ABC Transporters in Dynamic Macromolecular Assemblies

ATP-binding cassette (ABC) transporters constitute one of the largest families of integral membrane proteins, including importers, exporters, channels, receptors, and mechanotransducers, which fulfill a plethora of cellular tasks. ABC transporters are involved in nutrient uptake, hormone and xenobiotic secretion, ion and lipid homeostasis, antibiotic and multidrug resistance, and immunity, thus making them prime candidates for cellular regulation and pharmacological intervention. In recent years, numerous various structures of ABC transporters have been determined by X-ray crystallography or cryogenic electron microscopy. Structural and functional studies revealed that various auxiliary domains play key roles for the subcellular localization of ABC transporters and recruitment of regulatory factors. In this regard, the ABC transporter associated with antigen processing TAP stands out. In the endoplasmic reticulum membrane, TAP assembles the peptide-loading complex, which serves as a central checkpoint in adaptive immunity. Here, we discuss the various aspects of auxiliary domains for ABC transporter function with a particular emphasis on the structure of the peptide-loading complex, which is crucial for antigen presentation in adaptive immunity.     

Purification of Rat Ceruloplasmin: Characterization and Comparison with Human Ceruloplasmin

Rat ceruloplasmin was purified by a three-step column chromatography procedure, utilizing DEAE-Sepharose, Sepharose CL-6B, and CM-Sephadex A50 columns. The molecular weight of rat ceruloplasmin determined by a molecular sieve column was 124,000 daltons. An optical density ratio (610 nm/280 nm) of 0.051 and a molar extinction coefficient of 8600 were obtained. A decrease in lysine in rat ceruloplasmin compared with human ceruloplasmin could account for its reduced anodal mobility. Other differences in the amino acid sequence of the rat ceruloplasmin included an increase in methionine and cystine/cysteine, and a decrease in histidine, tyrosine and tryptophan.     

Building Macromolecular Assemblies by Information-driven Docking: INTRODUCING THE HADDOCK MULTIBODY DOCKING SERVER*

