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1.  Multipoint Binding of the SLP-76 SH2 Domain to ADAP Is Critical for Oligomerization of SLP-76 Signaling Complexes in Stimulated T Cells 
Molecular and Cellular Biology  2013;33(21):4140-4151.
The adapter molecules SLP-76 and LAT play central roles in T cell activation by recruiting enzymes and other adapters into multiprotein complexes that coordinate highly regulated signal transduction pathways. While many of the associated proteins have been characterized, less is known concerning the mechanisms of assembly for these dynamic and potentially heterogeneous signaling complexes. Following T cell receptor (TCR) stimulation, SLP-76 is found in structures called microclusters, which contain many signaling complexes. Previous studies showed that a mutation to the SLP-76 C-terminal SH2 domain nearly abolished SLP-76 microclusters, suggesting that the SH2 domain facilitates incorporation of signaling complexes into microclusters. S. C. Bunnell, A. L. Singer, D. I. Hong, B. H. Jacque, M. S. Jordan, M. C. Seminario, V. A. Barr, G. A. Koretzky, and L. E. Samelson, Mol. Cell. Biol., 26:7155–7166, 2006). Using biophysical methods, we demonstrate that the adapter, ADAP, contains three binding sites for SLP-76, and that multipoint binding to ADAP fragments oligomerizes the SLP-76 SH2 domain in vitro. These results were complemented with confocal imaging and functional studies of cells expressing ADAP with various mutations. Our results demonstrate that all three binding sites are critical for SLP-76 microcluster assembly, but any combination of two sites will partially induce microclusters. These data support a model whereby multipoint binding of SLP-76 to ADAP facilitates the assembly of SLP-76 microclusters. This model has implications for the regulation of SLP-76 and LAT microclusters and, as a result, T cell signaling.
doi:10.1128/MCB.00410-13
PMCID: PMC3811887  PMID: 23979596
2.  Spatial imaging of Zn and other elements in Huanglongbing-affected grapefruit by synchrotron-based micro X-ray fluorescence investigation 
Journal of Experimental Botany  2014;65(4):953-964.
Huanglongbing (HLB) is a highly destructive, fast-spreading disease of citrus, causing substantial economic losses to the citrus industry worldwide. Nutrient levels and their cellular distribution patterns in stems and leaves of grapefruit were analysed after graft-inoculation with lemon scions containing ‘Candidatus Liberibacter asiaticus’ (Las), the heat-tolerant Asian type of the HLB bacterium. After 12 months, affected plants showed typical HLB symptoms and significantly reduced Zn concentrations in leaves. Micro-XRF imaging of Zn and other nutrients showed that preferential localization of Zn to phloem tissues was observed in the stems and leaves collected from healthy grapefruit plants, but was absent from HLB-affected samples. Quantitative analysis by using standard references revealed that Zn concentration in the phloem of veins in healthy leaves was more than 10 times higher than that in HLB-affected leaves. No significant variation was observed in the distribution patterns of other elements such as Ca in stems and leaves of grapefruit plants with or without graft-inoculation of infected lemon scions. These results suggest that reduced phloem transport of Zn is an important factor contributing to HLB-induced Zn deficiency in grapefruit. Our report provides the first in situ, cellular level visualization of elemental variations within the tissues of HLB-affected citrus.
doi:10.1093/jxb/ert450
PMCID: PMC3935563  PMID: 24420564
Candidatus Liberibacter asiaticus; distribution; Huanglongbing; grapefruit; phloem; micro-XRF; zinc.
3.  Synthesis, Characterization and Direct Intracellular Imaging of Ultrasmall and Uniform Glutathione-Coated Gold Nanoparticles 
Gold nanoparticles (AuNPs) with core sizes below 2 nm and compact ligand shells constitute versatile platforms for the development of novel reagents in nanomedicine. Due to their ultrasmall size, these AuNPs are especially attractive in applications requiring delivery to crowded intracellular spaces in the cytosol and nucleus. For eventual use in vivo, ultrasmall AuNPs should ideally be monodisperse, since small variations in size may affect how they interact with cells and behave in the body. Here we report the synthesis of ultrasmall, uniform 144-atom AuNPs protected by p-mercaptobenzoic acid (Au144(pMBA)60) followed by ligand exchange with glutathione (GSH). Quantitative scanning transmission electron microscopy (STEM) reveals that the resulting GSH-coated AuNPs (Au(GSH)) have a uniform mass distribution with cores that contain 134 gold atoms on average. Particle size dispersity is analyzed by analytical ultracentrifugation, giving a narrow distribution of apparent hydrodynamic diameter of 4.0 ± 0.6 nm. To evaluate the nanoparticles' intracellular fate, the cell penetrating peptide TAT is attached non-covalently to Au(GSH), which is confirmed by fluorescence quenching and isothermal titration calorimetry. HeLa cells are then incubated with both Au(GSH) and the Au(GSH)-TAT complex, and imaged without silver enhancement of the AuNPs in unstained thin sections by STEM. This imaging approach enables unbiased detection and quantification of individual ultrasmall nanoparticles and aggregates in the cytoplasm and nucleus of the cells.
