A molecular ruler for measuring quantitative distance distributions

We report a novel molecular ruler for measurement of distances and distance distributions with accurate external calibration. Using solution X-ray scattering we determine the scattering interference between two gold nanocrystal probes attached site-specifically to a macromolecule of interest. Fourier transformation of the interference pattern provides a model-independent probability distribution for the distances between the probe centers-of-mass. To test the approach, we measure end-to-end distances for a variety of DNA structures. We demonstrate that measurements with independently prepared samples and using different X-ray sources are highly reproducible, we demonstrate the quantitative accuracy of the first and second moments of the distance distributions, and we demonstrate that the technique recovers complex distribution shapes. Distances measured with the solution scattering-interference ruler match the corresponding crystallographic values, but differ from distances measured previously with alternate ruler techniques. The X-ray scattering interference ruler should be a powerful tool for relating crystal structures to solution structures and for studying molecular fluctuations.     

Sepharose 4B as a matrix for affinity chromatography. A spin-labelling investigation using nitroxides as model ligands

Nitroxide spin labels were attached to CNBr-activated Sepharose 4B directly and through oligoglycines and oo-amino-carboxylic acids of varying length. The homogeneity of the carbohydrate environments of directly attached labels was investigated by measuring dipolar interactions between nitroxides as a function of solvation and of spin dilution with a diamagnetic analogue, as well as by electron exchange between the nitroxides and paramagnetic metal ions in solution. Only the exchange experiment revealed any inhomogeneity, suggesting that a small proportion of sites may be less accessible than the majority. The distances between sites were sufficiently small to allow, in principle, multiple-site interactions between quite small proteins in solution and immobilized ligands. Reorientation of the label at the matrix, characterized by the correlation time t, became more rapid with increasing spacer length n. For n > 12, the decrease in t was less pronounced. The two types of spacer behaved similarly. Thus an ideal spacer length for affinity separations is 12 atoms; this is in good agreement with data from a variety of affinity separations. The results of electron spin resonance studies of the effect of non-aqueous solvent on directly and indirectly labelled Sepharose 4B were used to suggest reasons why enzymes immobilized on Sepharose may be stabilized to denaturing solvents.     

Interhead distances in myosin attached to F-actin estimated by fluorescence energy transfer spectroscopy.

Fluorescence resonance energy transfer (FRET) spectroscopy has been used to determine distances between probes attached to the most reactive sulfhydryl (SH1) group on individual myosin "heads." We measured intramolecular and intermolecular interhead distances as well as the distance between one head of heavy meromyosin (HMM) mixed with subfragment-1 (S1) heads attached to F-actin under rigor conditions. The SH1 cysteine was specifically labeled with either a donor (5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid) or an acceptor probe (5-iodoacetamidofluorescein). In free solution, the distance between these probes was too large to allow significant FRET, but in the rigor complex with F-actin, intermolecular interhead distances between S1 molecules, HMM molecules, or S1 and HMM were determined to be 6.0-6.3 nm. The radial coordinate of the labels relative to F-actin was 5.0-6.4 nm. However, the intramolecular interhead distance in HMMs in which the two heads were labeled with D and A probes was estimated to be larger. The binding affinity of the second head of HMM(D/A) to F-actin may be reduced because of heterogeneous modification of the SH1 groups, such that the probability of single-head binding is increased.     

The structures of solvated methylmercury(II) chlolade,bromide and iodide in pyridine solution; Implications for aqueous solution

The structures of solvated methylmercury(II) halides in pyridine solution were determined by a large angle X-ray scattering technique. Near-linear CH₃HgX (X = Cl, Br and I) species solvated by two weakly-coordinated pyridine molecules are indirectly interpreted. Additional mercury-pyridine interactions, through van der Waals forces, are found at the sum of the van der Waals radii. The HgX bond distances in the methylmercury(II) halides are found to be 2.325(8), 2.480(3) and 2.649(3) Å for chloride, bromide and iodide, respectively. The HgC bond distances are assumed to be ∼2.08 Å. This interaction is indicated in the radial distribution functions. The bond distance between mercury and the two solvating pyridine molecules is ∼2.8 Å, e.g., 2.84(2) Å in methylmercury(II) bromide. The additional mercury interactions with roughly two pyridine molecules at the sum of van der Waals radii are revealed at around 3.15 Å. Comparison between Raman stretching vibrations and the solvated structures of methylmercury(II) complexes found in various solvents indicates a lower limit in solvent donor property for the formation of solvate bonds to mercury for the methylmercury(II) halides.     