Over the last years, large scale proteomics studies have generated a wealth of information of biomolecular complexes. Adding the structural dimension to the resulting interactomes represents a major challenge that classical structural experimental methods alone will have difficulties to confront. To meet this challenge, complementary modeling techniques such as docking are thus needed. Among the current docking methods, HADDOCK (High Ambiguity-Driven DOCKing) distinguishes itself from others by the use of experimental and/or bioinformatics data to drive the modeling process and has shown a strong performance in the critical assessment of prediction of interactions (CAPRI), a blind experiment for the prediction of interactions. Although most docking programs are limited to binary complexes, HADDOCK can deal with multiple molecules (up to six), a capability that will be required to build large macromolecular assemblies. We present here a novel web interface of HADDOCK that allows the user to dock up to six biomolecules simultaneously. This interface allows the inclusion of a large variety of both experimental and/or bioinformatics data and supports several types of cyclic and dihedral symmetries in the docking of multibody assemblies. The server was tested on a benchmark of six cases, containing five symmetric homo-oligomeric protein complexes and one symmetric protein-DNA complex. Our results reveal that, in the presence of either bioinformatics and/or experimental data, HADDOCK shows an excellent performance: in all cases, HADDOCK was able to generate good to high quality solutions and ranked them at the top, demonstrating its ability to model symmetric multicomponent assemblies. Docking methods can thus play an important role in adding the structural dimension to interactomes. However, although the current docking methodologies were successful for a vast range of cases, considering the variety and complexity of macromolecular assemblies, inclusion of some kind of experimental information (e.g. from mass spectrometry, nuclear magnetic resonance, cryoelectron microscopy, etc.) will remain highly desirable to obtain reliable results.Proteins are the wheels and millstones of the complex machinery that underlies human life. Catalyzing a huge diversity of chemical processes, proteins work in close association with other biomolecules: nucleic acids, sugars, lipids, and other proteins. This huge network of protein interactions enables the cell to respond quickly to changes in the environment, such as temperature, oxygen, or nutrient concentration. However, to fully understand this network, insights at the atomic level are needed.In the wake of the elucidation of the human genome (1, 2), many structural genomics projects are solving the structures of what is now becoming a considerable fraction of the human proteome (3). These projects are now moving to the next level, which is solving the atomic resolution structures of protein complexes. However, this is a challenge that is considerably greater than obtaining the structures of single proteins. First of all, a protein can take part in 10 interactions on average; thus, the number of complexes is expected to be at least an order of magnitude larger than the proteome, and their composition can even vary over time. Second, associations between subunits in protein complexes are often weak and reversible, which make purification and crystallization difficult. Finally, there are some very well studied classes of interactions, such as enzyme-inhibitor, antibody-antigen, and GTPase-GAP (GTPase-activating protein) interactions, but these classes represent binary interactions between proteins. In contrast, many of the most important functions in the cell are carried out by large, dynamic molecular assemblies, such as the ribosome, the proteasome, the spliceosome, RNA polymerases, and the nuclear pore complex (4, 5). For such assemblies, high resolution methods such as x-ray crystallography and NMR spectroscopy often provide atomic level information at the level of individual subunits or subcomplexes, but they typically encounter difficulties at the level of the full complex.Fortunately, low resolution information about protein complexes can often be obtained. Affinity purification (6, 7) followed by mass spectrometry is a high throughput technique to study the composition of a complex. However, dissociation inside the mass spectrometer can be a problem for transient or unstable complexes in which case chemical cross-linking can help. Once the composition of the complex is known, there is a variety of experimental techniques available to obtain structural information on the complex. The most detailed information can be gathered by using data obtained from various NMR experiments, for example chemical shift perturbations (8) or residual dipolar couplings (9); unfortunately, NMR is limited to complexes that are fairly small in size, making its applicability in the context of large assemblies less suited. Techniques that provide information about the shape of a protein complex, such as small angle x-ray scattering (SAXS),¹ cryoelectron tomography, and single molecule cryoelectron microscopy (cryo-EM), are more suited to characterize large complexes. Unfortunately, all of these techniques suffer from limitations in resolution that are either fundamental or caused by structural heterogeneities of the complex.A well known approach to obtain information on residues at an interface is site-directed mutagenesis (10). In principle, a loss of binding affinity indicates that the mutated residue mediates the interaction, although the reverse is not true. Also, one must take care of secondary effects, such as unfolding or conformational change caused by the mutation. Apart from that, very detailed information about interface residues can be obtained by extensive mutagenesis experiments, such as alanine scanning and double mutant cycles. Mass spectrometry offers the opportunity to get peptide level or residue level information about protein interfaces by accurate mass measurements of peptides from the protein complex, generated either a priori through proteolytic cleavage, or inside the mass spectrometer (MS/MS). For example, interface residues can be identified as residues that undergo slower hydrogen/deuterium exchange upon complex formation. This process can be monitored at the peptide level by mass spectrometry (or in smaller complexes, at the residue level by NMR), although this method is very sensitive to noise caused by conformational changes upon binding. In the same way, radical probe MS (RP-MS) uses differences in oxidation of residues by hydroxyl radicals generated in the mass spectrometer to identify interface residues. Finally, chemical cross-linking followed by MS can provide direct information about residue contact sites between different binding partners of the complex. Several cross-linking reagents can provide complementary information. However, it has been reported that the cross-linkers may disrupt the structure of the protein complex and that care should therefore be taken to interpret the results (11).There is a need for computational approaches to translate this low resolution information into atomic resolution models that can provide functional and mechanistic insights. One of the most promising approaches is docking, the prediction of the structure of a complex starting from the free, unbound structures of its constituents. In recent years, docking methods have made much progress in the blind prediction of the structure of protein complexes as seen in the recent rounds of the critical assessment of prediction of interactions (CAPRI) experiment (12, 13). Most docking methods are ab initio, which means that experimental data are not required. However, it is possible in several ab initio methods to use experimentally determined interface residues in the docking: in MolFit (14, 15) and ATTRACT (16, 17), it is possible to up-weight the interaction scores of interface residues; in ZDOCK (18, 19), it is possible to block non-interface residues; and in PatchDock (20, 21), ZDOCK, pyDock (22, 23), and several other methods, it is possible to filter the docking results based on experimental information. Next to purely ab initio approaches, there are also methods that make use of different types experimental information, for example PROXIMO (24), based on RP-MS data, and MultiFit (25), a hybrid fitting/docking approach based on electron microscopy data.A method that distinguishes itself from the variety of above mentioned docking approaches is HADDOCK (26–28). In HADDOCK, the docking can be driven by a variety of experimental data using information about interface, contacts, and relative orientations inside a complex simultaneously. Originally developed for NMR data, HADDOCK is able to deal with a large variety of experimental data as shown in 26–28) and “Materials and Methods” for more details.) HADDOCK has performed very well in translating these data into structures and structural models. More than 60 Protein Data Bank structures calculated using HADDOCK have been deposited to date as experimental structures in the Protein Data Bank (29). Moreover, HADDOCK has shown a strong performance in CAPRI. Finally, HADDOCK is a general purpose program that can integrate many kinds of data, but even with a single source of data it is able to perform as well as more specialized programs: for example, HADDOCK was able to closely reproduce the NMR-calculated E2A-HPr complex using only chemical shift perturbation data. For the ribonuclease S-protein-peptide complex (Protein Data Bank code 1J80 (30)) for which RP-MS data are available, PROXIMO was able to closely reproduce the crystal structure (root mean square deviation (r.m.s.d.) of the top scoring model from the reference crystal structure is 1.26 Å); using the same data, HADDOCK could get even closer with an r.m.s.d. of only 0.68 Å from the crystal structure (results not shown).

