The reaction of protein tryptophanyl residues with reduced nicotinamide adenine dinucleotide. Structure of a model adduct.

This paper describes in detail a simple, light producing and handling system which differs greatly from the commercial unit presently used by most investigators, and which overcomes a number of its disadvantages. The apparatus is designed so that it can easily be substituted for the light producing, dispersing, and collimating section of the commercial photoelectric scanners, with no further changes in the commercial scanners except the substitution of optical flats for the collimating and condensing lenses in the bottom and top, respectively, of the rotor chamber. The most outstanding feature of this new system is that the use of a faster monochromator ($\text{f}\text{75.3}$) and a cylindrical lens theoretically increases light intensity by up to 100 times that of the commercial scanner, under otherwise identical conditions. Other advantages include the following: (1) For collecting and collimating light, two pairs of mirrors give a precell optical system focused in the radial direction at all wavelengths of light; (2) these components and the lamp are located on a table outside the centrifuge, so virtually any size of lamp can be used; (3) the entire precell optical system is a self-contained unit which, within reasonable limits, may be moved to any desired location without markedly affecting the quality of the collimated light; and (4) the optical path length has been shortened, providing less dissipation of light energy. The principles behind the selection and design of the key components are discussed. New alignment procedures and apparatus developed to aid in fast, easy, accurate alignment are described and discussed. Several components for use with a system using a computer-controlled stepping motor scanner for collection of data are also described. This system is simple enough and is documented in sufficient detail so that other interested workers, even those with little or no optics experience, can duplicate and use the system.     

Sedimentation equilibrium studies on protein from kookaburra beak.

Fractionated samples of the soluble S-carboxymethyl proteins from kookaburra beak (Frenkel and Gillespie 1976) were examined by equilibrium sedimentation. The molecular weight was found to be 11,300 when the photoelectric scanning absorption optical system was employed and 13,700 when Rayleigh interference optics were used. Possible explanations for this difference are considered and it is concluded that it must arise from heterogeneity of the protein. Optical rotatory dispersion measurements indicate that the proteins probably exist as random coils in dilute aqueous buffer.     

Monitoring the homogeneity of adenovirus preparations (a gene therapy delivery system) using analytical ultracentrifugation

This study explores the capability of modern analytical ultracentrifugation (AUC) to characterize the homogeneity, under product formulation conditions, of preparations of adenovirus vectors used in gene therapy and to assess the lot-to-lot consistency of this unique drug product. We demonstrate that a single sedimentation velocity run on an adenovirus sample can detect and accurately quantify a number of different forms of virus particles and subvirus particles. These forms include (a) intact virus monomer particles, (b) virus aggregates, (c) empty capsids (ECs), and (d) smaller assembly intermediates or subparticles formed during normal or aberrant virus assembly (or as a result of damage to the intact adenovirus or EC material during all phases of virus production). This information, which is collected on adenovirus samples under the exact formulation conditions that exist in the adenovirus vial, is obtained by direct boundary modeling of the AUC data generated from refractometric and/or UV detection systems using the computer program SEDFIT developed by Peter Schuck. Although both detectors are useful, refractometric detection using the Rayleigh interferometer offers a key advantage for providing accurate concentration information due to the similar response factors for both protein and DNA and its insensitivity to light scattering effects. Additional AUC data obtained from analytical band sedimentation velocity and density gradient sedimentation equilibrium experiments in CsCl with UV detection were also generated. These results further support conclusions concerning the solution properties of adenovirus, the identity of the different virus species, and the overall capability of boundary sedimentation velocity analysis.     

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuumNot only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.     

