Method for Improved Fluorescence Corrections for Molar Mass Characterization by Multiangle Light Scattering
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Biochemistry & Molecular Biology↗
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Chlorophyllase from Arabidopsis thaliana Reveals an Emerging Model for Controlling Chlorophyll Hydrolysis
Chlorophyll (Chl) is one of Nature’s most complex pigments to biosynthesize and derivatize. This pigment is vital for survival and also paradoxically toxic if overproduced or released from a protective protein scaffold. Therefore, along with the mass production of Chl, organisms also invest in mechanisms to control its degradation and recycling. One important enzyme that is involved in these latter processes is chlorophyllase. This enzyme is employed by numerous photosynthetic organisms to hydrolyze the phytol tail of Chl. Although traditionally thought to catalyze the first step of Chl degradation, recent work suggests that chlorophyllase is instead employed during times of abiotic stress or conditions that produce reactive oxygen species. However, the molecular details regarding how chlorophyllases are regulated to function under such conditions remain enigmatic. Here, we investigate the Arabidopsis thaliana chlorophyllase isoform AtCLH2 using site-directed mutagenesis, mass spectrometry, dynamic light scattering, size-exclusion multiangle light scattering, and both steady-state enzyme kinetic and thermal stability measurements. Through these experiments, we show that AtCLH2 exists as a monomer in solution and contains two disulfide bonds. One disulfide bond putatively maps to the active site, whereas the other links two N-terminal Cys residues together. These disulfide bonds are cleaved by chemical or chemical and protein-based reductants, respectively, and are integral to maintaining the activity, stability, and substrate scope of the enzyme. This work suggests that Cys residue oxidation in chlorophyllases is an emerging regulatory strategy for controlling the hydrolysis of Chl pigments.
59 BASIC BIOLOGICAL SCIENCES↗
An atypical BRCT–BRCT interaction with the XRCC1 scaffold protein compacts human DNA Ligase IIIα within a flexible DNA repair complex
The XRCC1–DNA ligase IIIα complex (XL) is critical for DNA single-strand break repair, a key target for PARP inhibitors in cancer cells deficient in homologous recombination. Here, we combined biophysical approaches to gain insights into the shape and conformational flexibility of the XL as well as XRCC1 and DNA ligase IIIα (LigIIIα) alone. Structurally-guided mutational analyses based on the crystal structure of the human BRCT–BRCT heterodimer identified the network of salt bridges that together with the N-terminal extension of the XRCC1 C-terminal BRCT domain constitute the XL molecular interface. Coupling size exclusion chromatography with small angle X-ray scattering and multiangle light scattering (SEC-SAXS–MALS), we determined that the XL is more compact than either XRCC1 or LigIIIα, both of which form transient homodimers and are highly disordered. The reduced disorder and flexibility allowed us to build models of XL particles visualized by negative stain electron microscopy that predict close spatial organization between the LigIIIα catalytic core and both BRCT domains of XRCC1. Together our results identify an atypical BRCT–BRCT interaction as the stable nucleating core of the XL that links the flexible nick sensing and catalytic domains of LigIIIα to other protein partners of the flexible XRCC1 scaffold.
59 BASIC BIOLOGICAL SCIENCES↗
Elucidating lipid nanoparticle properties and structure through biophysical analyses
Designing lipid nanoparticle (LNP) delivery systems with specific targeting, potency and minimal side effects is crucial for their clinical use. However, traditional characterization methods, such as dynamic light scattering, cannot accurately quantify physicochemical properties of LNPs and how these are influenced by the lipid composition and mixing method. Here, we structurally characterize polydisperse LNP formulations by applying emerging solution-based biophysical methods that have higher resolution and provide biophysical data beyond size and polydispersity. These techniques include sedimentation velocity analytical ultracentrifugation, field-flow fractionation followed by multiangle light scattering and size-exclusion chromatography in line with synchrotron small-angle X-ray scattering. Here, we show that LNPs have intrinsic polydispersity in size, RNA loading and shape, which depend on both the formulation technique and the lipid composition. Lastly, we predict LNP transfection in vitro and in vivo by examining the relationship between mRNA translation and physicochemical characteristics. Solution-based biophysical methods will be essential for determining LNP structure–function relationships, facilitating the creation of new design rules for LNPs.
