Impaired assembly results in the accumulation of multiple HLA-C heavy chain folding intermediates

Class I MHC H chains assemble with beta2-microglobulin (beta2m) and are loaded with peptide Ags through multiple folding steps. When free of beta2m, human H chains react with Abs to linear epitopes, such as L31. Immunodepletion and coimmunoprecipitation experiments, performed in this study, detected a preferential association of L31-reactive, beta2m-free H chains with calnexin in beta2m-defective cells, and with calreticulin and TAP in beta2m-expressing cells. In beta2m-defective cells, the accumulation of calnexin-bound H chains stoichiometrically exceeded their overall accumulation, a finding that supports both chaperoning preferences and distinct sorting abilities for different class I folds. No peptide species, in a mass range compatible with that of the classical class I ligands, could be detected by mass spectrometry of acidic eluates from L31-reactive HLA-Cw1 H chains. In vitro assembly experiments in TAP-defective T2 cells, and in cells expressing an intact Ag-processing machinery, demonstrated that L31 H chains are not only free of, but also unreceptive to, peptides. L31 and HC10, which bind nearly adjacent linear epitopes of the alpha1 domain alpha helix, reciprocally immunodepleted free HLA-C H chains, indicating the existence of a local un-/mis-folding involving the N-terminal end of the alpha1 domain alpha helix and peptide-anchoring residues of the class I H chain. Thus, unlike certain murine free H chains, L31-reactive H chains are not the immediate precursors of conformed class I molecules. A model inferring their precursor-product relationships with other known class I intermediates is presented.     

Human blood plasma preparation for two-dimensional gel electrophoresis

Human plasma consists of mainly large proteins, which vary in terms of both composition and concentration with the physiological state of the individual. Alterations in protein concentrations reflect the current state of the individual's health and thus may be utilized as valuable biomarkers for a specific biological process or disease. Two-dimensional gel electrophoresis (2-DE) has proven to be a valuable method for the separation and comparison of complex protein mixtures, for example, from disease and healthy states, as this method provides information regarding the variation, relative quantities, and structures of the intact proteins. The procedures utilized for the preparation of samples for 2-DE are critical to the acquisition of high-quality results for the discovery of biomarkers. The objective of this study was to review the preparation methods of plasma for 2-DE, particularly those designed to improve the detection of proteins in low abundance in plasma on 2-DE. The use of anticoagulants and protease inhibitors during the collection of blood, the removal of abundant proteins using multicomponent immunodepletion system, and desalting procedure allow us to compile profiles of proteins occurring in low concentrations in the plasma and to improve the pattern generated during 2-DE.     

Investigations of the structural function of the J chain in human immunoglobulin M

Human IgM molecules were treated with Na(2)SO(3) or mercaptoethylamine in concentrations ranging from 2 to 14mm or 2 to 22mm respectively. The dissociation of IgM to IgM(s) varied from 0% to 100%. At the intermediate concentrations of either reagent the amount of freed J chains was less than expected. In an attempt to find an explanation for this, IgM was partially dissociated to IgM(s) with mercaptoethylamine. The IgM(s) isolated by gel filtration was divided according to the ascending and descending portions of the elution curve. These portions were treated with 24mm-mercaptoethylamine and analysed for the presence of J chains. Only the ascending portion contained free J chains. Thus, after mild reduction where not all the IgM molecules are dissociated to IgM(s), some J chains remain covalently attached to some IgM(s) molecules although most of the J chains are freed. It was concluded that the J chain could serve as a ;hitch' for IgM(s) molecules forming intact IgM.     

Physical and Biological Properties of Phage φ29 Deoxyribonucleic Acid

Deoxyribonucleic acid (DNA) molecules having a mean length of 5.8 mum were released from purified Bacillus subtilis bacteriophage phi29 with 2 m sodium perchlorate. Small 0.1 to 0.2-mum molecules were also detected in these DNA preparations. Since intact single chains annealed to form linear duplex molecules, phage phi29 DNA was found to be nonpermuted. The molecular weights of single chains of phi29 DNA were approximately half that of native DNA, as determined by analytical band sedimentation in CsCl, indicating that phi29 DNA is composed of two continuous polynucleotide chains. The molecular weight values of native and annealed phi29 DNA from sedimentation agreed with the molecular weight values obtained from electron microscopy. The infectivity of phi29 DNA was reduced to a low level by alkaline denaturation and was partially restored by annealing.     

