Tools for the Quantitative Analysis of Sedimentation Boundaries Detected by Fluorescence Optical Analytical Ultracentrifugation

Fluorescence optical detection in sedimentation velocity analytical ultracentrifugation allows the study of macromolecules at nanomolar concentrations and below. This has significant promise, for example, for the study of systems of high-affinity protein interactions. Here we describe adaptations of the direct boundary modeling analysis approach implemented in the software SEDFIT that were developed to accommodate unique characteristics of the confocal fluorescence detection system. These include spatial gradients of signal intensity due to scanner movements out of the plane of rotation, temporal intensity drifts due to instability of the laser and fluorophores, and masking of the finite excitation and detection cone by the sample holder. In an extensive series of experiments with enhanced green fluorescent protein ranging from low nanomolar to low micromolar concentrations, we show that the experimental data provide sufficient information to determine the parameters required for first-order approximation of the impact of these effects on the recorded data. Systematic deviations of fluorescence optical sedimentation velocity data analyzed using conventional sedimentation models developed for absorbance and interference optics are largely removed after these adaptations, resulting in excellent fits that highlight the high precision of fluorescence sedimentation velocity data, thus allowing a more detailed quantitative interpretation of the signal boundaries that is otherwise not possible for this system.     

Analytical Ultracentrifugation Sedimentation Velocity for the Characterization of Detergent-Solubilized Membrane Proteins Ca++-ATPase and ExbB

We have investigated the potential of new methods of analysis of sedimentation velocity (SV) analytical ultracentrifugation (AUC) for the characterization of detergent-solubilized membrane proteins. We analyze the membrane proteins Ca⁺⁺-ATPase and ExbB solubilized with DDM (dodecyl-β-d-maltoside). SV is extremely well suited for characterizing sample heterogeneity. DDM micelles (s _20w?=?3.1 S) and complexes (Ca⁺⁺-ATPase: s _20w?=?7.3 S; ExbB: s _20w?=?4 S) are easily distinguished. Using different detergent and protein concentrations, SV does not detect any evidence of self-association for the two proteins. An estimate of bound detergent of 0.9 g/g for Ca⁺⁺-ATPase and 1.5 g/g for ExbB is obtained from the combined analysis of SV profiles obtained using absorbance and interference optics. Combining s _20w with values of the hydrodynamic radius, R _s?=?5.5 nm for Ca⁺⁺-ATPase or R _s?=?3.4 nm for ExbB, allows the determination of buoyant molar masses, M _b. In view of their M _b and composition, Ca⁺⁺-ATPase and ExbB are monomers in our experimental conditions. We conclude that one of the main advantages of SV versus other techniques is the possibility to ascertain the homogeneity of the samples and to focus on a given complex even in the presence of other impurities or aggregates. The relative rapidity of SV measurements also allows experiments on unstable samples.     

Molecular Weights of CTLA-4 and CD80 by Sedimentation Equilibrium Ultracentrifugation

Proteins that are heavily glycosylated pose unique challenges in their biophysical characterization. In particular, molecular weight analysis is exacerbated by such glycosylation. For example, glyoproteins are refractory to careful mass spectrum analysis and often give anomalous retention times using size exclusion chromatography. We combine several approaches to characterize the molecular weights of the extracellular domains of the glycoproteins CTLA-4 and CD80 using carbohydrate analysis, electrospray mass spectrometry, size exclusion chromatography, and analytical ultracentrifugation. In addition, we have applied a method described previously, using sedimentation equilibrium analysis to calculate the contribution of carbohydrates to the molecular masses of CTLA-4 and CD80. It is important to understand the oligomeric states of these protein domains because the interaction between these lymphocyte receptors plays an important costimulatory role in the Th-cell antigenic response. It is thought that extracellular interactions between these receptors may regulate both the self-association of these receptor proteins and the oligomeric state of the heterocomplex; this regulation has important consequences for potentiating the signaling mechanism between Th-cells and antigen-presenting cells.     

Binding Affinities Controlled by Shifting Conformational Equilibria: Opportunities and Limitations

Conformational selection is an established mechanism in molecular recognition. Despite its power to explain binding events, it is hardly used in protein/ligand design to modulate molecular recognition. Here, we explore the opportunities and limitations of design by conformational selection. Using appropriate thermodynamic cycles, our approach predicts the effects of a conformational shift on binding affinity and also allows one to disentangle the effects induced by a conformational shift from other effects influencing the binding affinity. The method is assessed and applied to explain the contribution of a conformational shift on the binding affinity of six ubiquitin mutants showing different conformational shifts in six different complexes.     

Engineering of Bispecific Affinity Proteins with High Affinity for ERBB2 and Adaptable Binding to Albumin

The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein.     