Multi-wavelength anomalous diffraction using medium-angle X-ray solution scattering (MADMAX)

Proteins are dynamic molecules whose function in virtually all biological processes requires conformational motion. Direct experimental probes of protein structure in solution are needed to characterize these motions. Anomalous scattering from proteins in solution has the potential to act as a precise molecular ruler to determine the positions of specific chemical groups or atoms within proteins under conditions in which structural changes can take place free from the constraints of crystal contacts. In solution, anomalous diffraction has two components: a set of cross-terms that depend on the relative location of the anomalous centers and the rest of the protein, and a set of pure anomalous terms that depend on the distances between the anomalous centers. The cross-terms are demonstrated here to be observable and to provide direct information about the distance between the anomalous center and the center of mass of the protein. The second set of terms appears immeasurably small in the context of current experimental capabilities. Here, we outline the theory underlying anomalous scattering from proteins in solution, predict the anomalous differences expected on the basis of atomic coordinate sets, and demonstrate the measurement of anomalous differences at the iron edge for solutions of myoglobin and hemoglobin.     

Quantitative surface-enhanced resonance Raman scattering of phthalocyanine-labelled oligonucleotides

The evaluation of phthalocyanine labels for the surface-enhanced resonance Raman scattering (SERRS) detection of oligonucleotides is reported. Three phthalocyanine-labelled oligonucleotides were assessed, each containing a different metal centre. Detection limits for each labelled oligonucleotide were determined using two excitation frequencies where possible. Limits of detection as low as 2.8 × 10⁻¹¹mol.dm⁻³ were obtained which are comparable to standard fluorescently labelled probes used in previous SERRS studies. The identification of two phthalocyanine-labelled oligonucleotides without separation was also demonstrated indicating their suitability for multiplexing. This study extends the range of labels suitable for quantitative surface-enhanced resonance Raman scattering with silver nanoparticles and offers more flexibility and choice when considering SERRS for quantitative DNA detection.     

Small angle neutron scattering from lysozyme solutions in unsaturated and supersaturated states (SANS from lysozyme solutions)

Small angle neutron scattering (SANS) method was used to study lysozyme solutions, with particular interest in an understanding of the crystallization process at the initial stage. It is found that (1) in the unsaturated solution, the protein molecules aggregate with a continuous increase in size when NaCl concentration is increased, and (2) in the supersaturated solution, an irreversible change, superimposed on the former process, occurs when the supersaturation is realized. These facts indicate the usefulness of SANS in detecting changes of protein molecules in solution on the nanometer scale. The reliability of the SANS results are indicated by (1) comparing them with those of small angle X-ray scattering (SAXS), and (2) comparing the effect of D(2)O and H(2)O as solvent. Since the interparticle interaction is essential in the crystallization process and a simple Guinier plot analysis is not allowed, a more rigorous framework of analyzing data with interference function is developed, through which both average interparticle distance and particle size are estimated.     

Structural properties of human glycodelin A in water and in water-alcohol mixtures: a comparison with bovine beta-lactoglobulin A.

Human glycodelin A (GdA) is a glycoprotein that is highly homologous to bovine beta-lactoglobulin A (beta-LgA) because the amino acid sequences are 50-60% identical. The structural characteristics of human GdA and beta-LgA were compared in water and 2-propanol/water solutions. Circular dichroism spectra reveal that in water the two proteins have a very similar beta-sheet secondary structure. In the presence of 2-propanol/water mixtures (up to 50% v/v) the alpha-helix structure of both proteins increases. A further increase in the alcohol percentage of the solvent (up to 80% v/v 2-propanol) causes the formation of a new folded tertiary structure containing mainly beta-sheet features. Synchrotron radiation small angle X-ray scattering indicates that, in a neutral pH aqueous solution, GdA is a dimer. Its radius of gyration value (Rg), 25.1+/-0.4 A, is greater than that of beta-LgA (21.1+/-0.3 A), probably because of the contribution of polysaccharides bound to Asn-28 and Asn-63 residues of GdA. Conversely, small angle X-ray scattering and gel permeation chromatography data on GdA in 2-propanol have revealed a massive aggregation of the protein.     