Table I

Various experimental data that can be incorporated into HADDOCK

Measurement of Acute Phase Proteins in the Rat Brain: Contribution of Vascular Contents

A localized acute phase response occurs in the brain in Alzheimer's disease. Acute phase proteins have previously been measured in brain homogenates to quantify this response. The extent to which measurements of these proteins reflect brain parenchymal contents, as opposed to vascular contents, is unknown. In this study, the acute phase proteins ceruloplasmin (CP), complement factor 3 (C3), haptoglobin (HP), and albumin were measured in regional brain homogenates from phosphate buffered saline-perfused and sham-perfused rats (n = 7–9/group). Interleukin 1- (IL1-) and copper were also measured. Mean CP, C3, HP, and albumin concentrations in perfused specimens decreased by 94%, 88%, 90%, and 81% vs. sham-perfused specimens (all p < 0.001), while ILl- and copper were unchanged. These results suggest that acute phase protein measurements in brain homogenates reflect primarily vascular contents. However, IL1- and copper concentrations in brain homogenates are minimally influenced by vascular contents.     

The Differential Response of Proteins to Macromolecular Crowding

The habitat in which proteins exert their function contains up to 400 g/L of macromolecules, most of which are proteins. The repercussions of this dense environment on protein behavior are often overlooked or addressed using synthetic agents such as poly(ethylene glycol), whose ability to mimic protein crowders has not been demonstrated. Here we performed a comprehensive atomistic molecular dynamic analysis of the effect of protein crowders on the structure and dynamics of three proteins, namely an intrinsically disordered protein (ACTR), a molten globule conformation (NCBD), and a one-fold structure (IRF-3) protein. We found that crowding does not stabilize the native compact structure, and, in fact, often prevents structural collapse. Poly(ethylene glycol) PEG500 failed to reproduce many aspects of the physiologically-relevant protein crowders, thus indicating its unsuitability to mimic the cell interior. Instead, the impact of protein crowding on the structure and dynamics of a protein depends on its degree of disorder and results from two competing effects: the excluded volume, which favors compact states, and quinary interactions, which favor extended conformers. Such a viscous environment slows down protein flexibility and restricts the conformational landscape, often biasing it towards bioactive conformations but hindering biologically relevant protein-protein contacts. Overall, the protein crowders used here act as unspecific chaperons that modulate the protein conformational space, thus having relevant consequences for disordered proteins.     

Trapping Proteins within Gold Nanoparticle Assemblies: Dynamically Tunable Hot-spots for Nanobiosensing

The combination of stimuli-responsive materials with localized surface plasmon resonance nanotransducers provides new leverages in hot spot-based nanosensing. We introduce a simple and effective biodetection method based on the hydro-responsive property of (3-aminopropyl)-triethoxysilane (APTES). Gold nanoparticles were adsorbed onto hydro-responsive APTES thin film. The exposure of the film surface to an aqueous solution results in opening inter-particle gaps, allowing analyte binding. A subsequent drying of the sensor surface closes the gap by bringing the nanoparticles to the initial position, thereby trapping the analyte in the most sensitive regions (electromagnetic hot spots). In this reversible configuration, the generation and tuning of the hot spots are independent from both the presence of the analyte and the functionalization of the nanoparticles, which yields highly resolved coupled plasmon bands and provide a general and flexible nanosensing modality. Furthermore, the intensity of the hot spots can be easily and reversibly tuned to obtain picomolar sensitivity.     