36 MATERIALS SCIENCE↗
Solution characterization of the dynamic conjugative entry exclusion protein TraG
The R100 plasmid and the secretion system it encodes are representative of F-like conjugative type IV secretion systems for the transmission of mobile DNA elements in gram-negative bacteria, serving as a major contributor to the spread of antibiotic resistance in bacterial pathogens. The TraG protein of F-like systems consists of a membrane-bound N-terminal domain and a periplasmic C-terminal domain, denoted TraG*. TraG* is essential in preventing redundant DNA transfer through a process termed entry exclusion. In the donor cell, it interacts with TraN to facilitate mating pair stabilization; however, if a mating pore forms between bacteria with identical plasmids, TraG* interacts with its cognate TraS in the inner membrane of the recipient bacterium to prevent redundant donor–donor conjugation. Structural studies of TraG* from the R100 plasmid have revealed the presence of a dynamic region between the N- and C-terminal domains of TraG. Thermofluor, circular dichroism, collision-induced unfolding–mass spectrometry, and size exclusion chromatography linked to multiangle light scattering and small angle x-ray scattering experiments indicated an N-terminal truncation mutant displayed higher stability and less disordered content relative to full-length TraG*. The 45 N-terminal residues of TraG* are hypothesized to serve as part of a flexible linker between the two independently functioning domains.
59 BASIC BIOLOGICAL SCIENCES↗
Biochemical and structural analyses reveal that the tumor suppressor neurofibromin (NF1) forms a high-affinity dimer
Neurofibromin is a tumor suppressor encoded by the NF1 gene, which is mutated in Rasopathy disease neurofibromatosis type I. Defects in NF1 lead to aberrant signaling through the RAS–mitogen-activated protein kinase pathway due to disruption of the neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of most of the neurofibromin protein; to date, biochemical and structural data exist only for its GAP domain and a region containing a Sec-PH motif. To better understand the role of this large protein, here we carried out a series of biochemical and biophysical experiments, including size-exclusion chromatography–multiangle light scattering (SEC-MALS), small-angle X-ray and neutron scattering, and analytical ultracentrifugation, indicating that full-length neurofibromin forms a high-affinity dimer. We observed that neurofibromin dimerization also occurs in human cells and likely has biological and clinical implications. Analysis of purified full-length and truncated neurofibromin variants by negative-stain EM revealed the overall architecture of the dimer and predicted the potential interactions that contribute to the dimer interface. We could reconstitute structures resembling high-affinity full-length dimers by mixing N- and C-terminal protein domains in vitro. The reconstituted neurofibromin was capable of GTPase activation in vitro, and co-expression of the two domains in human cells effectively recapitulated the activity of full-length neurofibromin. Taken together, these results suggest how neurofibromin dimers might form and be stabilized within the cell.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
ULK complex organization in autophagy by a C-shaped FIP200 N-terminal domain dimer
The autophagy-initiating human ULK complex consists of the kinase ULK1/2, FIP200, ATG13, and ATG101. Hydrogen-deuterium exchange mass spectrometry was used to map their mutual interactions. The N-terminal 640 residues (NTD) of FIP200 interact with the C-terminal IDR of ATG13. Mutations in these regions abolish their interaction. Negative stain EM and multiangle light scattering showed that FIP200 is a dimer, while a single molecule each of the other subunits is present. The FIP200NTD is flexible in the absence of ATG13, but in its presence adopts the shape of the letter C ~20 nm across. The ULK1 EAT domain interacts loosely with the NTD dimer, while the ATG13:ATG101 HORMA dimer does not contact the NTD. Cryo-EM of the NTD dimer revealed a structural similarity to the scaffold domain of TBK1, suggesting an evolutionary similarity between the autophagy-initiating TBK1 kinase and the ULK1 kinase complex.
59 BASIC BIOLOGICAL SCIENCES↗
Nucleation and convection effects in protein crystal growth
The following activities are reported on: repartitioning of NaCl and protein impurities in lysozyme crystallization; dependence of lysozyme growth kinetics on step sources and impurities; facet morphology response to nonuniformities in nutrient and impurity supply; interactions in undersaturated and supersaturated lysozyme solutions; heterogeneity determination and purification of commercial hen egg white lysozyme; nonlinear response of layer growth dynamics in the mixed kinetics-bulk transport regime; development of a simultaneous multiangle light scattering technique; and x-ray topography of tetragonal lysozyme grown by the temperature-control technique.
