Exploring protein conformations in vitro and in cell with EPR distance measurements

The electron–electron double resonance (DEER) method, which provides distance distributions between two spin labels, attached site specifically to biomolecules (proteins and nucleic acids), is currently a well-recognized biophysical tool in structural biology. The most commonly used spin labels are based on nitroxide stable radicals, conjugated to the proteins primarily via native or engineered cysteine residues. However, in recent years, new spin labels, along with different labeling chemistries, have been introduced, driven in part by the desire to study structural and dynamical properties of biomolecules in their native environment, the cell. This mini-review focuses on these new spin labels, which allow for DEER on orthogonal spin labels, and on the state of the art methods for in-cell DEER distance measurements.     

Referential learning of French and Czech labels in African grey parrots (Psittacus erithacus): Different methods yield contrasting results

Some African grey parrots (Psittacus erithacus), the most famous being Pepperberg's parrot Alex, are able to imitate human speech and produce labels referentially. In this study, the aim was to teach ten African grey parrots from two laboratories to label items. Training three parrots from the first laboratory for several months with the Model/Rival method, developed by Pepperberg, in which two humans interact in front of the subject to demonstrate the use of a label, led to disappointing results. Similarly, seven parrots from the second laboratory, having been trained with several variants of Model/Rival attained little success. After the informal observation of the efficiency of other methods (i.e. learning to imitate labels either spontaneously or with specific learning methods and use of these labels referentially), four different teaching methods were tested with two birds: the Model/Rival; Repetition/Association which consisted of repeating a label and presenting the item only when the parrot produced the label; Intuitive in which the experimenter handled an item and repeated its name in front of the subject; Diffusion in which labels with either variable or flat intonation were played back daily to parrots. One bird learned three labels, one of which was used referentially, with the Repetition/Association method. He learned one label non-referentially with the Model/Rival but no labels were acquired using the other methods. The second bird did not learn any labels. This study demonstrates that different methods can be efficient to teach labels referentially and it suggests that rearing conditions and interindividual variability are important features when assessing learning ability of African grey parrots.     

Core–shell silver nanoparticles for optical labeling of cells

Silver nanoparticles have been modified with self-assembled monolayers of hydroxyl-terminated long chain thiols and encapsulated with a silica shell. The resulting core–shell nanoparticles were used as optical labels for cell analysis using flow cytometry and microscopy. The excitation of plasmon resonances in nanoparticles results in strong depolarized scattering of visible light, permitting detection at the single nanoparticle level. The nanoparticles were modified with neutravidin via epoxide–azide coupling chemistry, to which biotinylated antibodies targeting cell surface receptors were bound. The nanoparticle labels exhibited long-term stability in solutions with high salt concentrations without aggregation or silver etching. Labeled cells exhibited two orders of magnitude enhancement of the scattering intensity compared with unlabeled cells.     

13C magic angle spinning NMR study of the light-induced and temperature-dependent changes in Rhodobacter sphaeroides R26 reaction centers enriched in [4'-13C]tyrosine.

Solid-state 13C magic angle spinning (MAS) NMR has been used to investigate detergent-solubilized photosynthetic reaction centers of Rhodobacter sphaeroides R26, selectively enriched in [4-13C]-tyrosine. The reaction centers were frozen, in the dark and while subject to intense illumination, and studied at temperatures between approximately 215 and approximately 260 K. The signal consists of at least seven narrow lines superimposed on a broad doublet. The chemical shift anisotropy is similar to that for crystalline tyrosine. The two narrowest resonances, corresponding to signals from individual tyrosines, are 28 +/- 5 Hz wide, comparable to what is observed for quaternary carbons in linearly elastic organic solids. The line width as well as the chemical shift of these signals is essentially independent of temperature. This provides strong evidence for an unusually ordered, well-shielded, and structurally, electrostatically, and thermodynamically stable interior of the protein complex without structural heterogeneities. As the temperature is lowered, additional signal from the labels develops and the natural abundance resonances from the detergent broaden, providing evidence for considerable flexibility at the exterior of the protein complex and in the detergent belt at the higher temperatures. In addition, the NMR provides evidence for an electrostatically uniform and neutral complex, since the total dispersion in isotropic shifts for the labels is < 5 ppm and corresponds to electron density variations of less than 0.03 electronic equivalents with respect to tyrosine in the solid state or in solution. When the sample is frozen while subject to intense illumination, a substantial part of the protein is brought into the charge-separated state P.+QA.-. At least three sharp resonances, including the narrowest lines, are substantially reduced in intensity. It is argued that this effect is caused by the electronic spin density associated with the oxidized primary donor P.+. These results strongly suggest that the environment of the special pair is extremely rigid and question the role of protein conformational distortions during the primary photoprocess.     