UV microbeam irradiations of the mitotic spindle I. The UV-microbeam apparatus

Summary We describe the assembly of a UV microbeam microscope based on a Zeiss IM35 inverted microscope. The important UV transmitting elements are standard UV epifluorescence attachments available from Zeiss; the main modification involves fitting an adjustable slit in place of the field diaphragm. We describe how to align and focus the UV source for optimal irradiations. Our current version of this machine is also fitted with a monochromator and using monochromatic UV light, we can reproduceably create Areas of Reduced Birefringence in spindle fibres with ca. 2–3 s irradiations, while continually observing the fibres. The microscope is stable and easy to set up, allowing many consecutive experiments to be done, including multiple irradiations on the one cell. In conjunction with video image processing techniques, the cells can be observed continuously using polarising, Nomarski or other optical systems. Some preliminary observations demonstrating the versatility of the machine are described.Abbrevations ARB areas of reduced birefringence - MT microtubules - UV ultraviolet     

Direct analysis of solute self-association by sedimentation equilibrium

An improved procedure is described for the characterization of solute self-association by sedimentation equilibrium. Whereas previous statistical-mechanical approaches to allowance for the effects of thermodynamic nonideality have entailed tedious iteration because of their specification of activity coefficients in terms of the equilibrium concentrations of all species, such reliance upon knowledge of the solution composition is avoided by the adaptation of an alternative statistical-mechanical formulation [T. L. Hill and Y. D. Chen (1973) Biopolymers, Vol. 12, pp. 1285–1312] in which thermodynamic nonideality is expressed in terms of total solute concentration. The development of an analysis in terms of a relationship with total solute concentration as the experimental variable allows this attribute of the Adams-Fujita approach to be retained without sacrifice of statistical-mechanical rigor. Its use is illustrated by application to Rayleigh interferometric records of sedimentation equilibrium distributions reflecting α-chymotrypsin dimerization and lysozyme self-association. © 1996 John Wiley & Sons, Inc.     

Probing the oligomeric assemblies of pea porphobilinogen synthase by analytical ultracentrifugation

The enzyme porphobilinogen synthase (PBGS) can exist in different nonadditive homooligomeric assemblies, and under appropriate conditions, the distribution of these assemblies can respond to ligands such as metals or substrate. PBGS from most organisms was believed to be octameric until work on a rare allele of human PBGS revealed an alternate hexameric assembly, which is also available to the wild-type enzyme at elevated pH [Breinig, S., et al. (2003) Nat. Struct. Biol. 10, 757-763]. Herein, we establish that the distribution of pea PBGS quaternary structures also contains octamers and hexamers, using both sedimentation velocity and sedimentation equilibrium experiments. We report results in which the octamer dominates under purification conditions and discuss conditions that influence the octamer:hexamer ratio. As predicted by PBGS crystal structures from related organisms, in the absence of magnesium, the octameric assembly is significantly destabilized, and the oligomeric distribution is dominated largely by the hexameric assembly. Although the PBGS hexamer-to-octamer oligomeric rearrangement is well documented under some conditions, both assemblies are very stable (under AU conditions) in the time frame of our ultracentrifuge experiments.     

Dynamic equilibrium in histone assembly: self-assembly of single histones and histone pairs.

The assembly of acid-extracted, purified F2a1, F3, F2a2, and F2b histones and their six possible pairwise combination into organized structures has been studied by: (1) sedimentation velocity, (2) sedimentation equilibrium, (3) electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate after cross-linking the protein solution with dimethyl suberimidate, and (4) electron microscopy. Each of the purified histone fractions can renature and assemble into high molecular weight organized structures. This assembly is dependent on the ionic strength, protein concentration, and temperature of the solutions. The four histones studied assemble into structures of similar dimensions and shape. In each case the first structure observed is a bent rod with a diameter of 22 A. Conditions which favor assembly lead to formation of fibers with diameters of about 44 A. The conditions which lead to assembly into organized structures are similar for the arginine-rich histones, F2a1 and F3. Higher ionic strength is required for the assembly of the lysine-rich histones, F2a2 and F2b. Certain pairs of histones interact. Strong interactions among pairs of histones interfere with the self-assembly of single histones into large structures. Howver, increase in protein concentration or ionic stregth leads to formation of large molecular structures even in solutions of pairs of strongly interacting histones. These structures are similar to those obtained with single histones. The results suggest that aggregation and complexing of histones represent a reversible, ordered process of assembly. The various assembled forms are in a dynamic equilibrium. The final assembled form, which is similar in all cases, is dependent on the environmental conditions to which the histones are exposed. It is suggested that each of the assembled histone structures, regardless whether it is composed of a single histone or a pair of histones, can serve as a core around which the DNA can be wrapped.     

