Kenneth A. Rubinson

Small-angle neutron scattering and the errors in protein structures that arise from uncorrected background and intermolecular interactions

Small-angle neutron scattering (SANS) provides a unique method to probe soft matter in the 10–100 nm length scale in solutions. In order to determine the shape and size of biological macromolecular structures correctly with SANS, a background-subtracted, undistorted scattering curve must be measured, and the required accuracy and precision is especially needed at the short-length-scale limit. A true scattering curve is also needed to discern whether intermolecular interactions are present, which also are probed in the SANS experiment. This article shows how to detect intermolecular interactions so that subsequent structure modeling can be performed using only data that do not contain such contributions. It is also shown how control of many factors can lead to an accurate baseline, or background, correction for scattering from proteins, especially to account for proton incoherent scattering. Failure to make this background correction properly from proteins, polymers, nucleic acids and lipids can result in incorrect values for the calculated shapes and sizes of the molecules as well as the derived magnitudes of the intermolecular interactions.

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.

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.

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 Mg2+ 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. Proteins 2014; 82:2364–2374.

Practical corrections for p(H,D) measurements in mixed H2O/D2O biological buffers

Mixtures of light and heavy water are used in NMR, small-angle neutron scattering (SANS), growth media for producing deuterated biological molecules, and analytical methods such as hydrogen–deuterium exchange (HDX) mass spectrometry. It is common to measure the pH of these solutions with a combination glass electrode with all chambers filled with aqueous (H2O) potassium chloride solutions. In the daily measurement of samples containing mixtures of H2O with D2O in some ratio – call this measurement p(H,D) – we generally do not control for all of the contributions to the differences measured in carefully controlled electrochemical experiments. For example, the calibration solutions contain relatively low concentrations of the calibrant buffer with low or no added salt. Meanwhile the tested solutions can contain widely varying levels of any number of different salts as well as both polar and nonpolar organics and polymers and proteins. In this note, the p(H,D) behaviors of 50 mM solutions of five different buffers used in biological in vitro solutions were measured over the full range of H2O : D2O ratios in the open atmosphere. After calibration, pH measurements were made with the buffer solutions alone and with 100 mM KCl added to model a significant ionic strength difference. The solutions consisted of 1 : 1 volume mixtures of the acid and base forms of acetate, monobasic/dibasic phosphate, 2-amino-2-hydroxymethyl-propane-1,3-diol (tris), 2-amino-2-hydroxymethyl-propane-1,3-diol (HEPES), and glycine to span the common, full range for biological buffers. The pH values of the 1 : 1 mixtures mean that the measurements were, in fact, of their formal pKa values. Each of the buffers exhibited a unique pattern of behavior, and none of them exhibited a measured ΔpKa = pKDa − pKHa as large as 0.4, a value that has been suggested to be added to a pH measured in H2O to match the equivalent pD measured in D2O. The results do indicate that when a reasonable, required accuracy for pH measurement is ±0.1 units, three general guidelines apply: (1) where p(H,D) values are less than 8 for any D2O content, no correction is needed for the p(H,D) measurement when comparing it to pHH; (2) for less than 50% D2O, if the 8 8, any corrections required will depend on the specific conditions and the specific buffer. Outside of the range 4 < p(H,D) < 10 or for needed greater accuracy, any corrections required will depend on the specific conditions and the identity of the buffer.

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 unusual, slow redox properties of Cu, Ni, and Rh cobalamins

The rates of the redox processes for several metal cobalamin complexes are virtually zero which suggests that the use of other complexes to model the redox properties of vitamin B12 compounds is questionable.

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 D2O 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 polyelectrolytes 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.

Spin trapping experiments on nadh analogues and the role of radicals in carbonyl reductions

1 H N.m.r. and gas chromatographic analysis of the reactants and products show that the reaction of N-benzyl-1,4-dihydronicotinamide (NBDN), an analogue of NADH, with pyridine-2-carbaldehyde does not pass through a radical intermediate during either a thermal or a photolytic reaction; photolysis led to the removal of NBDN but not to the production of the reduced substrate and radicals detected with e.s.r. spectroscopy were shown to be artifacts.