Carbon-13 nuclear magnetic resonance spectroscopy of high-spin iron(III) prophyrin compounds

¹³C nuclear magnetic resonance spectra have been obtained for variety of high-spin iron(III) porphyrin compounds and corresponding μ-oxo-bridged dimeric species. Large hyperfine shifts and significant line broadening are observed. The monomeric exhibit hyperfine shifts which are downfield with te exception of an upfield shift for the meso-carbon atom. Possible unpaired spin delocalization mechanisms and prospects for observing ¹³C NMR porphyrin resonances in high-spin ferrihemoproteins are discussed. Spectra reported here provide strategy for incorporation of ¹³C labels in hemoproteins either by biosynthetic or chemical means. The vinyl-CH₂ resonances of iron(III) protoporphyrin IX located 260 parts per million downfield from tetramethylsilane are especially attractive from the standpoint of chemical labeling.     

Haisu: Hierarchically supervised nonlinear dimensionality reduction

We propose a novel strategy for incorporating hierarchical supervised label information into nonlinear dimensionality reduction techniques. Specifically, we extend t-SNE, UMAP, and PHATE to include known or predicted class labels and demonstrate the efficacy of our approach on multiple single-cell RNA sequencing datasets. Our approach, “Haisu,” is applicable across domains and methods of nonlinear dimensionality reduction. In general, the mathematical effect of Haisu can be summarized as a variable perturbation of the high dimensional space in which the original data is observed. We thereby preserve the core characteristics of the visualization method and only change the manifold to respect known or assumed class labels when provided. Our strategy is designed to aid in the discovery and understanding of underlying patterns in a dataset that is heavily influenced by parent-child relationships. We show that using our approach can also help in semi-supervised settings where labels are known for only some datapoints (for instance when only a fraction of the cells are labeled). In summary, Haisu extends existing popular visualization methods to enable a user to incorporate labels known a priori into a visualization, including their hierarchical relationships as defined by a user input graph.     

Electrochemical immunoassays

Immunoassays are routinely used to detect, specifically, low levels of many antigens. The trend away from the use of radioisotopic labels has resulted in a proliferation of alternative labels, many of which have electrochemical activity. The more successful of these assays have used enzyme labels, coupled with amperometric or potentiometric methods of detection of the products. A number of assays have also been designed which are specifically electrochemical in origin, not simply adaptations of currently used spectrophotometric methods. Much effort has been expended in developing a potentiometric immunoassay that measures the change in potential that should occur when an antibody binds to its antigen. The use of electroactive labels has resulted in a number of assays for drugs. The advantages of an enzyme-linked mediated assay for lidocaine, an antiarrhythmic drug, are discussed.     

1H-NMR investigation of the oxygenation of hemoglobin in intact human red blood cells.