Super-Resolution Long-Depth Focusing by Radially Polarized Light Irradiation Through Plasmonic Lens in Optical Meso-field

In this paper, we propose a novel plasmonic lens design consisting of an annular slit and concentric grooves. The simulation results show that under radially polarized illumination, a super-resolution long depth of focus (DOF) spot can be achieved in optical meso-field due to the constructive interference of scattered light by the concentric grooves. We also analyze the influence of depth-tuned annular grooves on focusing performance, including focal length, DOF, and full-width half-maximum. Moreover, focusing efficiency can be enhanced (～350 %) by introducing a circular metallic grating which surrounds the annular slit. This plasmonic lens has potential applications in nano-imaging and nano-photolithography.     

Characterization of weak protein dimerization by direct analysis of sedimentation equilibrium distributions: the INVEQ approach

Closer scrutiny has been accorded a recently reported procedure for characterizing weak protein dimerization by sedimentation equilibrium (INVEQ) in which the equilibrium distribution is analyzed as a dependence of radial distance on solute concentration rather than of solute concentration on radial distance. By demonstrating theoretically that the fundamental parameter derived from the analysis is simply the difference between the dimerization constant and the osmotic second virial coefficient for monomer-monomer interaction, this investigation refutes the original claim that independent estimates of these two parameters can be obtained by nonlinear curve fitting of the sedimentation equilibrium distribution. This criticism also applies to conventional analyses of sedimentation distributions by the commonly employed Beckman Origin and NONLIN software. Numerically simulated distributions are then analyzed to demonstrate limitations of the procedure and also to indicate a means of improving the reliability of the returned estimate of the dimerization constant. These features are illustrated by applying the original and revised analytical procedures to a sedimentation equilibrium distribution for alpha-chymotrypsin (pH 4.0, I 0.05 M).     

Determination of small differences in buoyant density of macromolecules by analytical density gradient equilibrium centrifugation.

A sensitive method is proposed for the determination of small differences between the buoyant densities of different species of monodisperse macromolecules by analytical density gradient equilibrium centrifugation. The procedure involves the measurement at sedimentation equilibrium of the bandwidths of the concentration distribution of the separate macromolecules and of a mixture of the different species. The difference in buoyant densities can then be estimated from the difference between the bandwidths.     

An experimental study of baseline reproducibility and its effect on high-speed sedimentation equilibrium data

Baseline variability is an important source of error in molecular weight determination with the high-speed sedimentation equilibrium method when Rayleigh interference optics are employed. Using Yphantis-style six-channel cell assemblies (1), we have measured this variability under several conditions at 32,000 rpm and below. These measurements have led to the conclusion that the baseline for speeds up to at least 32,000 rpm should be measured from a picture taken at low speed at the end of the high-speed experiment.     

Slit and Robo control cardiac cell polarity and morphogenesis

Basic aspects of heart morphogenesis involving migration, cell polarization, tissue alignment, and lumen formation may be conserved between Drosophila and humans, but little is known about the mechanisms that orchestrate the assembly of the heart tube in either organism. The extracellular-matrix molecule Slit and its Robo-family receptors are conserved regulators of axonal guidance. Here, we report a novel role of the Drosophila slit, robo, and robo2 genes in heart morphogenesis. Slit and Robo proteins specifically accumulate at the dorsal midline between the bilateral myocardial progenitors forming a linear tube. Manipulation of Slit localization or its overexpression causes disruption in heart tube alignment and assembly, and slit-deficient hearts show disruptions in cell-polarity marker localization within the myocardium. Similar phenotypes are observed when Robo and Robo2 are manipulated. Rescue experiments suggest that Slit is secreted from the myocardial progenitors and that Robo and Robo2 act in myocardial and pericardial cells, respectively. Genetic interactions suggest a cardiac morphogenesis network involving Slit/Robo, cell-polarity proteins, and other membrane-associated proteins. We conclude that Slit and Robo proteins contribute significantly to Drosophila heart morphogenesis by guiding heart cell alignment and adhesion and/or by inhibiting cell mixing between the bilateral compartments of heart cell progenitors and ensuring proper polarity of the myocardial epithelium.     

