Spectral Similarity: Full-Wavelength Range Calibration of Circular Dichroism Spectroscopy.

Circular dichroism (CD) measurements help characterize optically active molecules and higher-order biomolecular structures. The harmonization of interlaboratory CD measurements requires minimizing measurement uncertainties and determinate errors. Most CD measurements utilize a single-wavelength intensity measurement at a spectral peak to calibrate the intensities of the ultraviolet wavelength range. However, such a single-wavelength calibration is inherently less precise than using the CD instrument's spectrum over the entire measured spectral range. A more thorough and informative calibration can be achieved by remapping the spectrum into what we call a spectral similarity plot. This process allows a straightforward, quantitative evaluation of the shape congruence between two spectra over the full spectral range. While preliminary analyses suggest spectral similarity plots can be utilized with a variety of different spectrometry methods, here we illustrate the process applied to circular dichroism. Spectral similarity plots are highly sensitive to deviations due to differences in concentration, pathlength, source and detector properties, circular polarization balance, as well as wavelength nonlinearities and shifts. Deviations in these properties can be quantitated by a linear least squares fit of the remapped data. The remapping enables protocols to correct spectra toward congruence between two spectra. The spectra similarity comparison provides an objective, unambiguous test of the CD instrument quality when, for example, compared to a carefully calibrated system as shown in the examples described in the text.

Solution Structures of Bacillus anthracis Protective Antigen Proteins Using Small Angle Neutron Scattering and Protective Antigen 63 Ion Channel Formation Kinetics.

We are studying the structures of bacterial toxins that form ion channels and enable macromolecule transport across membranes. For example, the crystal structure of the Staphylococcus aureus α-hemolysin (α-HL) channel in its functional state was confirmed using neutron reflectometry (NR) with the protein reconstituted in membranes tethered to a solid support. This method, which provides sub-nanometer structural information, could also test putative structures of the Bacillus anthracis protective antigen 63 (PA63) channel, locate where B. anthracis lethal factor and edema factor toxins (LF and EF, respectively) bind to it, and determine how certain small molecules can inhibit the interaction of LF and EF with the channel. We report here the solution structures of channel-forming PA63 and its precursor PA83 (which does not form channels) obtained with small angle neutron scattering. At near neutral pH, PA83 is a monomer and PA63 a heptamer. The latter is compared to two cryo-electron microscopy structures. We also show that although the α-HL and PA63 channels have similar structural features, unlike α-HL, PA63 channel formation in lipid bilayer membranes ceases within minutes of protein addition, which currently precludes the use of NR for elucidating the interactions between PA63, LF, EF, and potential therapeutic agents.

Ion and water transport reasonably involves rotation and pseudorotation: measurement and modeling the temperature dependence of small-angle neutron scattering from aqueous SrI2.

X-ray and neutron scattering have provided insight into the short range (<8 Å) structures of ionic solutions for over a century. For longer distances, single scattering bands have, however, been seen. For the non-hydrolyzing salt SrI2 in aqueous (D2O) solution, a structure sufficient to scatter slow neutrons has been seen to persist down to a concentration of 0.1 mol L-1 where the measured average spacing between scatterers is over 20 Å. Theoretical studies of such long distance solution structures are difficult, and these difficulties are discussed. The width of the distribution in distances between the scatterers (ions, ion pairs, etc.) remains less than 10 Å, which approximates the average size of the ions and their first hydration shell. Here, we measure the temperature dependence from 10 °C to 90 °C of the small angle neutron scattering (SANS) by a 0.5 molar SrI2 solution in D2O and find that this surprisingly narrow distribution of the distances remains constant within experimental uncertainty. This structure of the ions in the solution appears to endure because changes in interion distances along any single spatial dimension require displacements near the size of a water molecule. Together, the experimental measurements support a rotatory mechanism for simultaneous ion transport and water countertransport. Since rotation minimizes displacement of the solution framework, it is suggested that water transport alone also involves rotation of multimolecular structures, and that the interpretation of single-molecule water rotation is confounded by pseudorotation that results from paired picosecond proton exchanges. It is pointed out that NMR-determined millisecond to microsecond proton exchange times of chelated-metal-ion bound waters and the much faster chelate rotational correlation times around 10 picoseconds, both of which require making and breaking of hydrogen bonds, are difficult to impossible to reconcile.

Why Proteins are Big: Length Scale Effects on Equilibria and Kinetics.