Using improved selective excitation methods for protein nuclear magnetic resonance (NMR), we have conducted measurements of the oxygenation of hemoglobin inside intact human red blood cells. The selective excitation methods use pulse shape-insensitive suppression of the water signal, while producing uniform phase excitation in the region of interest and, thus, are suitable for a wide variety of applications in vivo. We have measured the areas of 1H-NMR resonances of the hyperfine-shifted, exchangeable N delta H protons of the proximal histidine residues of the alpha- and beta-chains in deoxyhemoglobin (63 and 76 ppm downfield from the proton resonance of 2,2-dimethyl-2-silapentane-5-sulfonate (DSS), respectively), which are sensitive to the paramagnetic state of the iron, and for which the alpha- and beta-chain resonances are resolved, and from the ring current-shifted gamma 2-CH3 protons of the distal valine residues in oxyhemoglobin (2.4 ppm upfield from DSS), which are sensitive to the conformation of the heme pocket in the oxy state. We have found that the proximal histidine resonances are directly correlated with the degree of oxygenation of hemoglobin, whereas the distal valine resonances appear to be correlated with the conformation in the heme pocket that occurs after the binding of oxygen, in both the presence and absence of 2,3-diphosphoglycerate. In addition, from the proximal histidine resonances, we have observed a preference for the binding of oxygen to the alpha-chain (up to about 10%) of hemoglobin over the beta-chain in both the presence and absence of 2,3-diphosphoglycerate. These new results obtained in intact erythrocytes are consistent with our previous 1H-NMR studies on purified human normal adult hemoglobin. A unique feature of our 1H-NMR method is the ability to monitor the binding of oxygen specifically to the alpha- and beta-chains of hemoglobin both in solution and in intact red blood cells. This information is essential to our understanding of the molecular basis for the hemoglobin molecule serving as the oxygen carrier in vertebrates.     

New limits of sensitivity of site-directed spin labeling electron paramagnetic resonance for membrane proteins

Site-directed spin labeling electron paramagnetic resonance is a biophysical technique based on the specific introduction of spin labels to one or more sites in diamagnetic proteins, which allows monitoring dynamics and water accessibility of the spin-labeled side chains, as well as nanometer distances between two (or more) labels. Key advantages of this technique to study membrane proteins are addressed, with focus on the recent developments which will expand the range of applicability. Comparison with other biophysical methods is provided to highlight the strength of EPR as complementary tool for structural biology. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.     

Structure and dynamics of the Pf1 filamentous bacteriophage coat protein in micelles

The major coat protein of filamentous bacteriophage adopts its membrane-bound conformation in detergent micelles. High-resolution 1H and 15N NMR experiments are used to characterize the structure and dynamics of residues 30-40 in the hydrophobic midsection of Pf1 coat protein in sodium dodecyl sulfate micelles. Uniform and specific-site 15N labels enable the immobile backbone sites to be identified by their 1H/15N heteronuclear nuclear Overhauser effect and allow the assignment of 1H and 15N resonances. About one-third of the amide N-H protons in the protein undergo very slow exchange with solvent deuterons, which is indicative of sites in highly structured environments. The combination of results from 1H/15N heteronuclear correlation, 1H homonuclear correlation, and 1H homonuclear Overhauser effect experiments assigns the resonances to specific residues and demonstrates that residues 30-40 of the coat protein have a helical secondary structure.     

Simulation vs. reality: a comparison of in silico distance predictions with DEER and FRET measurements

Site specific incorporation of molecular probes such as fluorescent- and nitroxide spin-labels into biomolecules, and subsequent analysis by F?rster resonance energy transfer (FRET) and double electron-electron resonance (DEER) can elucidate the distance and distance-changes between the probes. However, the probes have an intrinsic conformational flexibility due to the linker by which they are conjugated to the biomolecule. This property minimizes the influence of the label side chain on the structure of the target molecule, but complicates the direct correlation of the experimental inter-label distances with the macromolecular structure or changes thereof. Simulation methods that account for the conformational flexibility and orientation of the probe(s) can be helpful in overcoming this problem. We performed distance measurements using FRET and DEER and explored different simulation techniques to predict inter-label distances using the Rpo4/7 stalk module of the M. jannaschii RNA polymerase. This is a suitable model system because it is rigid and a high-resolution X-ray structure is available. The conformations of the fluorescent labels and nitroxide spin labels on Rpo4/7 were modeled using in vacuo molecular dynamics simulations (MD) and a stochastic Monte Carlo sampling approach. For the nitroxide probes we also performed MD simulations with explicit water and carried out a rotamer library analysis. Our results show that the Monte Carlo simulations are in better agreement with experiments than the MD simulations and the rotamer library approach results in plausible distance predictions. Because the latter is the least computationally demanding of the methods we have explored, and is readily available to many researchers, it prevails as the method of choice for the interpretation of DEER distance distributions.     