Lipid-Composition-Mediated Forces Can Stabilize Tubular Assemblies of I-BAR Proteins

Collective action by inverse-Bin/Amphiphysin/Rvs (I-BAR) domains drive micron-scale membrane remodeling. The macroscopic curvature sensing and generation behavior of I-BAR domains is well characterized, and computational models have suggested various mechanisms on simplified membrane systems, but there remain missing connections between the complex environment of the cell and the models proposed thus far. Here, we show a connection between the role of protein curvature and lipid clustering in the relaxation of large membrane deformations. When we include phosphatidylinositol 4,5-bisphosphate-like lipids that preferentially interact with the charged ends of an I-BAR domain, we find clustering of phosphatidylinositol 4,5-bisphosphate-like lipids that induce a directional membrane-mediated interaction between membrane-bound I-BAR domains. Lipid clusters mediate I-BAR domain interactions and cause I-BAR domain aggregates that would not arise through membrane fluctuation-based or curvature-based interactions. Inside of membrane protrusions, lipid cluster-mediated interaction draws long side-by-side aggregates together, resulting in more cylindrical protrusions as opposed to bulbous, irregularly shaped protrusions.     

急性期蛋白的研究进展

急性期蛋白(如珠蛋白、血清淀粉样蛋白A、C-反应蛋白)是指机体受感染、创伤、炎症等应激原刺激下所产生变化的蛋白,又叫急性期反应物或应激敏感蛋白质.近年来研究表明:血清中急性期蛋白的浓度变化可为疾病的诊断、预防提供可靠的依据.已应用到被屠宰动物的疾病控制及目前亚临床动物疾病导致的畜禽出生率低等问题的监控上.本文就急性期蛋白的研究进展、临床应用及将来的发展趋势做一综述,为医学临床、科研、肉品安全等诸多方面的研究提供新的思路.     

The Acute Phase Protein Ceruloplasmin as a Non-Invasive Marker of Pseudopregnancy, Pregnancy, and Pregnancy Loss in the Giant Panda

After ovulation, non-pregnant female giant pandas experience pseudopregnancy. During pseudopregnancy, non-pregnant females exhibit physiological and behavioral changes similar to pregnancy. Monitoring hormonal patterns that are usually different in pregnant mammals are not effective at determining pregnancy status in many animals that undergo pseudopregnancy, including the giant panda. Therefore, a physiological test to distinguish between pregnancy and pseudopregnancy in pandas has eluded scientists for decades. We examined other potential markers of pregnancy and found that activity of the acute phase protein ceruloplasmin increases in urine of giant pandas in response to pregnancy. Results indicate that in term pregnancies, levels of active urinary ceruloplasmin were elevated the first week of pregnancy and remain elevated until 20–24 days prior to parturition, while no increase was observed during the luteal phase in known pseudopregnancies. Active ceruloplasmin also increased during ultrasound-confirmed lost pregnancies; however, the pattern was different compared to term pregnancies, particularly during the late luteal phase. In four out of the five additional reproductive cycles included in the current study where females were bred but no birth occurred, active ceruloplasmin in urine increased during the luteal phase. Similar to the known lost pregnancies, the temporal pattern of change in urinary ceruloplasmin during the luteal phase deviated from the term pregnancies suggesting that these cycles may have also been lost pregnancies. Among giant pandas in captivity, it has been presumed that there is a high rate of pregnancy loss and our results are the first to provide evidence supporting this notion.     

Circulating Microbial Products and Acute Phase Proteins as Markers of Pathogenesis in Lymphatic Filarial Disease

Lymphatic filariasis can be associated with development of serious pathology in the form of lymphedema, hydrocele, and elephantiasis in a subset of infected patients. Dysregulated host inflammatory responses leading to systemic immune activation are thought to play a central role in filarial disease pathogenesis. We measured the plasma levels of microbial translocation markers, acute phase proteins, and inflammatory cytokines in individuals with chronic filarial pathology with (CP Ag+) or without (CP Ag−) active infection; with clinically asymptomatic infections (INF); and in those without infection (endemic normal [EN]). Comparisons between the two actively infected groups (CP Ag+ compared to INF) and those without active infection (CP Ag− compared to EN) were used preliminarily to identify markers of pathogenesis. Thereafter, we tested for group effects among all the four groups using linear models on the log transformed responses of the markers. Our data suggest that circulating levels of microbial translocation products (lipopolysaccharide and LPS-binding protein), acute phase proteins (haptoglobin and serum amyloid protein-A), and inflammatory cytokines (IL-1β, IL-12, and TNF-α) are associated with pathogenesis of disease in lymphatic filarial infection and implicate an important role for circulating microbial products and acute phase proteins.     