Analysis of Antibody Aggregate Content at Extremely High Concentrations Using Sedimentation Velocity with a Novel Interference Optics

Monoclonal antibodies represent the most important group of protein-based biopharmaceuticals. During formulation, manufacturing, or storage, antibodies may suffer post-translational modifications altering their physical and chemical properties. Such induced conformational changes may lead to the formation of aggregates, which can not only reduce their efficiency but also be immunogenic. Therefore, it is essential to monitor the amount of size variants to ensure consistency and quality of pharmaceutical antibodies. In many cases, antibodies are formulated at very high concentrations > 50 g/L, mostly along with high amounts of sugar-based excipients. As a consequence, all routine aggregation analysis methods, such as size-exclusion chromatography, cannot monitor the size distribution at those original conditions, but only after dilution and usually under completely different solvent conditions. In contrast, sedimentation velocity (SV) allows to analyze samples directly in the product formulation, both with limited sample-matrix interactions and minimal dilution. One prerequisite for the analysis of highly concentrated samples is the detection of steep concentration gradients with sufficient resolution: Commercially available ultracentrifuges are not able to resolve such steep interference profiles. With the development of our Advanced Interference Detection Array (AIDA), it has become possible to register interferograms of solutions as highly concentrated as 150 g/L. The other major difficulty encountered at high protein concentrations is the pronounced non-ideal sedimentation behavior resulting from repulsive intermolecular interactions, for which a comprehensive theoretical modelling has not yet been achieved. Here, we report the first SV analysis of highly concentrated antibodies up to 147 g/L employing the unique AIDA ultracentrifuge. By developing a consistent experimental design and data fit approach, we were able to provide a reliable estimation of the minimum content of soluble aggregates in the original formulations of two antibodies. Limitations of the procedure are discussed.     

Protistan Grazing Analysis by Flow Cytometry Using Prey Labeled by In Vivo Expression of Fluorescent Proteins

Selective grazing by protists can profoundly influence bacterial community structure, and yet direct, quantitative observation of grazing selectivity has been difficult to achieve. In this investigation, flow cytometry was used to study grazing by the marine heterotrophic flagellate Paraphysomonas imperforata on live bacterial cells genetically modified to express the fluorescent protein markers green fluorescent protein (GFP) and red fluorescent protein (RFP). Broad-host-range plasmids were constructed that express fluorescent proteins in three bacterial prey species, Escherichia coli, Enterobacter aerogenes, and Pseudomonas putida. Micromonas pusilla, an alga with red autofluorescence, was also used as prey. Predator-prey interactions were quantified by using a FACScan flow cytometer and analyzed by using a Perl program described here. Grazing preference of P. imperforata was influenced by prey type, size, and condition. In competitive feeding trials, P. imperforata consumed algal prey at significantly lower rates than FP (fluorescent protein)-labeled bacteria of similar or different size. Within-species size selection was also observed, but only for P. putida, the largest prey species examined; smaller cells of P. putida were grazed preferentially. No significant difference in clearance rate was observed between GFP- and RFP-labeled strains of the same prey species or between wild-type and GFP-labeled strains. In contrast, the common chemical staining method, 5-(4,6-dichloro-triazin-2-yl)-amino fluorescein hydrochloride, depressed clearance rates for bacterial prey compared to unlabeled or RFP-labeled cells.     

Quantifying Protein-Protein Interactions of Peripheral Membrane Proteins by Fluorescence Brightness Analysis

Fluorescently labeled proteins that are found both in the cytoplasm and at the plasma membrane, such as peripheral membrane proteins, create stratified fluorescent layers that present a challenging environment for brightness studies with fluorescence fluctuation spectroscopy. The geometry of each layer along with fluorescence and brightness contributions from adjacent layers generates a convoluted raw brightness that conceals the underlying brightness of each individual layer. Because the brightness at a layer establishes the oligomeric state of the fluorescently labeled protein at said layer, we developed a method that connects the experimental raw brightness with the physical brightness at each layered compartment. The technique determines the oligomerization in each compartment from an axial intensity scan through the sample, followed by a fluorescence fluctuation spectroscopy measurement at each layer. We experimentally verify the technique with H-Ras-EGFP as a model system and determine its oligomeric state at both the plasma membrane and in the cytoplasm. Furthermore, we study the oligomerization of the Gag matrix domain of Human T-lymphotropic virus Type 1. The matrix domain targets the Gag polyprotein to the plasma membrane where, subsequently, viral assembly occurs. We determine the oligomerization of matrix in the cytoplasm and observe the onset of protein-protein interactions at the membrane. These observations shed light on the early assembly steps of the retrovirus.     