Analyses of sedimentation equilibrium data

A numerical procedure is presented which can quite adequately compute the molecular weight averages as a function of solute concentration from sedimentation equilibrium data for homogeneous systems and for monomer-dimer associating systems with a possible extension to heterogeneous systems where monotonic variation in the weight average molecular weight is observed such as in weakly associating or dissociating systems. The procedure utilizes the method of orthogonal polynomials for curve fitting which allows for a rapid determination of best fit with minimal round off error. The procedure is particularly applicable in cases where the concentration of solute at the meniscus can be considered to be neither appreciable and reasonably well determined as in low speed sedimentation equilibrium experiments, nor essentially zero as in high speed sedimentation equilibrium experiments where the calculations become somewhat more simplified. The use of moderate speed sedimentation equilibrium has the advantage of providing a more broad concentration distribution in the centrifuge cell which yields more extensive information concerning dissociating systems yet still provides results at low solute concentrations where most solutes can be considered to be behaving ideally.     

Sedimentation equilibrium analysis of interference optical data by systematic noise decomposition.

An analytical method is described for removal of systematic signal offsets from interference optical data of sedimentation equilibrium gradients. It is demonstrated that the time-invariant signal contributions can be extracted from hydrodynamic modeling of interference profiles acquired during the approach to sedimentation equilibrium. This method is based on a technique for the explicit algebraic calculation of time-invariant noise components from sedimentation data, recently described for the direct modeling of sedimentation velocity experiments (P. Schuck and B. Demeler, Biophys. J. 76, 2288-2296, 1999). The calculated systematic signal offset is very well defined by the experimental data, stable over time, and its calculation is robust and to a large extent independent of the hydrodynamic model. The calculated time-invariant signal can be used to reduce the systematic errors in the measured sedimentation equilibrium profiles by more than an order of magnitude. It is shown that the resulting net equilibrium fringe profiles after subtraction of the time-invariant noise component allow equilibrium analyses consistent with those obtained from absorbance profiles. However, due to a higher dynamic range and the higher number of data points, the parameters derived from the net interference analysis can exhibit significantly improved precision. The presented study demonstrates the feasibility and potential of this analytical method for full exploitation of the remarkable precision of the interference optical data acquisition system, allowing sedimentation equilibrium experiments at loading concentrations below 0.05 mg/ml.     

Hydrodynamic analysis of the human progesterone receptor A-isoform reveals that self-association occurs in the micromolar range

Human progesterone receptors exist as two functionally distinct isoforms, an 83 kDa A-receptor (PR-A) and a 99 kDa B-receptor (PR-B). The isoforms are identical except that PR-B has an additional 164 amino acids at its N-terminus. We have previously characterized the hydrodynamics and solution assembly energetics of PR-B [Heneghan, A. F., et al. (2005) Biochemistry 44, 9528-9537], and here we present an analysis of PR-A. At micromolar concentrations of the receptor, sedimentation velocity studies demonstrate that PR-A undergoes a concentration-dependent change in its sedimentation coefficient distribution, indicative of a self-associating system. Global analysis of data sets collected at multiple PR-A concentrations supports the presence of a hydrodynamically homogeneous 3.50 S monomer species in equilibrium with a 7.15 S dimer species. Sedimentation equilibrium analysis demonstrates that self-association can be rigorously described by a monomer-dimer assembly reaction and a dimerization free energy of -7.6 +/- 0.6 kcal/mol. Both the PR-A monomer and dimer are structurally asymmetric, although the extent of asymmetry is significantly decreased for the dimer, indicative of quaternary-induced hydrodynamic compaction. Limited proteolysis studies suggest that PR-A asymmetry arises from an ensemble of partially folded conformations within the N-terminal half of the molecule. Finally, comparison to our previous work on PR-B self-association energetics demonstrates that it dimerizes, under identical solution conditions, with an affinity at least 8-fold weaker than that of PR-A. Thus, residues unique to the B-isoform destabilize receptor assembly energetics. Importantly, the physical and chemical driving forces underlying isoform-specific dimerization suggest that B-unique amino acids modulate the energetics through an allosteric mechanism.     