Unveiling Contacts within Macromolecular Assemblies by Solving Minimum Weight Connectivity Inference (MWC) Problems*

Consider a set of oligomers listing the subunits involved in subcomplexes of a macromolecular assembly, obtained e.g. using native mass spectrometry or affinity purification. Given these oligomers, connectivity inference (CI) consists of finding the most plausible contacts between these subunits, and minimum connectivity inference (MCI) is the variant consisting of finding a set of contacts of smallest cardinality. MCI problems avoid speculating on the total number of contacts but yield a subset of all contacts and do not allow exploiting a priori information on the likelihood of individual contacts. In this context, we present two novel algorithms, MILP-W and MILP-W_B. The former solves the minimum weight connectivity inference (MWCI), an optimization problem whose criterion mixes the number of contacts and their likelihood. The latter uses the former in a bootstrap fashion to improve the sensitivity and the specificity of solution sets.Experiments on three systems (yeast exosome, yeast proteasome lid, human eIF3), for which reference contacts are known (crystal structure, cryo electron microscopy, cross-linking), show that our algorithms predict contacts with high specificity and sensitivity, yielding a very significant improvement over previous work, typically a twofold increase in sensitivity.The software accompanying this paper is made available and should prove of ubiquitous interest whenever connectivity inference from oligomers is faced.     

Quantitation of Acute Phase Proteins and Protein Electrophoresis in Monitoring the Acute Inflammatory Process in Experimentally and Naturally Infected Mice

Serologic screening for infectious disease in sentinel mice from rodent colonies is expensive and labor-intensive, often involving multiple assays for several different infectious agents. Previously, we established normal reference ranges for the protein fractions of several laboratory strains of mice by using a commercially available agarose system of protein electrophoresis. In the current study, we address protein fractionation and quantitation of acute phase proteins (APP) in mice experimentally infected with Sendai virus or mouse parvovirus. We further investigate this methodology by using samples from sentinel mice from colonies with endemic infection. All study groups showed significant increases in γ globulins. Various other protein fractions showed mild variable changes; significant differences were not detected for individual APP. These results contrast the significant changes observed in APP and protein electrophoresis by using the standard methods of inducing inflammatory responses through injection of complete Freund adjuvant or LPS. These present data suggest that although quantitation of individual APP may not be helpful, γ globulin levels may reflect infection in laboratory mice and provide a possible adjunct to traditional screening methods.Abbreviations: APP, acute phase protein; CFA, complete Freund adjuvant; CRP, C-reactive protein; MPV, mouse parvovirus; SAA, serum amyloid A; SAP, serum amyloid PSophisticated technologies including serology, culture, histology, and PCR are available to evaluate laboratory animals for the presence of infectious disease.⁴⁸ These analyses, albeit expensive and labor-intensive, are necessary to ensure that laboratory rodents are free from infectious agents that can interfere with research. In both human and veterinary medicine, the quantitation of acute phase proteins (APP) has been proposed to have diagnostic and prognostic utility to study disease and infection.^{2,11,14,21,26,40} APP are blood proteins primarily synthesized by hepatocytes as part of the complex systemic response termed the acute phase response. The acute phase response is part of the early defense or innate immune system, which is triggered by various stimuli, including trauma, infection, stress, neoplasia, and inflammation. The acute phase response has been referred to as the ‘molecular thermometer,’ whereby quantitation of specific APP might reflect the response to the triggering event.^10,14,40,44 To this point, several studies have been conducted in companion, laboratory, and large animals profiling changes in APP after experimental and natural infection.^{17,18,38,40,45,50}Mice have several major APP that may reflect acute and chronic inflammatory processes including C-reactive protein (CRP), haptoglobin, serum amyloid P (SAP), and serum amyloid A (SAA).^20,47 ELISA assays for these proteins are commercially available. A broader view of the sum of APP changes and the overall acute phase response is obtained through the use of protein electrophoresis.³³ This technique uses an agarose gel to separate protein fractions into albumin, α1 globulins, α2 globulins, β globulins, and γ globulins. Protein electrophoresis does not quantitate single proteins but rather groups of proteins that are mediators of acute inflammatory process. α1 globulins include α1 antitrypsin and α1 acid glycoprotein; α2 globulins include α2 macroglobulin and haptoglobin; β globulins include transferrin, SAA, and CRP, and γ globulins are composed primarily of IgG.³³ Many diagnostic and prognostic uses of protein electrophoresis in veterinary medicine have been reported.^1,4,12,25,33 Although rarely diagnostic of a particular disease, protein electrophoresis is helpful for the detection of acute and chronic inflammatory processes and stimulation of humoral immunity.^12,15,33APP have been proposed to be valuable biochemical markers of stress, infection, and pain in laboratory animals.^14,42 Previously, we established normal reference ranges for the protein fractions of several laboratory strains of mice by using a commercially available agarose system of protein electrophoresis.⁵⁴ The primary goal of the current project was to study the potential changes in APP and protein fractions in laboratory mice after experimental infection with viral pathogens. These data were compared to those generated by using traditional means of inducing acute inflammation with the injection of LPS and complete Freund adjuvant (CFA). In addition, we addressed the possible application of protein fractionation and quantitation of APP by using samples from sentinel mice from colonies with endemic infection.     