Identification and observation of alkyl proton resonances of the amino-terminal residues of bovine neurophysins. Evidence for conformational differences between neurophysin-I and neurophysin-II.

Analysis of the 220 MHz proton magnetic resonance spectra of bovine neurophysins-I and -II and of the effects of pH and succinylation of these spectra has allowed identification of the -CH3 proton resonances of the amino-terminal alanine of both proteins and of the -CH3 resonances of methionine-2 of neurophysin-II. The alanine -CH3 resonance of neurophysin-I is a sharp doublet at all pH values between 1 and 10.5 indicating relatively few restrictions on its mobility. By contrast, the -CH3 resonances of the amino-terminal alanine and methionine-2 of neurophysin-II undergo pH-dependent changes in broadening compatible with the formation of an intramolecular salt-bridge at neutral pH between the protonated alpha-amino and an unprotonated side chain carboxyl. The results suggest that differeces in the properties of the two proteins are partially mediated by conformational differences involving their amino-terminal sequences. The potential usefulness of the amino-terminal resonances as n.m.r. 'reporter' signals is additionally demonstrated by studies of the effects of spin labels on the neurophysin-I amino-terminal alanine resonance; these studies place the amino-terminus of neurophysin-I approximately 14 A from residue 3 of peptides bound to the strong neurophysin hormone-binding site.     

15N labeling of oligodeoxynucleotides for NMR studies of DNA-ligand interactions.

The amino protons of 15N-labeled DNA were studied as a possible structural probe in NMR investigations of the interaction of DNA with various ligands. Since the imino protons are located in the center of the double helix, and variations of their chemical shift values are difficult to interpret in terms of structural changes, these probes are not very useful. Instead, amino protons are located in the major or minor groove of the DNA and are often directly involved in the binding of a ligand. For a selective probing 4-15NH2-2'-deoxycytidine and 6-15NH2-2'-deoxyadenosine were obtained by chemical synthesis. The labeled nucleosides were introduced in distinct positions of oligodeoxynucleotides by large-scale DNA synthesis. Direct 15N NMR and 1H-15N multiple quantum NMR were applied to detect the corresponding 15N labels or protons attached to the 15N labels. Chemical shift values for the cytidine and the adenosine amino nitrogen and proton resonances of a symmetric 18 base pair lac operator sequence are reported.     

Identification of individual protein–ligand NOEs in the limit of intermediate exchange

Interactions of proteins with small molecules or other macromolecules play key roles in many biological processes and in drug action, and NMR is an excellent tool for their structural characterization. Frequently, however, line broadening due to intermediate exchange completely eliminates the signals needed for measuring specific intermolecular NOEs. This limits the use of NMR for detailed structural studies in such kinetic situations. Here we show that an optimally chosen excess of ligand over protein can reduce the extent of line broadening for both the ligand and the protein. This makes observation of ligand resonances possible but reduces the size of the measurable NOEs due to the residual line broadening and the non-stoichiometric concentrations. Because the solubility of small molecule drug leads are often limited to high micromolar concentrations, protein concentrations are restricted to even lower values in the low micromolar range. At these non-stoichiometric concentrations and in the presence of significant residual line broadening, conventional NOESY experiments very often are not sensitive enough to observe intermolecular NOEs since the signals inverted by the NOESY preparation pulse sequence relax prior to significant NOE build up. Thus, we employ methods related to driven NOE spectroscopy to investigate protein–ligand interactions in the intermediate exchange regime. In this approach, individual protein resonances are selectively irradiated for up to five seconds to build up measurable NOEs at the ligand resonances. To enable saturation of individual protein resonances we prepare deuterated protein samples selectively protonated at a few sites so that the 1D ¹H spectrum of the protein is resolved well enough to permit irradiation of individual protein signals, which do not overlap with the ligand spectrum. This approach is suitable for measuring a sufficiently large number of protein–ligand NOEs that allow calculation of initial complex structures, suitable for structure-based optimization of primary drug leads obtained from high-throughput screening. The method was applied to measure individual intermolecular NOEs between the anti-apoptotic protein Bcl-xL at 25 μM and a “first generation” small-molecule ligand, for which the spectrum is entirely broadened at stoichiometric concentrations. This approach is general and can also be used to characterize protein–protein or protein–nucleic-acid complexes.     