doi:10.1002/smll.201200071
PMCID: PMC3715615  PMID: 22517616
gold nanoparticle; nanomedicine; cellular uptake; STEM; cell penetrating peptide
4.  A Bayesian Approach for Quantifying Trace Amounts of Antibody Aggregates by Sedimentation Velocity Analytical Ultracentrifugation 
The AAPS journal  2008;10(3):481-493.
Sedimentation velocity analytical ultracentrifugation (SV-AUC) has become an important tool for the characterization of the purity of protein therapeutics. The work presented here addresses a need for methods orthogonal to size-exclusion chromatography for ensuring the reliable quantitation of immunogenic oligomers, for example, in antibody preparations. Currently the most commonly used approach for SV-AUC analysis is the diffusion-deconvoluted sedimentation coefficient distribution c(s) method, previously developed by us as a general purpose technique and implemented in the software SEDFIT. In both practical and theoretical studies, different groups have reported a sensitivity of c(s) for trace oligomeric fractions well below the 1% level. In the present work we present a variant of c(s) designed for the purpose of trace detection, with customized Bayesian regularization. The original c(s) method relies on maximum entropy regularization providing the most parsimonious distribution consistent with the data. In the present paper, we use computer simulations of an antibody system as example to demonstrate that the standard maximum entropy regularization, due to its design, leads to a theoretical lower limit for the detection of oligomeric traces and a consistent underestimate of the trace populations by ∼0.1% (dependent on the level of regularization). This can be overcome with a recently developed Bayesian extension of c(s) (Biomacromolecules (2007), 8, 2011-2024), utilizing the known regions of sedimentation coefficients for the monomer and oligomers of interest as prior expectation for the peak positions in the distribution. We show that this leads to more clearly identifiable and consistent peaks and lower theoretical limits of quantization by approximately an order of magnitude for some experimental conditions. Implications for the experimental design of SV-AUC and practical detection limits are discussed.
doi:10.1208/s12248-008-9058-z
PMCID: PMC2696691  PMID: 18814037
sedimentation velocity; analytical ultracentrifugation; trace aggregates; hydrodynamic separation; size-distribution; Bayesian analysis
6.  From plant surface to plant metabolism: the uncertain fate of foliar-applied nutrients 
The application of agrochemical sprays to the aerial parts of crop plants is an important agricultural practice world-wide. While variable effectiveness is often seen in response to foliar treatments, there is abundant evidence showing the beneficial effect of foliar fertilizers in terms of improving the metabolism, quality, and yields of crops. This mini-review is focused on the major bottlenecks associated with the uptake and translocation of foliar-applied nutrient solutions. A better understanding of the complex scenario surrounding the ultimate delivery of foliar-applied nutrients to sink cells and organs is essential for improving the effectiveness and performance of foliar fertilizers.
doi:10.3389/fpls.2013.00289
PMCID: PMC3728483  PMID: 23914198
apoplast; cuticle; foliar fertilizers; foliar uptake; leaf; foliar sprays; nutrient mobility; plant surfaces
7.  Analysis of high-affinity assembly for AMPA receptor amino-terminal domains 
The Journal of General Physiology  2012;139(5):371-388.