Proteins are polymers, and yet the language used in describing their thermodynamics and kinetics is most often that of small molecules. Using the terminology and mathematical descriptions of small molecules impedes understanding why proteins have evolved to be big in comparison. Many properties of the proteins should be interpreted as polymer behavior, and these arise because of the longer length scale of polymer dimensions. For example, entropic rubber elasticity arises only because of polymer properties, and understanding the separation of entropic and enthalpic contributions shows that the entropic contributions mostly reside within the polymer and enthalpy originates mostly at the site of small-molecule binding. Recognizing the physical chemistry of polymers in descriptions of proteins' structure and function can add clarity to what might otherwise appear to be confusing or even paradoxical behavior. Two of these paradoxes include, first, highly selective binding that is, nevertheless, weak, and, second, small perturbations of an enzyme that cause large changes in reaction rates. Further, for larger structures such as proteins every thermodynamic measurement depends on the length scale of the structure. One reason is that the larger molecule can control up to thousands of waters resulting in collective movements with kcal sums of single-calorie-per-molecule solvent energy changes. In addition, the nature of covalent polypeptides commonly leads to multiple binding-i.e., multivalency-and the benefits of multivalent binding can be assessed semiquantitatively drawing from understanding the chelate effect in coordination chemistry. Such approaches clarify the origins, inter alia, of many low energies of protein denaturation, which lie in the range of only a few kcal mol-1, and the difficulties in finding the structures of proteins in the multiple substates postulated within complex kinetic schemes. These models involving longer length scales can be used to elucidate why such observed behavior occurs, and can provide insight and clarity where the phenomena modeled employing experimentally inseparable translational, vibrational, and rotational entropy along with charge, dipole moment, hydration, hydrogen bonding, and van der Waals energies together obscure such origins. The short-distance, long distance separation does not include explaining any enzymatic lowering of activation energies due to stabilization of the intermediate(s) along the reaction path. However, well known small-molecule methods that treat electrostatics and bonding can be used to explain the local chemistry that contributes most of the changes in enthalpy and activation enthalpy for the process.

Submultiple Data Collection to Explore Spectroscopic Instrument Instabilities Shows that Much of the "Noise" is not Stochastic.

As has long been understood, the noise on a spectrometric signal can be reduced by averaging over time, and the averaged noise is expected to decrease as t1/2, the square root of the data collection time. However, with contemporary capability for fast data collection and storage, we can retain and access a great deal more information about a signal train than just its average over time. During the same collection time, we can record the signal averaged over much shorter, equal, fixed periods. This is, then, the set of signals over submultiples of the total collection time. With a sufficiently large set of submultiples, the distribution of the signal's fluctuations over the submultiple periods of the data stream can be acquired at each wavelength (or frequency). From the autocorrelations of submultiple sets, we find only some fraction of these fluctuations consist of stochastic noise. Part of the fluctuations are what we call "fast drift", which is defined as drift over a time shorter than the complete measurement period of the average spectrum. In effect, what is usually assumed to be stochastic noise has a significant component of fast drift due to changes of conditions in the spectroscopic system. In addition, we show that the extreme values of the fluctuation of the signals are usually not balanced (equal magnitudes, equal probabilities) on either side of the mean or median without an inconveniently long measurement time; the data is almost inevitably biased. In other words, the unbalanced data is collected in an unbalanced manner around the mean, and so the median provides a better measure of the true spectrum. As is shown here, by using the medians of these distributions, the signal-to-noise of the spectrum can be increased and sampling bias reduced. The effect of this submultiple median data treatment is demonstrated for infrared, circular dichroism, and Raman spectrometry.

Heparin's solution structure determined by small-angle neutron scattering.

Heparin is a linear, anionic polysaccharide that is widely used as a clinical anticoagulant. Despite its discovery 100 years ago in 1916, the solution structure of heparin remains unknown. The solution shape of heparin has not previously been examined in water under a range of concentrations, and here is done so in D2 O solution using small-angle neutron scattering (SANS). Solutions of 10 kDa heparin-in the millimolar concentration range-were probed with SANS. Our results show that when sodium concentrations are equivalent to the polyelectrolyte's charge or up to a few hundred millimoles higher, the molecular structure of heparin is compact and the shape could be well modeled by a cylinder with a length three to four times its diameter. In the presence of molar concentrations of sodium, the molecule becomes extended to nearly its full length estimated from reported X-ray measurements on stretched fibers. This stretched form is not found in the presence of molar concentrations of potassium ions. In this high-potassium environment, the heparin molecules have the same shape as when its charges were mostly protonated at pD ≈ 0.5, that is, they are compact and approximately half the length of the extended molecules.

The relative diffusive transport rate of SrI2 in water changes over the nanometer length scale as measured by coherent quasielastic neutron scattering.