Optimization of immunolabeled plasmonic nanoparticles for cell surface receptor analysis

Noble metal nanoparticles hold great potential as optical contrast agents due to a unique feature, known as the plasmon resonance, which produces enhanced scattering and absorption at specific frequencies. The plasmon resonance also provides a spectral tunability that is not often found in organic fluorophores or other labeling methods. The ability to functionalize these nanoparticles with antibodies has led to their development as contrast agents for molecular optical imaging. In this review article, we present methods for optimizing the spectral agility of these labels. We discuss synthesis of gold nanorods, a plasmonic nanoparticle in which the plasmonic resonance can be tuned during synthesis to provide imaging within the spectral window commonly utilized in biomedical applications. We describe recent advances in our group to functionalize gold and silver nanoparticles using distinct antibodies, including EGFR, HER-2 and IGF-1, selected for their relevance to tumor imaging. Finally, we present characterization of these nanoparticle labels to verify their spectral properties and molecular specificity.     

Nuclear magnetic resonance observation and dynamics of specific amide protons in T4 lysozyme

We have produced T4 lysozyme using a bacterial expression system which allows efficient incorporation of isotopically labeled amino acids in lysozyme. By using conditions that repress the expression of various transaminases, we have incorporated 15N-labeled amino acid into the five phenylalanine residues of the protein. The relatively large spin--spin coupling (87 +/- 3 Hz) between the 15N nucleus and the phenylalanine amide protons may then be exploited in a variety of ways to selectively observe the five phenylalanine amide proton resonances. These include a simple "echo difference" technique which displays the amide proton resonances in one dimension and a "forbidden echo" technique [Bax, A., Griffey, R. H., & Hawkins, B.L. (1983) J. Magn. Reson. 55, 301-335] which gives two-dimensional information allowing the proton and 15N chemical shifts of each amide to be determined. With these approaches, all five phenylalanine amide protons give resolved resonances. Deuterium exchange experiments demonstrate that three of the five resonances are slow to exchange (half-times of about 1 week at pH 5.5 and 4 degrees C) while the other two are rapid with complete exchange in hours or less. These observations correlate well with the secondary structure of the protein which shows three residues in alpha-helical regions and two residues in surface-exposed environments. This approach of isotopic substitution on nitrogen or carbon atoms is of general utility and should allow virtually any proton on a protein of molecular weight 20 000 or thereabout to be selectively observed.     

Resonance assignments in the proton-NMR spectrum of carbonmonoxy hemoglobin by two-dimensional methods