Rosenberger, Franz↗
Interactions in Undersaturated and Supersaturated Lysozyme Solutions: Static and Dynamic Light Scattering Results
We have performed multiangle static and dynamic light scattering studies of lysozyme solutions at pH=4.7. The Rayleigh ratio R(sub g) and the collective diffusion coefficient D(sub c) were determined as function of both protein concentration c(sub p) and salt concentration c(sub s) with two different salts. At low salt concentrations, the scattering ratio K(sub c)(sub p)/R(sub theta) and diffusivity increased with protein concentration above the values for a monomeric, ideal solution. With increasing salt concentration this trend was eventually reversed. The hydrodynamic interactions of lysozyme in solution, extracted from the combination of static and dynamic scattering data, decreased significantly with increasing salt concentration. These observations reflect changes in protein interactions, in response to increased salt screening, from net repulsion to net attraction. Both salts had the same qualitative effect, but the quantitative behavior did not scale with the ionic strength of the solution. This indicates the presence of salt specific effects. At low protein concentrations, the slopes of K(sub c)(sub p)/R(sub theta) and D(sub c) vs c(sub p) were obtained. The dependence of the slopes on ionic strength was modeled using a DLVO potential for colloidal interactions of two spheres, with the net protein charge Z(sub e) and Hamaker constant A(sub H) as fitting parameters. The model reproduces the observed variations with ionic strength quite well. Independent fits to the static and dynamic data, however, led to different values of the fitting parameters. These and other shortcomings suggest that colloidal interaction models alone are insufficient to explain protein interactions in solutions.
Muschol, Martin↗
Determination of particle number concentration for biological particles using AF4-MALS: Dependencies on light scattering model and refractive index
Not Available
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Most of the rhamnogalacturonan-I from cultured Arabidopsis cell walls is covalently linked to arabinogalactan-protein
To characterize a purified rhamnogalacturonan-I (RG-I) containing both RG-I and arabinogalactan-protein (AGP) types of glycosyl residues, an AGP-specific β-1,3-galactanase that can cleave the AG backbone and release the AG sidechain was applied to this material. Carbohydrate analysis and NMR spectroscopy verified that the galactanase-released carbohydrate consists of RG-I covalently attached to the AG sidechain, proving a covalent linkage between RG-I and AGP. Size exclusion chromatography-multiangle light scattering-refractive index detection revealed that the galactanase-released RG-I has an average molecular weight of 41.6 kDa, which, together with the percentage of pectic sugars suggests an RG-I-AGP comprising one AGP covalently linked to two RG-I glycans. Carbohydrate analysis and NMR results of the RG-I-AGP, the galactanase-released glycans, and the RG lyase-released glycans demonstrated that the attached RG-I glycans are decorated with α-1,5-arabinan, β-1,4-galactan, xylose, and 4-O-Me-xylose sidechains. Furthermore, our measurement suggests that the covalently linked RG-I-AGP is the major component of the traditionally prepared RG-I.