X-ray and neutron scattering have been used to provide insight into the structures of ionic solutions for over a century, but the probes have covered distances shorter than 8 Å. For the non-hydrolyzing salt SrI2 in aqueous solution, a locally ordered lattice of ions exists that scatters slow neutrons coherently down to at least 0.1 mol L(-1) concentration, where the measured average distance between scatterers is over 18 Å. To investigate the motions of these scatterers, coherent quasielastic neutron scattering (CQENS) data on D2O solutions with SrI2 at 1, 0.8, 0.6, and 0.4 mol L(-1) concentrations was obtained to provide an experimental measure of the diffusive transport rate for the motion between pairs of ions relative to each other. Because CQENS measures the motion of one ion relative to another, the frame of reference is centered on an ion, which is unique among all diffusion measurement methods. We call the measured quantity the pairwise diffusive transport rate Dp. In addition to this ion centered frame of reference, the diffusive transport rate can be measured as a function of the momentum transfer q, where q = (4π/λ)sin θ with a scattering angle of 2θ. Since q is related to the interion distance (d = 2π/q), for the experimental range 0.2 Å(-1)≤q≤ 1.0 Å(-1), Dp is, then, measured over interion distances from 40 Å to ≈6 Å. We find the measured diffusional transport rates increase with increasing distance between scatterers over the entire range covered and interpret this behavior to be caused by dynamic coupling among the ions. Within the model of Fickian diffusion, at the longer interionic distances Dp is greater than the Nernst-Hartley value for an infinitely dilute solution. For these nm-distance diffusional transport rates to conform with the lower, macroscopically measured diffusion coefficients, we propose that local, coordinated counter motion of at least pairs of ions is part of the transport process.

One-dimensional ionic self-assembly in a fluorous solution: the structure of tetra-n-butylammonium tetrakis[3,5-bis(perfluorohexyl)phenyl]borate in perfluoromethylcyclohexane by small-angle neutron scattering (SANS).

Fluorous liquids are the least polarizable condensed phases known, and their nonpolar members form solutions with conditions the closest to being in vacuo. A soluble salt consisting of a large fluorophilic anion, tetrakis[3,5-bis(perfluorohexyl)phenyl]borate, and its counterion, tetra-n-butylammonium, dissolved in perfluoromethylcyclohexane produces ionic solutions with extremely low conductivity. These solutions were subjected to small-angle neutron scattering (SANS) to ascertain the solute structure. At concentrations of 9% mass fraction, the fluorophilic electrolyte forms straight, long (>160 Å) self-assembled structures that are, in essence, long, homogeneous cylinders. Molecular models were made assuming a requirement for electroneutrality on the shortest length scale possible. This shows a structure formed from a stack of alternating anions and cations, and the structures fit the experimental scattering well. At the lower concentration of 1%, the stacks of ion pairs are shorter and eventually break up to form solitary ion pairs in the solution. These characteristics suggest such conditions provide an interesting new way to form long, self-assembling ionic nanostructures with single-molecule diameters in free solution onto which various moieties could be attached.

The solution structure of full-length dodecameric MCM by SANS and molecular modeling.

The solution structure of the full-length DNA helicase minichromosome maintenance protein from Methanothermobacter thermautotrophicus was determined by small-angle neutron scattering (SANS) data together with all-atom molecular modeling. The data were fit best with a dodecamer (dimer of hexamers). The 12 monomers were linked together by the B/C domains, and the adenosine triphosphatase (AAA+) catalytic regions were found to be freely movable in the full-length dodecamer both in the presence and absence of Mg(2+) and 50-meric single-stranded DNA (ssDNA). In particular, the SANS data and molecular modeling indicate that all 12 AAA+ domains in the dodecamer lie approximately the same distance from the axis of the molecule, but the positions of the helix-turn-helix region at the C-terminus of each monomer differ. In addition, the A domain at the N-terminus of each monomer is tucked up next to the AAA+ domain for all 12 monomers of the dodecamer. Finally, binding of ssDNA does not lock the AAA+ domains in any specific position, which leaves them with the flexibility to move both for helicase function and for binding along the ssDNA.

Structure determination of functional membrane proteins using small-angle neutron scattering (sans) with small, mixed-lipid liposomes: native beef heart mitochondrial cytochrome c oxidase forms dimers.

The low-resolution three-dimensional structure of purified native beef heart mitochondrial cytochrome c oxidase (COX) in asolectin unilamellar liposomes has been measured by small-angle neutron scattering under the conditions where the protein remains fully functional. From a neutron scattering perspective, the use of mixed-lipid liposomes provided for a more homogeneous matrix than can be achieved using a single lipid. As a result, the measurements were able to be performed under conditions where the liposome scattering was essentially eliminated (contrast-matched conditions). The protein structure in the membrane was modeled as a simple parallelepiped with side lengths of (59 × 70 × 120) Å with uncertainties, respectively, (11, 12, 20 Å). The molecular mass calculated for a typical protein with this volume is estimated to be (410 ± 124) kDa, which indicates the mass of a COX dimer. The longest dimension has some uncertainty due to intermolecular scattering contributing to the data. Nevertheless, that length was estimated using an average protein density and the known dimer molecular mass. Using the same cross sectional dimensions for the structure, the length is estimated to be 120 Å. However, the measured scattering curve of the dimer in the liposome differs significantly from that calculated from the X-ray structure of the dimer in a crystal of mixed micelles (PDB 3AG1). The calculated SANS scattering from the crystal structure was fit with a parallelepiped, measuring (59 × 101 × 129) Å with fitting uncertainties, respectively, (2, 3, 3 Å). Our results suggest that COX is a functional dimer when reconstituted into mixed-lipid liposomes.