Navigated laser capture microdissection as an alternative to direct histological staining for proteomic analysis of brain samples

Proteomic analysis of the brain is complicated by the need to obtain cells from specific anatomical regions, or nuclei. Laser capture microdissection (LCM) is a technique that is precise enough to dissect single cells within a tissue section, and thus could be useful for isolating specific brain nuclei for analysis. However, we and others have previously demonstrated that histological staining protocols used to guide LCM have detrimental effects on protein separation by two-dimensional electrophoresis (2-DE). Here we describe a new LCM method called navigated LCM. This microdissection method uses fixed but unstained tissue as starting material and thus enables us to avoid artifacts induced by tissue staining. By comparing 2-DE results obtained from fixed, unstained LCM brain tissue samples to those obtained from manually dissected samples, we demonstrated that this microdissection process gave similar protein recovery rates and similar resolution of protein spots on 2-DE gels. Moreover, matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis of selected spots from gels derived from control and fixed, LCM samples revealed that the fixation-LCM process had no effect on protein identification. Navigated LCM of tissue sections is therefore a practical and powerful method for performing proteomic studies in specifically defined brain regions.     

Fluorescence correlation spectroscopy of intact nuclear pore complexes

No methods proposed thus far have the sensitivity to measure the transport of single molecules through single nuclear pore complexes (NPCs) in intact cells. Here we demonstrate that fluorescence correlation spectroscopy (FCS) combined with real-time tracking of the center of mass of single NPCs in live, unperturbed cells allows us to detect the transport of single molecules in a reference system of a pore with high temporal (millisecond) and spatial (limited by diffraction) resolution. We find that the transport of the classical receptor karyopherin-β1 (Kapβ1) is regulated so as to produce a peculiar distribution of characteristic times at the NPC. This regulation, which is spatially restricted to the pore, depends on the properties and metabolic energy of Kapβ1. As such, this method provides a powerful tool for studying nucleocytoplasmic shuttling at the nanometer scale under physiological conditions.     

Direct imaging of DNA motif sequences with encoded nanoparticles

We present a method for encoded tagging and imaging of short nucleic acid motif chains (oligomotifs) using selective hybridization of heterogeneous Au nanoparticles (Au-NP). The resulting encoded NP string is thus representative of the underlying motif sequence. As the NPs are much more massive than the motifs, the motif chain order can be directly observed using scanning electron microscopy. Using this technique we demonstrate direct sequencing of oligomotifs in single DNA molecules consisting of four 100-nt motif chains tagged with four different types of NPs. The method outlined is a precursor for a high density direct sequencing technology.     

Polyethylene glycol fractionation improved detection of low-abundant proteins by two-dimensional electrophoresis analysis of plant proteome

Poor detection of low-abundant proteins is a common problem in two-dimensional electrophoresis (2-DE) for separation of proteins in a proteome analysis. This is attributed partially, at least, to the existence of high-abundant proteins, e.g. ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in plants. They engage a large proportion of the whole-cell proteins and thus prevent low-abundant proteins from being up-taken by immobilized pH gradient (IPG) strip, consequently making the latter poorly detectable by 2-DE. In this work, we report a straightforward protocol for preparation of whole-cell proteins through differential polyethylene glycol (PEG) precipitation aiming at elimination of Rubisco from plant protein samples. In comparison with 2-DE analysis of protein samples prepared using a conventional TCA/acetone method, a relatively high reproducibility of proteins was achieved using a PEG fractionation protocol in terms of protein yield and protein species. As expected, the large subunit of Rubisco was precipitated predominantly in the 16% PEG fraction. This allowed proteins of the Rubisco-containing fraction to be analyzed separately from those of other PEG fractions. After taking into account the overlapping protein spots among 2-DE gels of all fractions through image and statistical analyses, we detected with this protocol a total 5077 protein spots, among which ca. 80% are proteins undetectable with the TCA/acetone method, while the rest of proteins exhibited a significant increase in their abundance. This protocol was developed using Arabidopsis as a source of protein and thus may also be applicable to protein preparations of other plants.     