Multiple-Wavelength Focusing and Demultiplexing Plasmonic Lens Based on Asymmetric Nanoslit Arrays

A multiple-wavelength focusing and demultiplexing plasmonic lens based on asymmetric nanoslit arrays is designed. The nanoslit arrays are perforated in a gold film and act as metal–insulator–metal plasmonic waveguides. By manipulating the widths of the slit arrays, the plasmonic lens can concentrate two incident plane wave beams to two separated focal points corresponding to their wavelengths. The full wave simulation is performed to verify the designed lens. This work provides a way to design more compact and integrated wavelength-division multiplexing plasmonic devices for nanophotonic communication and spectral imaging.     

Association properties of betaB1- and betaA3-crystallins: ability to form heterotetramers

As major constituents of the mammalian lens, beta-crystallins associate into dimers, tetramers, and higher-order complexes to maintain lens transparency and refractivity. A previous study has shown that dimerization of betaB2- and betaA3-crystallins is energetically highly favored and entropically driven. While heterodimers further associate into higher-order complexes in vivo, a significant level of reversibly associated tetrameric crystallin has not been previously observed in vitro. To enhance our understanding of the interactions between beta-crystallins, we characterized the association of betaB1-crystallin, a major component of large beta-crystallin complexes (beta-high), with itself and with betaA3-crystallin. Mouse betaB1-crystallin and human betaA3-crystallin were expressed in Escherichia coli and purified chromatographically. Their association was then characterized using size-exclusion chromatography, native gel electrophoresis, isoelectric focusing, and analytical sedimentation equilibrium centrifugation. When present alone, each beta-crystallin associates into homodimers; however, no tetramer formation is seen. Once mixing has taken place, formation of a heterocomplex between betaB1- and betaA3-crystallins is observed using size-exclusion chromatography, native gel electrophoresis, isoelectric focusing, and sedimentation equilibrium. In contrast to results previously obtained after betaB2- and betaA3-crystallins had been mixed, mixed betaB1- and betaA3-crystallins show a dimer-tetramer equilibrium with a K d of 1.1 muM, indicating that these two beta-crystallins associate predominantly into heterotetramers in vitro. Thus, while each purified beta-crystallin associates only into homodimers and under the conditions studied mixed betaB2- and betaA3-crystallins form a mixture of homo- and heterodimers, mixed betaB1- and betaA3-crystallins associate predominantly into heterotetramers in equilibrium with heterodimers. These findings suggest a unique role for betaB1-crystallin in promoting higher-order crystallin association in the lens.     

Light-difference detector for reading Rayleigh fringe patterns from the ultracentrifuge

We have developed a dual-photocell, light-difference detector, easily attached to a comparator screen, which provides rapid and direct location of fringe centers from Rayleigh interferograms without the need for digital micrometers or measurement of optical densities. The device provides a pulse for digital micrometers which triggers the printing of fringe centers during manual movement of the stage, providing a two-thirds saving in time. From evaluation of sedimentation equilibrium patterns it was found that the precision of (1) concentration measurement is 0.003 fringes and (2) molecular weight determinations is several parts per thousand.     

Characterization of dimer subunits of intermediate filament proteins

The fundamental subunit of the various types of intermediate-sized filaments (IF) has been shown to be a tetramer that is thought to represent a double dimer, i.e. an array of two laterally packed coiled-coils of alpha-helices. The two-chain state of intact IF proteins had up to this point not been isolated and characterized as has been done for other fibrous alpha-helical coiled-coil proteins. Using buffers containing 3 M-guanidinium hydrochloride we prepared dimers by depolymerization of IF or by reconstitution from fully denatured molecules. Dimers of desmin (from chicken gizzard), vimentin (from bovine lens tissue and cultured human fibroblasts) and the neurofilament protein NF-L (from bovine brain) as well as in vitro formed homodimers of human and rat cytokeratins numbers 8 (A), 18 (D) and 19 ("40K"), are characterized by ultracentrifugation techniques (sedimentation velocity and equilibrium), electron microscopy and chemical cross-linking. The results show that IF proteins from discrete complexes of two polypeptide chains in parallel orientation and probably in coiled-coil configuration, which apparently have a high tendency to further associate into double dimers. Implications of these results for concepts of IF organization and IF protein assembly are discussed.