Supramolecular structure of self-assembling alamethicin analog studied by ESR and PELDOR

Three analogs of alamethicin F50/5, labelled with the TOAC (='2,2,6,6-tetramethylpiperidin-1-oxyl-4-amino-4-carboxylic acid') spin label at positions 1 (Alm1), 8 (Alm8), and 16 (Alm16), resp., were studied by Electron-Spin-Resonance (ESR) and Pulsed Electron-Electron Double-Resonance (PELDOR) techniques in solvents of different polarity to investigate the self-assembly of amphipathic helical peptides in membrane-mimicking environments. In polar solvents, alamethicin forms homogeneous solutions. In the weakly polar chloroform/toluene 1 : 1 mixture, however, this peptide forms aggregates that are detectable at 293 K by ESR in liquid solution, as well as by PELDOR in frozen, glassy solution at 77 K. In liquid solution, free alamethicin molecules and their aggregates show rotational-mobility correlation times tau(r) of 0.87 and 5.9 ns, resp. Based on these values and analysis of dipole-dipole interactions of the TOAC labels in the aggregates, as determined by PELDOR, the average number N of alamethicin molecules in the aggregates is estimated to be less than nine. A distance-distribution function between spin labels in the supramolecular aggregate was obtained. This function exhibits two maxima: a broad one at a distance of 3.0 nm, and a wide one at a distance of ca. 7 nm. A molecular-dynamics (MD)-based model of the aggregate, consisting of two parallel tetramers, each composed of four molecules arranged in a 'head-to-tail' fashion, is proposed, accounting for the observed distances and their distribution.     

Dynamics may significantly influence the estimation of interatomic distances in biomolecular X-ray structures

Atomic positions obtained by X-ray crystallography are time and space averages over many molecules in the crystal. Importantly, interatomic distances, calculated between such average positions and frequently used in structural and mechanistic analyses, can be substantially different from the more appropriate time-average and ensemble-average interatomic distances. Using crystallographic B-factors, one can deduce corrections, which have so far been applied exclusively to small molecules, to obtain correct average distances as a function of the type of atomic motion. Here, using 4774 high-quality protein X-ray structures, we study the significance of such corrections for different types of atomic motion. Importantly, we show that for distances shorter than 5 Å, corrections greater than 0.5 Å may apply, especially for noncorrelated or anticorrelated motion. For example, 14% of the studied structures have at least one pair of atoms with a correction of ≥ 0.5 Å in the case of noncorrelated motion. Using molecular dynamics simulations of villin headpiece, ubiquitin, and SH3 domain unit cells, we demonstrate that the majority of average interatomic distances in these proteins agree with noncorrelated corrections, suggesting that such deviations may be truly relevant. Importantly, we demonstrate that the corrections do not significantly affect stereochemistry and the overall quality of final refined X-ray structures, but can provide marked improvements in starting unrefined models obtained from low-resolution X-ray data. Finally, we illustrate the potential mechanistic and biological significance of the calculated corrections for KcsA ion channel and show that they provide indirect evidence that motions in its selectivity filter are highly correlated.     

Features of the Secondary Emission Enhancement Near Plasmonic Gold Film

Plasmonic gold films (PGF) prepared by vacuum deposition of gold onto quartz slides possess unique property to enhance electromagnetic signal in the near field. Spectral tuning of PGF’s plasmon band to resonance with the electronic spectra of adsorbed molecules provides selective enhancement of fluorescence or surface-enhanced Raman scattering in the far field. Plasmon-enhanced fluorescence (PEF) of mitoxantrone (mitox) as a function of the distance between gold surface and adsorbed molecules for different polarization and incidence angle of exciting light is analyzed in this work. Spectrophotometric data reveal that probability of localized plasmon excitation in gold grains increases with growth of incidence angle for s-polarized and decrease for p-polarized excitation. This fact correlates well with oblate shape of gold particles detected by Atomic force microscope. However, the fluorescence intensity of dyes deposited at fixed distance from gold surface increase with angle of incidence of p-polarized light more noticeably than for s-polarized one. Nevertheless, the behavior of mitox PEF signal upon p-polarized laser excitation and different angle of incidence are similar in appearance to such phenomenon as selective photoelectric effect. According to this observation, the near-field interactions between plasmons and molecule as possible mechanism of PEF is discussed.     