Role of beta 2-microglobulin in the intracellular transport and surface expression of murine class I histocompatibility molecules

We have examined the requirement for beta 2-microglobulin (beta 2m) in the intracellular transport of murine class I histocompatibility molecules to the cell surface. R1E cells that are defective in the synthesis of beta 2m were transfected with either the class I H-2Kb or H-2Db genes alone, or together with the beta 2m gene. Kb or Db heavy chains synthesized in the presence of beta 2m were transported rapidly through the cell and expressed efficiently at the cell surface. In the absence of beta 2m, no "free" Kb heavy chains were detectable at the cell surface and their intracellular transport was blocked at an early stage. In contrast, a significant quantity of "free" Db heavy chains could be detected at the cell surface as we have reported previously. However, we have shown here that defects in intracellular transport were apparent in that the majority (approximately 70%) of newly synthesized Db heavy chains accumulated intracellularly and were degraded. Therefore, although Kb and Db heavy chains differ in their abilities to be expressed at the cell surface in the absence of beta 2m they both require association with beta 2m for efficient intracellular transport. In addition, R1E cells transfected with a deletion construct of the Kb gene expressed a truncated molecule lacking the alpha 3 extracellular domain (Kb-3) at the cell surface, but, like free Db, most newly synthesized Kb-3 molecules accumulated intracellularly. The free Kb, Kb-3, and Db heavy chains were not recognized by most mAb specific for Kb and Db, respectively. Therefore, even the transported forms of free Db and Kb-3 were not native in conformation, which is surprising given the current view that correct folding is essential for intracellular transport. Interestingly, the free Db and Kb-3 heavy chains that reached the cell surface differed in their detergent binding properties from those retained within the cell. This suggests that the transported heavy chains may have folded differently thus allowing their export to the cell surface.     

Aluminum modifies the viscosity of filamentous actin solutions as measured by optical displacement microviscometry

Two-dimensional electrophoresis (2-DE) is a highly resolving technique for arraying proteins by isoelectric point and molecular mass. To date, the resolving ability of 2-DE for protein separation is unsurpassed, thus ensuring its use as the fundamental separation method for proteomics. When immobilized pH gradients (IPGs) are used for isoelectric focusing in the first dimension, excellent reproducibility and high protein load capacity can be achieved. While this has been beneficial for separations of soluble and mildly hydrophobic proteins, separations of membrane proteins and other hydrophobic proteins with IPGs have often been poor. Stimulated by the growing interest in proteomics, recent developments in 2-DE methodology have been aimed at rectifying this situation. Improvements have been made in the area of protein solubilization and sample fractionation, leading to a revamp of traditional approaches for 2-DE of membrane proteins. This review explores these developments.     

Linear response theory: An alternative to PB and GB methods for the analysis of molecular dynamics trajectories?

We explore the use of classical Linear Response Theory (LRT) as an alternative strategy to the use of Molecular Mechanics/Poisson-Boltzmann strategies to compute the solvation free energy of macromolecules from molecular dynamics simulations using an explicit representation of solvent. The method reproduces well the free energy of solvation of standard amino acid side chains, small peptides, and proteins. The use of a fully discrete representation of solvent avoids the possible problems of continuum models to represent the solvation of systems containing tightly bound water molecules.     

Covalent attachment of sulfhydryl-specific, electron spin resonance spin-labels to Fab' fragments of murine monoclonal antibodies that recognize human platelet membrane glycoproteins. Development of membrane protein specific spin probes

A general method for the production of high-affinity, nitroxide-labeled, protein-specific spin probes is described in this paper. Fab' fragments are generated from protein-specific, murine monoclonal antibodies by pepsin digestion and mild reduction with cysteine. The free sulfhydryl group located in the carboxy-terminal region of these molecules and produced de novo by this manipulation is then alkylated by reaction with 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO-maleimide), thereby generating spin-labeled Fab' fragments of these monoclonal antibodies. Two prototypic monoclonal antibodies were tested, each specific for a different integral membrane glycoprotein of human blood platelets. The results indicate that Fab' spin probes generated by this method retain the ability to bind to these glycoproteins within the membrane of intact platelets. These reagents thus represent probes that can be generally used to monitor integral membrane protein mobility on the surface of the intact cell.     