Analytical ultracentrifugation (AUC) and steady-state fluorescence anisotropy were used to measure the equilibrium dissociation constant (Kd) for formation of dimers by the amino-terminal domains (ATDs) of the GluA2 and GluA3 subtypes of AMPA receptor. Previous reports on GluA2 dimerization differed in their estimate of the monomer–dimer Kd by a 2,400-fold range, with no consensus on whether the ATD forms tetramers in solution. We find by sedimentation velocity (SV) analysis performed using absorbance detection a narrow range of monomer–dimer Kd values for GluA2, from 5 to 11 nM for six independent experiments, with no detectable formation of tetramers and no effect of glycosylation or the polypeptide linker connecting the ATD and ligand-binding domains; for GluA3, the monomer–dimer Kd was 5.6 µM, again with no detectable tetramer formation. For sedimentation equilibrium (SE) experiments, a wide range of Kd values was obtained for GluA2, from 13 to 284 nM, whereas for GluA3, the Kd of 3.1 µM was less than twofold different from the SV value. Analysis of cell contents after the ∼1-week centrifuge run by silver-stained gels revealed low molecular weight GluA2 breakdown products. Simulated data for SE runs demonstrate that the apparent Kd for GluA2 varies with the extent of proteolysis, leading to artificially high Kd values. SV experiments with fluorescence detection for GluA2 labeled with 5,6-carboxyfluorescein, and fluorescence anisotropy measurements for GluA2 labeled with DyLight405, yielded Kd values of 5 and 11 nM, consistent with those from SV with absorbance detection. However, the sedimentation coefficients measured by AUC using absorbance and fluorescence systems were strikingly different, and for the latter are not consistent with hydrodynamic protein models. Thus, for unknown reasons, the concentration dependence of sedimentation coefficients obtained with fluorescence detection SV may be unreliable, limiting the usefulness of this technique for quantitative analysis.
doi:10.1085/jgp.201210770
PMCID: PMC3343374  PMID: 22508847
8.  The molecular refractive function of lens γ-crystallins 
Journal of molecular biology  2011;411(3):680-699.
γ-crystallins constitute the major protein component in the nucleus of the vertebrate eye lens. Present at very high concentrations, they exhibit extreme solubility and thermodynamic stability to prevent scattering of light and the formation of cataracts. However, functions beyond this structural role have remained mostly unclear. Here, we calculate molecular refractive index increments of crystallins. We show that all lens γ-crystallins have evolved a significantly elevated molecular refractive index increment, which is far above those of most proteins, including non-lens members of the βγ-crystallin family from different species. The same trait has evolved in parallel in crystallins of different phyla, including in the S-crystallins of cephalopods. A high refractive index increment can lower the crystallin concentration required to achieve a suitable refractive power of the lens, and thereby reduce their propensity to aggregate and form cataract. To produce a significant increase of the refractive index increment, a substantial global shift in the amino acid composition is required, which can naturally explain the highly unusual amino acid composition of γ-crystallins and their functional homologues. This function provides a new perspective for interpreting their molecular structure.
doi:10.1016/j.jmb.2011.06.007
PMCID: PMC3146585  PMID: 21684289
crystallin; protein structure function; protein refractive index; excluded volume
9.  The Boundary Structure in the Analysis of Reversibly Interacting Systems by Sedimentation Velocity 
Methods (San Diego, Calif.)  2011;54(1):16-30.
Sedimentation velocity (SV) experiments of heterogeneous interacting systems exhibit characteristic boundary structures that can usually be very easily recognized and quantified. For slowly interacting systems, the boundaries represent concentrations of macromolecular species and they can be interpreted directly with population models based solely on the mass action law. For fast reactions, migration and chemical reactions are coupled, and different, but equally easily discernable boundary structures appear. However, these features have not been commonly utilized for data analysis, for the lack of an intuitive and computationally simple model. The recently introduced effective particle theory (EPT) provides a suitable framework. Here, we review the motivation and theoretical basis of EPT, and explore practical aspects for its application. We introduce an EPT-based design tool for SV experiments of heterogeneous interactions in the software SEDPHAT. As a practical tool for the first step of data analysis, we describe how the boundary resolution can be further improved in c(s) with a Bayesian adjustment of maximum entropy regularization to the case of heterogeneous interactions between molecules that have been previously studied separately. This can facilitate extracting the characteristic boundary features by integration of c(s) and their assembly into isotherms as a function of total loading concentrations, which are fitted with EPT in a second stage. Methods for addressing concentration errors in isotherms are discussed. Finally, in an experimental model system of alpha-chymotrypsin interacting with soybean trypsin inhibitor, we show that EPT provides an excellent description of the experimental sedimentation boundary structure of fast interacting systems.
doi:10.1016/j.ymeth.2011.01.010
PMCID: PMC3090504  PMID: 21315155
10.  Density Contrast Sedimentation Velocity for the Determination of Protein Partial-Specific Volumes 
PLoS ONE  2011;6(10):e26221.