Small angle X-ray scattering study on bovine porphobilinogen synthase (5-aminolaevulinate dehydratase)

The quaternary structure of the native (zinc) porphobilinogen synthase (5-amino-laevulinate dehydratase) from bovine liver and its lead-substituted derivative is studied in solution by small angle X-ray scattering. In spite of the profound inhibitory effect of lead ions in the enzyme they do not produce a change in the quaternary structure detectable by small angle X-ray scattering. The most important molecular parameters of the native enzyme were found to be: radius of gyration Rg = 4.04 +/- 0.04 nm and maximum dimension Dmax = 12.0 +/- 0.5 nm. The corresponding values for the lead derivative are: Rg = 4.26 +/- 0.1 nm and Dmax = 12.5 +/- 0.5 nm. The quaternary structure of the enzyme in solution is described by a model, which fits the experimental scattering and distance distribution function.     

Distance and dynamics determination by W-band DEER and W-band ST-EPR

To explore high-field EPR in biological applications we have compared measurements of dynamics with X-band (9 GHz) and W-band (94 GHz) saturation transfer EPR (ST-EPR) and distance determination by X and W-band DEER. A fourfold increase of sensitivity was observed for W-band ST-EPR compared with X-band. The distance measurements at both fields showed very good agreement in both the average distances and in the distance distributions. Multifrequency EPR thus provides an additional experimental dimension to facilitate extraction of distance populations. However, the expected orientational selectivity of W-band DEER to determine the relative orientation of spins has not been realized, most likely because of the large orientational disorder of spin labels on the protein surface.     

Influence of the 33 kDa manganese-stabilizing protein on the structure and substrate accessibility of the oxygen-evolving complex of photosystem II

The 33 kDa manganese-stabilizing extrinsic protein binds to the lumenal side of photosystem II (PS II) close to the Mn(4)Ca cluster of the oxygen-evolving complex, where it limits access of small molecules to the metal site. Our previous finding that the removal of this protein did not alter the magnetic coupling regime within the manganese cluster, measured by electron spin-echo envelope modulation [Gregor, W., and Britt, R. D. (2000) Photosynth. Res. 65, 175-185], prompted us to examine whether this accessibility control is also true for substrate water, using the same pulsed EPR technique. Comparing the deuteron modulation of the S(2)-state multiline signal of PS II membranes, equilibrated with deuterated water (D(2)O) after removal or retention of the 33 kDa protein, we observed no change in the number and the distance of deuterons magnetically coupled to manganese, indicating that the number and distance of water molecules bound to the manganese cluster are independent of bound 33 kDa protein in the S(1) state, in which the sample was poised prior to cryogenic illumination. A simple modulation depth analysis revealed a distance of 2.5-2.6 A between the closest deuteron and manganese. These results are in agreement with our refined X-ray absorption analysis. The manganese K-edge positions, reflecting their oxidation states, and the extended X-ray absorption fine structure amplitudes and distances between the manganese ions and their oxygen and nitrogen ligands (1.8, 2.7, and 3.3-3.4 A) were independent of bound 33 kDa protein.     

Small-angle X-ray scattering study of the (Fab′)2 fragment of the human immunoglobulin Kol

The conformation of the (Fab′)₂ fragment of the human immunoglubulin Kol has been investigated in solution by small angle X-ray scattering. The following molecular parameters were determined: radius of gyration 4.10 ± 0.05 nm; maximum distance 14.0 ± 0.5 nm and hydrated volume 150 ± 8 nm³. A model of the fragment is presented, which fits these experimental data and shows good agreement with the distance distribution function in real space and the scattering curve in reciprocal space. We have to assume that the (Fab′)₂ fragment has many different conformations in solution. The method of small-angle X-ray scattering only allows the determination of an average conformation which is very similar within the resolution of the method to the static structure determined in the crystal.     

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.     

Lysozyme viscoelastic matrices in tetramethylurea/water media: a small angle X-ray scattering study

Semi-solid viscoelastic matrices produced out of lysozyme in organic/aqueous media [tetramethylurea (TMU)/water] were characterized by small angle X-ray scattering (SAXS). The scattering curves were modeled in their form and interference factors. Radii of gyration of scattering particles were found to undergo a dramatic increase from 14 A in water to approximately 44 A in the matrices. Average correlation distances d=155 A were consistently verified for the scattering particles in the matrices, irrespective of solvent composition (in the 0.6相似文献   

Reactions and structures in the gallium(III)/indium(III)-N,N-dimethylthioformamide systems