59 BASIC BIOLOGICAL SCIENCES↗
Effects of Microheterogeneity in Hen Egg-White Lysozyme Crystallization
In earlier sodium dodecylsulfate polyacylamide gel electrophoresis (SDS-PAGE) studies it has been found that commonly utilized commercial hen egg-white lysozyme (HEWL) preparations contained 0.2-0.4 mol% covalently bound dimers. Here it is shown, using high-performance capillary electrophoresis (HPCE), that HEWL contains, in addition, two differently charged monomers in comparable amounts. To explore the origin of these microheterogeneous contaminants, purified HEWL (PHEWL) has been oxidized with hydrogen peroxide (0.0026-0.88 M) at various pH levels between 4.5 and 12.0. Optical densitometry of oxidized PHEWL (OHEWL) bands in SDS PAGE gels shows that hydrogen peroxide at 0.88 M in acetate buffer pH 4.5 increased the amount of dimers about sixfold over that in commercial HEWL. OHEWL had, in addition to one of the two monomer forms found in HEWL and PHEWL, three other differently charged monomer forms, each of them representing about 25% of the preparation. SDS-PAGE analysis of OHEWL yielded two closely spaced dimer bands with M(sub r) = 28 000 and 27 500. In addition, larger HEWL oligomers with M, = 1.7 million and 320 000 were detected by gel-filtration fast protein liquid chromatography with multiangle laser light scattering detection. Non-dissociating PAGE in large pore size gels at pH 4.5 confirmed the presence of these large oligomers in HEWL and OHEWL. Increased microheterogeneity resulted in substantial effects on crystal growth and nucleation rate. On addition of 10 microgram(exp -1) mg ml(exp -1) OHEWL to 32 mg ml(exp -1) HEWL crystallizing solutions, both the number and size of forming crystals decreased roughly proportionally to the concentration of the added microheterogeneity. The same effect was observed in HEWL solutions on addition of 0.03-9,3 M Hydrogen peroxide. Repartioning of the dimer during crystallzation aat various temperatures between 277 and 293 K was analyzed by SDS-PAGE. The crystals contained <= 25 % weight by volume of the oligomers in the solution, with no apparent temperature dependence of the repartioning.
Thomas, B. R.↗
Sensitivity of Multiangle, Multispectral Polarimetric Remote Sensing Over Open Oceans to Water-Leaving Radiance: Analyses of RSP Data Acquired During the MILAGRO Campaign
For remote sensing of aerosol over the ocean, there is a contribution from light scattered underwater. The brightness and spectrum of this light depends on the biomass content of the ocean, such that variations in the color of the ocean can be observed even from space. Rayleigh scattering by pure sea water, and Rayleigh-Gans type scattering by plankton, causes this light to be polarized with a distinctive angular distribution. To study the contribution of this underwater light polarization to multiangle, multispectral observations of polarized reflectance over ocean, we previously developed a hydrosol model for use in underwater light scattering computations that produces realistic variations of the ocean color and the underwater light polarization signature of pure sea water. In this work we review this hydrosol model, include a correction for the spectrum of the particulate scattering coefficient and backscattering efficiency, and discuss its sensitivity to variations in colored dissolved organic matter (CDOM) and in the scattering function of marine particulates. We then apply this model to measurements of total and polarized reflectance that were acquired over open ocean during the MILAGRO field campaign by the airborne Research Scanning Polarimeter (RSP). Analyses show that our hydrosol model faithfully reproduces the water-leaving contributions to RSP reflectance, and that the sensitivity of these contributions to Chlorophyll a concentration [Chl] in the ocean varies with the azimuth, height, and wavelength of observations. We also show that the impact of variations in CDOM on the polarized reflectance observed by the RSP at low altitude is comparable to or much less than the standard error of this reflectance whereas their effects in total reflectance may be substantial (i.e. up to >30%). Finally, we extend our study of polarized reflectance variations with [Chl] and CDOM to include results for simulated spaceborne observations.
Chowdhary, Jacek↗
Polarimetric sensitivity of light-absorbing carbonaceous aerosols over ocean: A theoretical assessment
Visible-light-absorbing carbonaceous aerosols within the boundary layer affect the radiance and polarization states of the radiation at the top of the atmosphere. Remote sensing from suborbital and satellite-based platforms utilizes these radiance and polarization signals to retrieve the key properties of these aerosols. Recent retrieval algorithms have shown a progressive trend toward including multi-angular and multi-spectral polarimetric measurements to produce better retrieval accuracy in comparison to those using measurements based on a single viewing angle. Here, we perform a theoretical investigation of the top of atmosphere (TOA) radiance-related reflectance factor (bidirectional reflectance factor (BRF)) and the two types of polarimetry-related factors (polarized bidirectional reflectance factor (pBRF) and the degree of linear polarization (DoLP)) for different types of atmospheric light-absorbing carbonaceous aerosols as a function of particle size distribution. We selected three polarimetric bands corresponding to those utilized by NASA's Airborne Multiangle SpectroPolarimetric Imager (AirMSPI)—near-UV (470 nm), visible (660 nm), and near-infrared (865 nm)—for our simulations which were performed over ocean surface using the successive order of scattering (SOS) algorithm coupled to a Lorenz-Mie aerosol optics model. The analysis of particle phase matrix elements indicates a close relationship between the angular dependencies of DoLP and associated phase matrix components at the shortest polarimetric band (470 nm). Using Jacobian analysis, we find that the radiance- and polarimetry-related reflectance factors of weakly light-absorbing aerosols, such as brown carbon, are more sensitive to changes in particle size and imaginary refractive index in comparison with those of black carbon, which is strongly light-absorbing. Our results suggest that the DoLP data could be used by future retrieval algorithms for reliably estimating microphysical properties of absorbing carbonaceous aerosols with imaginary refractive index less than 0.4.