The partial-specific volume of proteins is an important thermodynamic parameter required for the interpretation of data in several biophysical disciplines. Building on recent advances in the use of density variation sedimentation velocity analytical ultracentrifugation for the determination of macromolecular partial-specific volumes, we have explored a direct global modeling approach describing the sedimentation boundaries in different solvents with a joint differential sedimentation coefficient distribution. This takes full advantage of the influence of different macromolecular buoyancy on both the spread and the velocity of the sedimentation boundary. It should lend itself well to the study of interacting macromolecules and/or heterogeneous samples in microgram quantities. Model applications to three protein samples studied in either H2O, or isotopically enriched H218O mixtures, indicate that partial-specific volumes can be determined with a statistical precision of better than 0.5%, provided signal/noise ratios of 50–100 can be achieved in the measurement of the macromolecular sedimentation velocity profiles. The approach is implemented in the global modeling software SEDPHAT.
doi:10.1371/journal.pone.0026221
PMCID: PMC3197611  PMID: 22028836
11.  Climate Change Affects Winter Chill for Temperate Fruit and Nut Trees 
PLoS ONE  2011;6(5):e20155.
Background
Temperate fruit and nut trees require adequate winter chill to produce economically viable yields. Global warming has the potential to reduce available winter chill and greatly impact crop yields.
Methodology/Principal Findings
We estimated winter chill for two past (1975 and 2000) and 18 future scenarios (mid and end 21st century; 3 Global Climate Models [GCMs]; 3 greenhouse gas emissions [GHG] scenarios). For 4,293 weather stations around the world and GCM projections, Safe Winter Chill (SWC), the amount of winter chill that is exceeded in 90% of all years, was estimated for all scenarios using the “Dynamic Model” and interpolated globally. We found that SWC ranged between 0 and about 170 Chill Portions (CP) for all climate scenarios, but that the global distribution varied across scenarios. Warm regions are likely to experience severe reductions in available winter chill, potentially threatening production there. In contrast, SWC in most temperate growing regions is likely to remain relatively unchanged, and cold regions may even see an increase in SWC. Climate change impacts on SWC differed quantitatively among GCMs and GHG scenarios, with the highest GHG leading to losses up to 40 CP in warm regions, compared to 20 CP for the lowest GHG.
Conclusions/Significance
The extent of projected changes in winter chill in many major growing regions of fruits and nuts indicates that growers of these commodities will likely experience problems in the future. Mitigation of climate change through reductions in greenhouse gas emissions can help reduce the impacts, however, adaption to changes will have to occur. To better prepare for likely impacts of climate change, efforts should be undertaken to breed tree cultivars for lower chilling requirements, to develop tools to cope with insufficient winter chill, and to better understand the temperature responses of tree crops.
doi:10.1371/journal.pone.0020155
PMCID: PMC3101230  PMID: 21629649
12.  Direct interaction of the mouse cytomegalovirus m152/gp40 immunoevasin with RAE-1 isoforms† 
Biochemistry  2010;49(11):2443-2453.
Cytomegaloviruses (CMVs) are ubiquitous species-specific viruses that establish acute, persistent, and latent infections. Both human and mouse CMVs encode proteins that inhibit the activation of natural killer (NK) cells by downregulating cellular ligands for the NK cell activating receptor, NKG2D. The MCMV glycoprotein m152/gp40 downregulates the surface expression of RAE-1 in order to avoid NK cell control in vivo. So far it is unclear if there is a direct interaction between m152 and RAE-1, and if so, if m152 interacts differentially with the five identified RAE-1 isoforms, which are expressed as two groups in MCMV-susceptible or resistant mouse strains. To address these questions, we expressed and purified the extracellular domains of RAE-1 and m152, and performed size exclusion chromatography binding assays as well as analytical ultracentrifugation and isothermal titration calorimetry to characterize these interactions quantitatively. We further evaluated the role of full-length and naturally glycosylated m152 and RAE-1 in cotransfected HEK293T cells. Our results confirmed that m152 binds RAE-1 directly, relatively tightly (Kd < 5 μM), and with 1:1 stoichiometry. The binding is quantitatively different depending on particular RAE-1 isoforms, corresponding to the susceptibility to downregulation by m152. A PLWY motif found in RAE-1β, although contributing to its affinity for m152, does not influence the affinity of RAE-1 γ or δ, suggesting that other differences contribute to the RAE-1/m152 interaction. Molecular modeling of the different RAE-1 isoforms suggests a potential site for the m152 interaction.