The structure of the N,N-dimethylthioformamide (DMTF) solvated gallium(III) ion has been determined in solution by means of extended X-ray absorption fine structure (EXAFS) spectroscopy. The gallium(III) ion is four-coordinate in tetrahedral fashion with a mean Ga-S bond distance of 2.233(2) Å in DMTF solution. At the dissolution of indium(III) perchlorate or trifluoromethanesulfonate in DMTF coordinated solvent molecules are partly reduced to sulfide ions, and a tetrameric complex with the composition [In₄S₄(SHN(CH₃)₂)₁₂]⁴⁺ is formed. The structure of the solid tetrameric complex in the perchlorate salt was solved with single crystal X-ray diffraction. Four indium(III) ions and four sulfide ions form a highly symmetric heterocubane structure where each indium binds three bridging sulfide ions and each sulfide ion binds three indium(III) ions with a mean In-S bond distance of 2.584(1) Å, and S-In-S angles of 90.3(1)°. Each indium(III) additionally binds three DMTF molecules at significantly longer mean In-S bond distance, 2.703(1) Å; the S-In-S angles are in the range 80.3-90.4°. Large angle X-ray scattering data on a DMTF solution of indium(III) trifluoromethanesulfonate show that the same tetrameric species characterized in the solid state is also present in solution, whereas the EXAFS measurements only give information about the In-S bond distances due to the short core hole lifetime.     

Native atomic burials, supplemented by physically motivated hydrogen bond constraints, contain sufficient information to determine the tertiary structure of small globular proteins

We investigate the possibility that atomic burials, as measured by their distances from the structural geometrical center, contain sufficient information to determine the tertiary structure of globular proteins. We report Monte Carlo simulated annealing results of all-atom hard-sphere models in continuous space for four small proteins: the all-beta WW-domain 1E0L, the alpha/beta protein-G 1IGD, the all-alpha engrailed homeo-domain 1ENH, and the alpha + beta engineered monomeric form of the Cro protein 1ORC. We used as energy function the sum over all atoms, labeled by i, of |R(i) - R(i) (*)|, where R(i) is the atomic distance from the center of coordinates, or central distance, and R(i) (*) is the "ideal" central distance obtained from the native structure. Hydrogen bonds were taken into consideration by the assignment of two ideal distances for backbone atoms forming hydrogen bonds in the native structure depending on the formation of a geometrically defined bond, independently of bond partner. Lowest energy final conformations turned out to be very similar to the native structure for the four proteins under investigation and a strong correlation was observed between energy and distance root mean square deviation (DRMS) from the native in the case of all-beta 1E0L and alpha/beta 1IGD. For all alpha 1ENH and alpha + beta 1ORC the overall correlation between energy and DRMS among final conformations was not as high because some trajectories resulted in high DRMS but low energy final conformations in which alpha-helices adopted a non-native mutual orientation. Comparison between central distances and actual accessible surface areas corroborated the implicit assumption of correlation between these two quantities. The Z-score obtained with this native-centric potential in the discrimination of native 1ORC from a set of random compact structures confirmed that it contains a much smaller amount of native information when compared to a traditional contact Go potential but indicated that simple sequence-dependent burial potentials still need some improvement in order to attain a similar discriminability. Taken together, our results suggest that central distances, in conjunction to physically motivated hydrogen bond constraints, contain sufficient information to determine the native conformation of these small proteins and that a solution to the folding problem for globular proteins could arise from sufficiently accurate burial predictions from sequence followed by minimization of a burial-dependent energy function.     

X-Ray Characterization of Melanins—I

The intrinsic local structure characterization of natural sepia melanin and L-dopa and tyrosine synthetic melanin powder has been carried out by X-ray diffraction using synchrotron radiation. The derived structure factor, S(q), shows six significant diffuse peaks within the q-range from 0.3 Å^-1 to 16 Å^-1 in the reciprocal space (q= (4π sin θ)/λ, 2θ is the scattering angle). The Fourier transform of S(q), which yields the radial distribution function (RDF), gives us information in real space of a 1.42 Å distance averaged over the C-C, C-O and C-N bond lengths as well as peaks at 2.40-2.41 Å, 3.67-3.71 Å and 4.67-4.70 Å discrete neighbor distances. There is a great similarity in the scattering intensity profiles of the natural and synthetic melanins indicating that the synthetically prepared material may be essentially similar to “real” melanin in its local atomic arrangements. An evidence of a prepeak at q = 0.45 Å^-1 has been confirmed which indicates a preferred length scale of ～ 13-20 Å that corresponds to the initial particle size in colloidal melanin solutions.