Quantifying Protein-Protein Interactions of Peripheral Membrane Proteins by Fluorescence Brightness Analysis

Fluorescently labeled proteins that are found both in the cytoplasm and at the plasma membrane, such as peripheral membrane proteins, create stratified fluorescent layers that present a challenging environment for brightness studies with fluorescence fluctuation spectroscopy. The geometry of each layer along with fluorescence and brightness contributions from adjacent layers generates a convoluted raw brightness that conceals the underlying brightness of each individual layer. Because the brightness at a layer establishes the oligomeric state of the fluorescently labeled protein at said layer, we developed a method that connects the experimental raw brightness with the physical brightness at each layered compartment. The technique determines the oligomerization in each compartment from an axial intensity scan through the sample, followed by a fluorescence fluctuation spectroscopy measurement at each layer. We experimentally verify the technique with H-Ras-EGFP as a model system and determine its oligomeric state at both the plasma membrane and in the cytoplasm. Furthermore, we study the oligomerization of the Gag matrix domain of Human T-lymphotropic virus Type 1. The matrix domain targets the Gag polyprotein to the plasma membrane where, subsequently, viral assembly occurs. We determine the oligomerization of matrix in the cytoplasm and observe the onset of protein-protein interactions at the membrane. These observations shed light on the early assembly steps of the retrovirus.     

Resolution and Properties of Two High Affinity Cyclic Adenosine 3':5'-monophosphate-Binding Proteins from Wheat Germ

A high affinity cAMP-binding protein (cABP II) was purified to homogeneity from wheat germ. The apparent molecular weight of cABP II, as determined from gel exclusion chromatography, is 5.2 × 10⁵ (at low ionic strength) and 2.8 × 10⁵ (at high ionic strength). One polypeptide subunit (molecular weight, 80,000) was resolved by polyacrylamide gel electrophoresis of cABP II under subunit dissociating conditions. The purification protocol employed resolves cABP II from a distinct, less acidic cAMP-binding protein (cABP I). The K_d values for cAMP are about 10⁻⁶ molar and 10⁻⁷ molar for cABP II and cABP I, respectively. The cAMP-binding sites of cABP I and cABP II have a marked adenine-analog specificity, binding adenine, adenosine, adenine-derived nucleosides and nucleotides and a variety of adenine derivatives having cytokinin activity. While cABP II is phosphorylated in reactions catalyzed by endogenous protein kinases, there is no evidence for modulation of these cABP II-protein kinase interactions by cAMP.     

High Affinity Binding of Indium and Ruthenium Ions by Gastrins

The peptide hormone gastrin binds two ferric ions with high affinity, and iron binding is essential for the biological activity of non-amidated forms of the hormone. Since gastrins act as growth factors in gastrointestinal cancers, and as peptides labelled with Ga and In isotopes are increasingly used for cancer diagnosis, the ability of gastrins to bind other metal ions was investigated systematically by absorption spectroscopy. The coordination structures of the complexes were characterized by extended X-ray absorption fine structure (EXAFS) spectroscopy. Changes in the absorption of gastrin in the presence of increasing concentrations of Ga³⁺ were fitted by a 2 site model with dissociation constants (K_d) of 3.3 x 10⁻⁷ and 1.1 x 10⁻⁶ M. Although the absorption of gastrin did not change upon the addition of In³⁺ ions, the changes in absorbance on Fe³⁺ ion binding in the presence of indium ions were fitted by a 2 site model with K_d values for In³⁺ of 6.5 x 10⁻¹⁵ and 1.7 x 10⁻⁷ M. Similar results were obtained with Ru³⁺ ions, although the K_d values for Ru³⁺ of 2.6 x 10⁻¹³ and 1.2 x 10⁻⁵ M were slightly larger than observed for In³⁺. The structures determined by EXAFS all had metal:gastrin stoichiometries of 2:1 but, while the metal ions in the Fe, Ga and In complexes were bridged by a carboxylate and an oxygen with a metal-metal separation of 3.0–3.3 Å, the Ru complex clearly demonstrated a short range Ru—Ru separation, which was significantly shorter, at 2.4 Å, indicative of a metal-metal bond. We conclude that gastrin selectively binds two In³⁺ or Ru³⁺ ions, and that the affinity of the first site for In³⁺ or Ru³⁺ ions is higher than for ferric ions. Some of the metal ion-gastrin complexes may be useful for cancer diagnosis and therapy.     