doi:10.1021/bi902130j
PMCID: PMC2840211  PMID: 20166740
13.  A global analysis of the comparability of winter chill models for fruit and nut trees 
Many fruit and nut trees must fulfill a chilling requirement to break their winter dormancy and resume normal growth in spring. Several models exist for quantifying winter chill, and growers and researchers often tacitly assume that the choice of model is not important and estimates of species chilling requirements are valid across growing regions. To test this assumption, Safe Winter Chill (the amount of winter chill that is exceeded in 90% of years) was calculated for 5,078 weather stations around the world, using the Dynamic Model [in Chill Portions (CP)], the Chilling Hours (CH) Model and the Utah Model [Utah Chill Units (UCU)]. Distributions of the ratios between different winter chill metrics were mapped on a global scale. These ratios should be constant if the models were strictly proportional. Ratios between winter chill metrics varied substantially, with the CH/CP ratio ranging between 0 and 34, the UCU/CP ratio between −155 and +20 and the UCU/CH ratio between −10 and +5. The models are thus not proportional, and chilling requirements determined in a given location may not be valid elsewhere. The Utah Model produced negative winter chill totals in many Subtropical regions, where it does not seem to be useful. Mean annual temperature and daily temperature range influenced all winter chill ratios, but explained only between 12 and 27% of the variation. Data on chilling requirements should always be amended with information on the location and experimental conditions of the study in which they were determined, ideally including site-specific conversion factors between winter chill models. This would greatly facilitate the transfer of such information across growing regions, and help prepare growers for the impact of climate change.
doi:10.1007/s00484-010-0352-y
PMCID: PMC3077742  PMID: 20730614
Chilling Hours; Chilling requirement; Chill Portions; Dynamic Model; Utah Model; Winter chill
14.  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 vacuum
Not 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.
doi:10.3791/1530
PMCID: PMC3157848  PMID: 19893484
15.  On the analysis of sedimentation velocity in the study of protein complexes 
European Biophysics Journal   2009;38(8):1079-1099.
Sedimentation velocity analytical ultracentrifugation has experienced a significant transformation, precipitated by the possibility of efficiently fitting Lamm equation solutions to the experimental data. The precision of this approach depends on the ability to account for the imperfections of the experiment, both regarding the sample and the instrument. In the present work, we explore in more detail the relationship between the sedimentation process, its detection, and the model used in the mathematical data analysis. We focus on configurations that produce steep and fast-moving sedimentation boundaries, such as frequently encountered when studying large multi-protein complexes. First, as a computational tool facilitating the analysis of heterogeneous samples, we introduce the strategy of partial boundary modeling. It can simplify the modeling by restricting the direct boundary analysis to species with sedimentation coefficients in a predefined range. Next, we examine factors related to the experimental detection, including the magnitude of optical aberrations generated by out-of-focus solution columns at high protein concentrations, the relationship between the experimentally recorded signature of the meniscus and the meniscus parameter in the data analysis, and the consequences of the limited radial and temporal resolution of the absorbance optical scanning system. Surprisingly, we find that large errors can be caused by the finite scanning speed of the commercial absorbance optics, exceeding the statistical errors in the measured sedimentation coefficients by more than an order of magnitude. We describe how these effects can be computationally accounted for in SEDFIT and SEDPHAT.
doi:10.1007/s00249-009-0514-1
PMCID: PMC2755746  PMID: 19644686
Analytical ultracentrifugation; Hydrodynamics; Direct boundary modeling; Lamm equation
16.  A Bayesian Approach for Quantifying Trace Amounts of Antibody Aggregates by Sedimentation Velocity Analytical Ultracentrifugation 
The AAPS Journal  2008;10(3):481-493.