Electrophoretic characteristics of monoclonal immunoglobulin G of different subclasses

Monoclonal IgG are commonly observed in various B cell disorders, of which multiple myeloma is the most clinically relevant. In a series of serum samples, we identified by immunofixation 73 monoclonal IgG, including 63 IgG(1), 4 IgG(2), 5 IgG(3), and 1 IgG(4). The light chains were of kappa type in 45 cases, and of lambda type in 28 cases. These monoclonal IgG were further characterized by high resolution two-dimensional polyacrylamide gel electrophoresis (2-DE) in various isoelectric focusing conditions, as well as by 3-DE (2-DE of the proteins extracted from agarose after serum protein agarose electrophoresis). After 2-DE, 38 out of 73 monoclonal gamma chains (52%) were visualized using immobilized pH 3-10 gradients for isoelectric focusing. In 6 cases (8%), gamma chains were only detected using alkaline immobilized pH 6-11 gradients. In 3 cases (4%), 3-DE revealed monoclonal gamma chains hidden by polyclonal gamma chains. Finally, in 26 cases (36%), no monoclonal gamma chains were clearly visualized. Sixty-one monoclonal light chains (84%) were detected using immobilized pH 3-10 gradients, whereas 12 (16%) were not. Monoclonal gamma chains and light chains were highly heterogeneous in terms of pI and M(r). However, a statistically significant correlation (P<0.05) was observed between the position of the monoclonal IgG in agarose gel and the pI of their heavy and light chains (R=0.733, multiple linear regression). Because of the extreme diversity of their heavy and light chains, it appears that a classification of monoclonal IgG based only on their electrophoretic properties is not possible.     

Application of two-dimensional electrophoresis for monitoring gastrointestinal digestion of milk

Summary. Two-dimensional electrophoresis (2-DE) was used for tracing in vivo gastrointestinal digestion of milk proteins in a rapid model system with rats. Contents of stomach and small intestine from digestion trials with rats given a single dose of milk powder were recovered after 1 hour. They were then subjected to 2-DE (IEF and SDS-PAGE). 2-DE showed undigested proteins in a MW range 13.0–66.0 kDa in stomach and 13.0–25.0 kDa in the small intestine, thus indicating that milk proteins are slowly digested. This approach may shed light on pattern of protein digestion and mechanism of amino acid and peptide assimilation.     

A blue dive: from ‘blue fingers’ to ‘blue silver’. A comparative overview of staining methods for in-gel proteomics

Gel-based proteomics are the most useful method for protein separation, even when compared with gel-free proteomics. Proteomic analysis by 2D gel electrophoresis (2-DE) with immobilized pH gradients is in turn the best approach to large-scale protein-expression screening. Spots visualization is pivotal for protein identification by mass spectrometry. Commonly used staining methods with excellent mass spectrometry compatibility are coomassie brilliant blue (CBB) or fluorescent dyes. In this study, an implementation of ‘blue silver’ colloidal CBB staining, characterized by high sensitivity and immediate low background, is discussed. The sensitivity of classical, colloidal and ‘blue silver’ CBB staining methods was compared on monodimensional and 2-DE gels. The implementation of the ‘blue silver’ method performs better, provided the physical state of the micelles is respected. An example of a 2-DE of human urine treated with combinatorial peptide ligand libraries demonstrates that implemented ‘blue silver’ can evidence the complexity of the sample.     