In-situ characterization of self-assembled monolayers of water-soluble oligo(ethylene oxide) compounds.

In-situ spectroscopic ellipsometry (SE) was utilized to examine the formation of the self-assembled monolayers (SAMs) of the water-soluble oligo(ethylene oxide) [OEO] disulfide [S(CH(2)CH(2)O)(6)CH(3)](2) {[S(EO)(6)](2)} and two analogous thiols - HS(CH(2)CH(2)O)(6)CH(3) {(EO)(6)} and HS(CH(2))(3)O(CH(2)CH(2)O)(5)CH(3) {C(3)(EO)(5)} - on Au from aqueous solutions. Kinetic data for all compounds follow simple Langmuirian models with the disulfide reaching a self-limiting final state (d=1.2nm) more rapidly than the full coverage final states of the thiol analogs (d=2.0nm). The in-situ ellipsometric thicknesses of all compounds were found to be nearly identical to earlier ex-situ ellipsometric measurements suggesting similar surface coverages and structural models in air and under water. Exposure to bovine serum albumin (BSA) shows the self-limiting (d=1.2nm) [S(EO)(6)](2) SAMs to be the most highly protein resistant surfaces relative to bare Au and completely-formed SAMs of the two analogous thiols and octadecanethiol (ODT). When challenged with up to near physiological levels of BSA (2.5mg/mL), protein adsorption on the final state [S(EO)(6)](2) SAM was only 3% of that which adsorbed to the bare Au and ODT SAMs.

Self-assembled monolayers of an oligo(ethylene oxide) disulfide and its corresponding thiol assembled from water: characterization and protein resistance.

Self-assembled monolayers (SAMs) of the disulfide [S(CH2CH2O)6CH3]2 ([S(EO)6]2) on Au from 95% ethanol and from 100% water are described. Spectroscopic ellipsometry and reflection-absorption infrared spectroscopy indicate that the [S(EO)6]2 films are similar to the disordered films of HS(CH2CH2O)6CH3 ((EO)6) and HS(CH2)3O(CH2CH2O)5CH3 (C3EO5) at their protein adsorption minima. The [S(EO)6]2 SAMs exhibit constant film thickness (d) of 1.2 +/- 0.2 nm over long immersion times (up to 20 days) and do not attain the highly ordered, 7/2 helical structure of the (EO)6 and C3EO5 SAMs (d = 2.0 nm). Exposure of these self-limiting [S(EO)6]2 SAMs to bovine serum albumin show high resistance to protein adsorption.

Single-molecule mass spectrometry in solution using a solitary nanopore.

We introduce a two-dimensional method for mass spectrometry in solution that is based on the interaction between a nanometer-scale pore and analytes. As an example, poly(ethylene glycol) molecules that enter a single alpha-hemolysin pore cause distinct mass-dependent conductance states with characteristic mean residence times. The conductance-based mass spectrum clearly resolves the repeat unit of ethylene glycol, and the mean residence time increases monotonically with the poly(ethylene glycol) mass. This technique could prove useful for the real-time characterization of molecules in solution.

Control of protein adsorption: molecular level structural and spatial variables.

The adsorption of fibrinogen (Fb) and bovine serum albumin onto polycrystalline Au coated with HS(CH2)3O(CH2CH2O)5CH3 was determined by surface plasmon resonance from bare Au (0% coverage) to the complete ( approximately 100% coverage) self-assembled monolayer (SAM). Both proteins exhibit similar adsorption curves with common onset ( approximately 60% coverage) and range ( approximately 60% to 80% coverage) of minimal protein adsorption. Reflection-absorption infrared spectroscopic data show that widespread order is not present in the films over this range of coverage, indicating loosely packed, bound oligomers that are uniformly distributed and fully screen the underlying substrate. On the basis of our data, we propose a mechanism of protein rejection by oligo(ethylene oxide) (OEO)-modified surfaces in terms of changes in free energy (DeltaGsystem; system = protein + surface) due to oligomer conformational constriction over an area greater than the contact area. Minimal protein adsorption corresponds to the maximum DeltaGsystem for a given compression. This controlled study of protein adsorption provides insights into the molecular level understanding of protein adsorption unavailable from previous polymer and comparative SAM studies.