscale distance measurements with double electron-electron resonance (DEER) were in excellent agreement with distances obtained by molecular modeling. Binding of BPTI also led to a slight change in distances between labels at C97 but not at E90C. While the shorter distance in the tetramer increased, the larger diagonal distance decreased. These findings can be explained by a widening of the tetrameric structure upon substrate binding much like the opening of two pairs of scissors.

Effect of Ionic Liquids on the Solution Structure of Human Serum Albumin

The effect of several ionic liquids (ILs) on the solution structure of human serum albumin (HSA) is revealed by continuous wave electron paramagnetic resonance (EPR) spectroscopy and nanoscale distance measurements with double electron-electron resonance (DEER) spectroscopy. HSA, the most abundant protein in human blood, is able to bind and transport multiple fatty acids (FAs). Using spin-labeled FA, the uptake of the FA by the protein and their spatial distribution in the protein can be monitored. The FA distribution provides an indirect yet effective way to characterize the structure of the protein in solution. Addition of imidazolium-based ILs to an aqueous solution of HSA/FA conjugates is accompanied by significant destabilization and unfolding of the protein's tertiary structure. In contrast, HSA maintains its tertiary structure when choline dihydrogenphosphate (dhp) is added. The comparison of FA distance distributions in HSA with and without choline dhp surprisingly revealed that with this IL, the FA anchoring units are in better agreement with the crystallographic data. Furthermore, the FA entry point distribution appears widened and more asymmetric than in pure buffer. These results indicate that choline dhp as a cosolvent may selectively stabilize HSA conformations closer to the crystal structure out of the overall conformational ensemble.

Probing How Counterion Structure and Dynamics Determine Polyelectrolyte Solutions Using EPR Spectroscopy

In this review article, we describe how methods of electron paramagnetic resonance (EPR) spectroscopy were used to investigate polyion–counterion interactions in polyelectrolyte solutions. This subject is usually treated experimentally by light, X-ray, or neutron scattering techniques. It is shown that a large arsenal of EPR spectroscopic methods–from various sophisticated methods of line shape analysis of continuous-wave EPR, via electron spin echo envelope modulation, nanoscale distance measurements through double electron–electron resonance to high-field pulse EPR–can be used to characterize the intrinsically complicated structures formed in polyelectrolyte solutions. We show that even polymer physical models such as scaling relations can be tested in this way. The distinguishing feature with respect to the numerous scattering studies in this area is that EPR techniques are local methods, and by employing spin-carrying (i.e., EPR-active) probe ions, it is possible to examine polyelectrolytes from the counterions’ point of view.

Monitoring Simultaneous Distance and Orientation Changes in Discrete Dimers of DNA Linked Gold Nanoparticles.

Important optical properties of discrete pairs of DNA tethered gold nanoparticles, including their scattering cross-section and resonance wavelength, depend both on the dimer structure and the refractive index of their immediate environment. We show that far-field polarization microscopy aids the optical identification and interpretation of structural changes including hinge motions and nanoscale distance changes in individual assemblies. Grecco and Martinez have shown in their theoretical work that the interparticle separation dependent polarization anisotropy of discrete nanoparticle dimers enables nanoscale distance measurements (Optics Express 2006, 14, 8716 - 8721). Here we implement this approach experimentally and evaluate measured polarization anisotropies in the framework of a dipolar coupling model. We use polarization sensitive darkfield microscopy to resolve simultaneous distance and orientation changes during the compaction of discrete pairs of DNA tethered gold nanoparticles by fourth generation polyamidoamino (PAMAM) dendrimers. The relative contributions from interparticle separation and refractive index variations to changes in the light polarization and scattering intensity are quantified and compared.

Dynamic phase shifts in nanoscale distance measurements by double electron electron resonance (DEER)

The off-resonant pump pulse used in double electron electron resonance (DEER) measurements produces dynamic phase shifts that are explained here by simple analytic and vector descriptions of the full range of signal behaviors observed during DEER measurements, including: large phase shifts in the signal; changes in the position and shape of the detected echo; and changes in the signal intensity. The dynamic phase shifts depend on the width, amplitude and offset frequency of the pump pulse. Isolated radicals as well as pairs or clusters of dipolar-coupled radicals have the same dynamic phase shift that is independent of pump pulse delay in a typical measurement. A method of calibrating both the pump pulse offset frequency and the pump pulse field strength is outlined. A vector model is presented that explains the dynamic phase shifts in terms of precessing magnetization that is either spin locked or precessing about the effective pump field during the pump pulse. Implications of the dynamic phase shifts are discussed as they relate to setting up, calibrating and interpreting the results of DEER measurements.