Sedimentation velocity analytical ultracentrifugation (SV-AUC) has become an important tool for the characterization of the purity of protein therapeutics. The work presented here addresses a need for methods orthogonal to size-exclusion chromatography for ensuring the reliable quantitation of immunogenic oligomers, for example, in antibody preparations. Currently the most commonly used approach for SV-AUC analysis is the diffusion-deconvoluted sedimentation coefficient distribution c(s) method, previously developed by us as a general purpose technique and implemented in the software SEDFIT. In both practical and theoretical studies, different groups have reported a sensitivity of c(s) for trace oligomeric fractions well below the 1% level. In the present work we present a variant of c(s) designed for the purpose of trace detection, with customized Bayesian regularization. The original c(s) method relies on maximum entropy regularization providing the most parsimonious distribution consistent with the data. In the present paper, we use computer simulations of an antibody system as example to demonstrate that the standard maximum entropy regularization, due to its design, leads to a theoretical lower limit for the detection of oligomeric traces and a consistent underestimate of the trace populations by ∼0.1% (dependent on the level of regularization). This can be overcome with a recently developed Bayesian extension of c(s) (Brown et al., Biomacromolecules, 8:2011–2024, 2007), utilizing the known regions of sedimentation coefficients for the monomer and oligomers of interest as prior expectation for the peak positions in the distribution. We show that this leads to more clearly identifiable and consistent peaks and lower theoretical limits of quantization by approximately an order of magnitude for some experimental conditions. Implications for the experimental design of SV-AUC and practical detection limits are discussed.
doi:10.1208/s12248-008-9058-z
PMCID: PMC2696691  PMID: 18814037
analytical ultracentrifugation; Bayesian analysis; hydrodynamic separation; sedimentation velocity; size-distribution; trace aggregates
17.  A new adaptive grid-size algorithm for the simulation of sedimentation velocity profiles in analytical ultracentrifugation 
Computer physics communications  2008;178(2):105-120.
Analytical ultracentrifugation allows one to measure in real-time the concentration gradients arising from the application of a centrifugal force to macromolecular mixtures in solution. In the last decade, the ability to efficiently solve the partial differential equation governing the ultracentrifugal sedimentation and diffusion process, the Lamm equation, has spawned significant progress in the application of sedimentation velocity analytical ultracentrifugation for the study of biological macromolecules, for example, the characterization of protein oligomeric states and the study of reversible multi-protein complexes in solution. The present work describes a numerical algorithm that can provide an improvement in accuracy or efficiency over existing algorithms by more than one order of magnitude, and thereby greatly facilitate the practical application of sedimentation velocity analysis, in particular, for the study of multi-component macromolecular mixtures. It is implemented in the public domain software SEDFIT for the analysis of experimental data.
doi:10.1016/j.cpc.2007.08.012
PMCID: PMC2267755  PMID: 18196178
protein interactions; analytical ultracentrifugation; finite element methods; size distributions
18.  Two Interferons Alpha Influence Each Other During Their Interaction With the Extracellular Domain of Human Type I Interferon Receptor Subunit 2 
Biochemistry  2007;46(50):14638-14649.
The interaction between two human interferons alpha (IFN-αs) and the extracellular (EC) domain of human type I IFN receptor subunit 2 (IFNAR2) was analyzed. Previous experiments using Daudi cells showed that IFN-α21b and some IFN-α hybrids (made from IFN-α2c and 21b) competed poorly for the IFN-α2b binding site. This study examined the causes of the poor competition between these IFN-αs. IFN-α2c and the IFN hybrid CM3 {IFN-α21b(1-75)(81-95)/IFN-α2c(76-80)(96-166), Y86K} were selected for this study based on their cell binding and biological properties. Competitive binding ELISA, native electrophoresis followed by Western blot, electrospray ionization mass spectrometry (ESI-MS), surface plasmon resonance biosensor (SPR) analysis, as well as neutralization of antiproliferative activities on Daudi cells in the presence of soluble IFNAR2-EC show evidence that each of the described IFN-α subtypes affected the binding of the other IFN-α to IFNAR2-EC by affecting the stability of the complex, i.e. dissociation of the complex. Moreover, native electrophoresis with different IFNAR2-EC mutants showed that IFN-α2c and CM3 utilize different amino acids in the binding domain of IFNAR2-EC. In addition to that, analytical ultracentrifugation (AUC) revealed differences in the oligomeric state of the two studied interferons. Our results demonstrated that two individual IFN-αs interact differentially with IFNAR2-EC and influence each other during this interaction. This study contributes to the understanding of the mutual interaction between multiple IFN-α subtypes during the competition for binding to the receptor.
doi:10.1021/bi7012036
PMCID: PMC2570179  PMID: 18027911
19.  Using prior knowledge in the determination of macromolecular size distributions by analytical ultracentrifugation 
Biomacromolecules  2007;8(6):2011-2024.