refractive index↗
A preview of a microgravity laser light scattering instrument
The development of a versatile, miniature, modular light scattering instrument to be used in microgravity is described. The instrument will measure microscopic particles in the size range of thirty angstroms to above three microns. This modular instrument permits several configurations, each optimized for a particular experiment. In particular, a multiangle instrument will probably be mounted in a rack in the Space Shuttle and on the Space Station. It is possible that a Space Shuttle glove-box and a lap-top computer containing a correlator card can be used to perform a number of experiments and to demonstrate the technology needed for more elaborate investigations.
Meyer, W. V.↗
Development of a versatile laser light scattering instrument
A versatile laser light scattering (LLS) instrument is developed for use in microgravity to measure microscopic particles of 30 A to above 3 microns. Since it is an optical technique, LLS does not affect the sample being studied. A LLS instrument built from modules allows several configurations, each optimized for a particular experiment. The multiangle LLS instrument can be mounted in the rack in the Space Shuttle and on Space Station Freedom. It is possible that a Space Shuttle glove-box and a lap-top computer containing a correlator card can be used to perform a number of experiments and to demonstrate the technology needed for more elaborate investigations. This offers simple means of flying a great number of experiments without the additional requirements of full-scale flight hardware experiments.
Meyer, William V.↗
Theoretical and Observational Determination of Global and Regional Radiation Budget, Forcing and Feedbacks at the Top-of-Atmosphere and Surface
Report consists of: 1. List of accomplishments 2. List of publications 3. Abstracts of published or submitted papers and 4. Subject invention disclosure. The accomplishments of the grant listed are: 1. Improved the third-order turbulence closure in cloud resolving models to remove the liquid water oscillation. 2. Used the University of California-Los Angeles (UCLA) large-eddy simulation (LES) model to provide data for radiation transfer testing. 3. Revised shortwave k-distribution models based on HITRAN 2000. 4. Developed a gamma-weighted two-stream radiative transfer model for radiation budget estimate applications. 5. Estimated the effect of spherical geometry to the earth radiation budget. 6. Estimated top-of-atmosphere irradiance over snow and sea ice surfaces. 7. Estimated the aerosol direct radiative effect at the top of the atmosphere. 8. Estimated the top-of-atmosphere reflectance of the clear-sky molecular atmosphere over ocean. 9. Developed and validated new set of Angular Distribution Models for the CERES TRMM satellite instrument (tropical) 10. Developed and validated new set of Angular Distribution Models for the CERES Terra satellite instrument (global) 11. Quantified the top-of-atmosphere direct radiative effect of aerosols over global oceans from merged CERES and MODIS observations 12 Clarified the definition of TOA flux reference level for radiation budget studies 13. Developed new algorithm for unfaltering CERES measured radiances 14. Used multiangle POLDER measurements to produce narrowband angular distribution models and examine the effect of scene identification errors on TOA albedo estimates 15. Developed and validated a novel algorithm called the Multidirectional Reflectance Matching (MRM) model for inferring TOA albedos from ice clouds using multi-angle satellite measurements. 16. Developed and validated a novel algorithm called the Multidirectional Polarized Reflectance Matching (MPRM) model for inferring particle shapes from ice clouds using multi-angle polarized satellite measurements. 17. Developed 4 advanced light scattering models including the three-dimensional (3D) uniaxial perfectly matched layer (UPML) finite-difference time-domain (FDTD) model. 18. Develop sunglint in situ measurement and study reflectance distribution in the sunglint area. 19. Lead a balloon-borne radiometer TOA albedo validation effort. 20. Developed a CERES surface UVB, UVA, and UV index product.
Loeb, Norman G.↗