Variations of Some Enzyne and Coenzyme Concentrations in Nornal Human Erythrocytes Fractionated by Velocity Sedimentation

Abstract

The concentrations of glucose-6-phosphate dehydrogenase, adenosine triphosphate and 2,3-diphosphoglycerate were measured in various fractions of normal human erythrocytes separated by velocity sedimentation in isotonic saline and sucrose buffer based on their tendency to aggregate. The levels of 2,3-diphosphoglycerate varied most drastically in fresh erythrocytes, being lowest in fast sedimenting cells and highest in slowly sedimenting cells. This result implies that the 2,3-diphosphoglycerate concentrations is not constant in all normal human erythrocytes and that 2,3-diphosphoglycerate may be involved in the mechanism of red cell clumping in vitro.     

阳离子胶体金标记活细胞阴离子位点的双光子荧光成像及其应用

使用阳离子胶体金标记中国仓鼠卵巢细胞(CHO-K1)的阴离子位点,并采用双光子荧光显微成像和荧光寿命成像技术记录活细胞的阴离子场分布.阳离子胶体金是纳米量级金微粒与多聚L-赖氨酸的结合物,金纳米微粒在超短激光脉冲的照射下可以产生高度局域化的光热效应.当飞秒激光脉冲聚焦在细胞膜上标记的金纳米微粒时会产生这种纳米尺度的微光热效应,并在不影响细胞活性的前提下暂时提高细胞膜的通透性.基于这种效应,使用聚焦的飞秒激光脉冲三维扫描照射CHO-K1细胞,将分子质量为10ku的荧光探针大分子异硫氰酸荧光素葡聚糖(fluorescein isothioeyanate-dextran, FITC-D)递送到CHO-K1细胞的内部,并用双光子荧光图像记录其递送的过程.使用流式细胞仪分析不同实验条件下FITC-D的转导率和细胞死亡率的关系.     

牛1gG高亲和力受体的分子克隆及序列分析

报道编码牛 Ig G高亲和力受体 ( bovine Ig G Fc receptor I,bo FcγR )的全长序列 .从牛肺巨噬细胞 c DNA文库中克隆的该片段全长 1 .4kb,其中的 ORF为 1 0 50 bp,共编码包括信号肽、胞外域、穿膜区和胞内区在内的 349个氨基酸 ,含有 5个潜在的 N-连接糖基化位点 .与人和鼠的 Ig G高亲和力受体 ( hu FcγR 和 mo FcγR )相比 ,其核苷酸同源性分别为 80 %和 69% ,氨基酸同源性分别为 66%和 55% .研究表明 ,人、牛和鼠的 3种 Ig G高亲和力受体的单体 Ig G结合域高度保守     

牛IgG高亲和力受体的分子克隆及序列分析

报道编码牛IgG高亲和力受体(bovineIgGFcreceptorI,boFcγRI)的全长序列.从牛肺巨噬细胞cDNA文库中克隆的该片段全长1.4kb,其中的ORF为1050bp,共编码包括信号肽、胞外域、穿膜区和胞内区在内的349个氨基酸,含有5个潜在的N-连接糖基化位点.与人和鼠的IgG高亲和力受体(huFcγRI和mohγRI)相比,其核苷酸同源性分别为80%和69%,氨基酸同源性分别为66%和55%.研究表明,人、牛和鼠的3种IgG高亲和力受体的单体IgG结合域高度保守.     

高效亲和膜色谱快速分析及小量制备蛋白质

以甲基丙烯酸缩水甘油酯纤维素复合膜为基质,分别以蛋白A(Protein A)、人免疫球蛋白G(HIgG)、三嗪染料(Cibacron blue F3GA)、亚胺二乙酸铜离子为配基,用不同方法制备了适合于分析及小量制备的高效亲和膜色谱介质,并对高效亲和柱的基本性能及其应用于各种相应蛋白的定量测定情况进行了考察。利用这种方法可以针对不同的目标蛋白及所存在的环境采用不同的配基,对各种蛋白的定量测定及小量制备可达到较为满意的结果。     

牛1gG高亲和力受体的分子克隆及序列分析

报道编码牛 Ig G高亲和力受体 ( bovine Ig G Fc receptor I,bo FcγR )的全长序列 .从牛肺巨噬细胞 c DNA文库中克隆的该片段全长 1 .4kb,其中的 ORF为 1 0 50 bp,共编码包括信号肽、胞外域、穿膜区和胞内区在内的 349个氨基酸 ,含有 5个潜在的 N-连接糖基化位点 .与人和鼠的 Ig G高亲和力受体 ( hu FcγR 和 mo FcγR )相比 ,其核苷酸同源性分别为 80 %和 69% ,氨基酸同源性分别为 66%和 55% .研究表明 ,人、牛和鼠的 3种 Ig G高亲和力受体的单体 Ig G结合域高度保守