应用差异蛋白质组学方法分析作物化感作用的分子机理

试验旨在分析运用分子标记技术(QTL)和差异蛋白组学技术研究作物化感作用分子机理的差异性。首先运用差异蛋白组学技术探讨在生物胁迫(稗草)下水稻化感作用潜力变化的内在分子机理。分别用稗草和水稻的根系分泌物培养切自一株5叶龄化感水稻P I312777植株并经恢复的2个分蘖。7d后,提取处理和对照相同叶位叶片的全蛋白质并进行双向电泳,每张电泳胶片上获得800多个电泳胶点,其中差异表达的蛋白质点有4个。采用M ALD I-TOF-M S对各差异蛋白质点进行肽质量指纹图谱分析,经过SW ISS-PROT数据库查询,结果表明化感水稻P I312777在稗草胁迫下的特异蛋白分别与苯丙氨酸氨解酶(PAL)、硫还原型蛋白(T rx-m)、3-羟基-3-甲基戊二酰辅酶A还原酶(HM GR)和过氧化物酶(POD)相匹配。根据编码以上4个差异蛋白质的DNA序列,发现编码以上4个差异蛋白的基因分别位于水稻染色体4、7、8和12上的特定克隆位点,这就是与化感作用相关基因。前人也运用QTL方法开展作物化感作用的分子机理研究,但由于所采用的供体材料、受体植物及对表型性状的评价方法等的不同,定位结果存在较大的差异。综合比较两种方法后认为,运用差异蛋白组学技术分析水稻化感作用的分子机理,比QTL技术更加直接和深入。因为比较胁迫处理和对照植物组织的2-DE图谱将能鉴定出由表达候选基因编码的胁迫蛋白质,氨基酸残基序列的测定将揭示那些功能与胁迫性状密切相关的蛋白质,这种编码的基因就是兼具功能与表达的候选基因。     

Subunit interactions of skeletal muscle myosin and myosin subfragment 1. Evidence for heavy chain-alkali light chain association-dissociation equilibrium

Modification of the free alkali light chains of myosin by iodoacetylation results in a much lower extent of exchange into myosin subfragment 1 by the thermal hybridization procedure (Burke, M., and Sivaramakrishnan, M. (1981) Biochemistry 20, 5908-5913). As reported by others (Wagner, P. D., and Stone, D. B. (1983) J. Biol. Chem. 258, 8876-8882), free alkali light chains modified by iodoacetate at their single sulfhydryl residue exhibit minimal exchange into intact myosin. However, when unmodified alkali light chain is used to probe for exchange, close to the theoretical limit of exchange is observed for subfragment 1, and significant levels of exchange are found for myosin. It appears that modification of the free alkali light chain alters the structure of the protein, and this causes either a marked reduction in its affinity for the heavy chain or in its ability to enter the light chain binding site. This conclusion is supported by tryptic digestions done on the unmodified and modified free light chains where it is found that the latter is degraded at a much faster rate, indicating a more open structure for the modified protein. The observation that alkali light chain exchanges into myosin when unmodified alkali light chains are used indicates that the presence of the associated 5,5'-dithiobis-(2-nitrobenzoic acid) light chains does not preclude the reversible dissociation of this subunit from myosin under ionic and temperature conditions approaching the physiological state.     

Tmp21, a novel MHC-I interacting protein, preferentially binds to Β2-microglobulin-free MHC-I heavy chains

MHC-I molecules play a critical role in immune surveillance against viruses by presenting peptides to cytotoxic T lymphocytes. Although the mechanisms by which MHC-I molecules assemble and acquire peptides in the ER are well characterized, how MHC-I molecules traffic to the cell surface remains poorly understood. To identify novel proteins that regulate the intracellular transport of MHC-I molecules, MHC-I-interacting proteins were isolated by affinity purification, and their identity was determined by mass spectrometry. Among the identified MHC-I-associated proteins was Tmp21, the human ortholog of yeast Emp24p, which mediates the ER-Golgi trafficking of a subset of proteins. Here, we show that Tmp21 binds to human classical and non-classical MHC-I molecules. The Tmp21-MHC-I complex lacks Β(2)-microglobulin, and the number of the complexes is increased when free MHC-I heavy chains are more abundant. Taken together, these results suggest that Tmp21 is a novel protein that preferentially binds to Β(2)-microglobulin-free MHC-I heavy chains.