A Robust and Versatile Automated Glycoanalytical Technology for Serum Antibodies and Acute Phase Proteins: Ovarian Cancer Case Study

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Highlights

• •Quantitative high-throughput glycoanalytical technology as a diagnostic tool for ovarian cancer detection.
• •Multiplexed approach harnessing N-glycan data for six glycoproteins from a single biological sample.
• •Detailed characterization of human serum N-glycans from antibodies IgG, IgM and IgA and acute phase proteins transferrin, haptoglobin and alpha-1-antitrypsin.
• •Structural differences in antibody and acute phase protein glycosylation for mechanistic insights.

     

Study of Interaction of Ceruloplasmin with Serprocidins

This paper describes formation of complexes of ceruloplasmin (CP) with such proteins of the serprocidin family as azurocidin (CAP37), neutrophilic elastase (NE), cathepsin G (CG), and proteinase 3 (PR3). We present evidence that serprocidins form complexes with CP at a molar ratio 1: 1. Phenylmethylsulfonyl fluoride, a serine protease inhibitor, did not prevent the interaction of serprocidins with CP in the course of SDS-free disc electrophoresis. CP affected the activities of NE, CG, and PR3 as a competitive inhibitor with K _i ≈ 1 μM. Inhibitory effect of CP depended on ionic strength of the solution and was negligible at NaCl concentrations above 300 mM. In the mode of competitive inhibitors serprocidins suppressed oxidase activity of CP towards p-phenylenediamine. CAP37 displayed the strongest inhibitory effect (K _i ≈20 nM). Upon adding various serprocidins to human, rat, rabbit, dolphin, dog, horse, and mouse plasma only CAP37 would form a complex with CP. Synthetic peptide RKARPRQFPRRR (5–13, 61–63 CAP37) displaced CAP37 from its complex with CP. Adding CAP37 to the triple complex formed by CP, lactoferrin, and myeloperoxidase resulted in displacement of the latter from the complex. The dissociation constant of CAP37 with immobilized CP was 13 nM. Therefore, among serprocidins CAP37 can be regarded as the specific partner of CP.     

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology ¹. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes ^{2, 3}. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice ⁴, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast ⁵. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes ⁶, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.     

Circadian Rhythm of Acute Phase Proteins under the Influence of Bright/Dim Light during the Daytime

We investigated the influence of two different light intensities, dim (100 lx) and bright (5,000 lx), during the daytime on the circadian rhythms of selected acute phase proteins of C‐reactive protein (CRP), α1‐acid glycoprotein (AGP), α1‐antichymotrypsin (ACT), transfferin (TF), α2‐macroglobulin (α2‐m), haptoglobin (HP), and ceruloplasmin (CP). Serum samples were collected from 7 healthy volunteers at 4 h intervals during two separate single 24 h spans during which they were exposed to the respective light intensity conditions. A circadian rhythm was detected only in ACT concentration in the bright light condition. The concentration of ACT, a positive acute phase protein (APP), increased (significantly significant differences in the ACT concentration were detected at 14:00 and 22:00 h) and AGP showed a tendency to be higher under the daytime bright compared to dim light conditions. There were no significant differences between the time point means under daytime dim and bright light conditions for α2‐M, AGP, Tf, Cp, or Hp. The findings suggest that some, but not all, APP may be influenced by the environmental light intensity.