Analytical ultracentrifugation has reemerged as a widely used tool for the study of ensembles of biological macromolecules to understand, for example, their size-distribution and interactions in free solution. Such information can be obtained from the mathematical analysis of the concentration and signal gradients across the solution column and their evolution in time generated as a result of the gravitational force. In sedimentation velocity analytical ultracentrifugation, this analysis is frequently conducted using high resolution, diffusion-deconvoluted sedimentation coefficient distributions. They are based on Fredholm integral equations, which are ill-posed unless stabilized by regularization. In many fields, maximum entropy and Tikhonov-Phillips regularization are well-established and powerful approaches that calculate the most parsimonious distribution consistent with the data and prior knowledge, in accordance with Occam’s razor. In the implementations available in analytical ultracentrifugation, to date, the basic assumption implied is that all sedimentation coefficients are equally likely, and that the information retrieved should be condensed to the least amount possible. Frequently, however, more detailed distributions would be warranted by specific detailed prior knowledge on the macromolecular ensemble under study, such as, the expectation of the sample to be monodisperse or paucidisperse, or the expectation for the migration to establish a bimodal sedimentation pattern based on Gilbert & Jenkins’ theory for the migration of chemically reacting systems. So far, such prior knowledge has remained largely unused in the calculation of the sedimentation coefficient or molecular weight distributions, or was only applied as constraints. In the present paper, we examine how prior expectations can be built directly into the computational data analysis, conservatively in a way that honors the complete information of the experimental data, whether or not consistent with the prior expectation. Consistent with analogous results in other fields, we find that use of available prior knowledge can have a dramatic effect on the resulting molecular weight, sedimentation coefficient and size-and-shape distributions, and significantly increase both their sensitivity and resolution. Further, the use of multiple alternative priors allows to probe the range of possible interpretations consistent with the data.
doi:10.1021/bm070193j
PMCID: PMC1994561  PMID: 17521163
Analytical ultracentrifugation; sedimentation velocity; sedimentation equilibrium; maximum entropy; Fredholm integral equations; size-distribution; regularization
20.  Studying multi-site binary and ternary protein interactions by global analysis of isothermal titration calorimetry data in SEDPHAT: Application to adaptor protein complexes in cell signaling 
Multi-site interactions and the formation of ternary or higher-order protein complexes are ubiquitous features of protein interactions. Cooperativity between different ligands is a hallmark for information transfer, and frequently critical for the biological function. We describe a new computational platform for the global analysis of isothermal titration calorimetry (ITC) data for the study of binary and ternary multi-site interactions, implemented as part of the public domain multi-method analysis software SEDPHAT. The global analysis of titrations performed in different orientations was explored, and the potential for unraveling cooperativity parameters in multi-site interactions was assessed in theory and experiment. To demonstrate the practical potential and limitations of global analyses of ITC titrations for the study of cooperative multi-protein interactions, we have examined the interactions of three proteins critical for signal transduction after T-cell activation, LAT, Grb2, and SOS. We have shown previously that multivalent interactions between these three molecules promote the assembly of large multi-protein complexes important for T cell receptor activation. By global analysis of the heats of binding observed in sets of ITC injections in different orientations, which allowed us to follow the formation of binary and ternary complexes, we observed negative and positive cooperativity that may be important to control the pathway of assembly and disassembly of adaptor protein particles.
doi:10.1110/ps.062558507
PMCID: PMC1794685  PMID: 17192587
ITC; multi-protein complexes; cooperativity; signal transduction; adaptor protein; SOS1; LAT; Grb2; protein interactions; reversible associations; ITC (isothermal titration calorimetry); ΔH (enthalpy change of binding); KA (binding association constant); LAT (linker for activation of T-cells); Sos1 (son of sevenless homolog 1); Grb2 (growth factor receptor-bound protein 2); CAII (bovine carbonic anhydrase II); CBS (4-carboxybenzenesulfonamide)

Results 1-20 (20)