Proton-nuclear-magnetic-resonance spectroscopy is a powerful tool for investigating the solution structure of biopolymers provided that a substantial number of proton resonances are assigned in the spectrum. For large proteins the assignments have usually been made by the comparative one-dimensional NMR investigations of the parent and derivative proteins in different physicochemical conditions. In this paper, we show that the more powerful two-dimensional methods could be successfully applide to proteins of the size of human adult hemoglobin (Mr = 64,500). J-Correlated and NOE-correlated spectroscopy, together with topological relationships in the known crystalline structure, enabled us to assign a large number of resonances. The majority of the assigned resonances correspond to the heme substituents and to amino acids in the heme pockets of the two subunits. These results thus provide an extensive set of intrinsic probes for mapping the conformation of the ligand-binding site and its functional changes. Comparison of the observed ring-current shifts of the assigned resonances with those calculated from the known crystallographic coordinates suggests a close similarity between the heme-pocket tertiary conformation in solution and in the crystalline state. A significant difference was noted for Leu141 in beta subunits which, in solution, appears to have stronger contacts with the heme groups than in the crystalline form. The present results also demonstrate that two-dimensional-NMR methods could be successfully applied to the investigation of the structure of large biomolecules in solution (Mr less than or equal to 65,000).     

Study of the tryptophan residues of lysozyme using 1H nuclear magnetic resonance.

The identification and complete assignment of the C-2 and N-1 proton nuclear magnetic resonances (NMR) of the six tryptophan residues of hen lysozyme are reported. Identification of the resonances required a detailed examination of the spectra of the protein in H2O and in 2H2O, and involved the application of spin-echo and Carr-Purcell-Meiboom-Gill pulse sequences. Assignment was achieved by observing the effects on the NMR spectra of performing specific chemical modifications, of binding paramagnetic species (lanthanide ions and spin labels), of binding inhibitors and protons and of carrying out solvent exchange experiments. The problems involved in completion of assignment are fully discussed. In the course of performing experiments to make assignments, several interesting aspects of the behaviour of the tryptophan residues in the protein structure were observed and are discussed.     

1H-Nuclear magnetic-resonance studies on glycophorin and its carbohydrate-containing tryptic peptides.

The proton nuclear magnetic resonance (1H-NMR) spectra of glycophorin and its tryptic sialoglycopeptides were investigated. From the intensities of the assigned resonances it was concluded that all of the residues in the sialoglycopeptides are sufficiently mobile in conformation to give sharp resonances, while in glycophorin this is true for only approximately 80% of the peptide backbone. The resonances of the central sequence of some 20 of the hydrophobic residues are strongly broadened. This region is probably that of alpha-helical structure which is known to aggregate. The linewidths and intensities of the resonances are not, or only slightly, affected by changing the ionic strength, temperature or by carboxymethylation of the Met-81 residue in glycophorin. Glycophorin was found to bind about 100 mol sodium dodecylsulphate/mol protein as derived from studies on linebroadening of the latter's C-3 to C-11 methylene resonances. The bound dodecyl-sulphate probably increases the mobilities of the hydrophobic residues in the protein as these resonance intensities are increased by the binding. The carbohydrate chains in glycophorin were conformationally mobile; no evidence was found for tight carbohydrate-protein interactions. The relevance of flexible carbohydrate chains in membrane glycoproteins is discussed in relation to cell surface chemistry.     

Detection of microarray-hybridized oligonucleotides with magnetic beads

The efficiency of hybridization analysis with oligonucleotide microarrays depends heavily on the method of detection. Conventional methods based on labeling nucleic acids with fluorescent, chemiluminescent, enzyme, or radioactive reporters suffer from a number of serious drawbacks which demand development of new detection techniques. Here, we report two new approaches for detection of hybridization with oligonucleotide microarrays employing magnetic beads as active labels. In the first method streptavidin-coated magnetic beads are used to discover biotin-labeled DNA molecules hybridized with arrayed oligonucleotide probes. In the second method biotin-labeled DNA molecules are bound first to the surface of magnetic beads and then hybridized with arrayed complementary strands on bead-array contacts. Using a simple low-power microscope with a dark-field illumination and a pair of complementary primers as a model hybridization system we evaluated sensitivity, speed, and cost of the new detection method and compared its performance with the detection techniques employing enzyme and fluorescent labels. It was shown that the detection of microarray-hybridized DNA with magnetic beads combines low cost with high speed and enhanced assay sensitivity, opening a new way to routine hybridization assays which do not require precise measurements of DNA concentration.