Improvement of protein stability in protein microarrays

Protein stability in microarrays was improved using protein stabilizers. PEG 200 at 30% (w/v) was the most efficient stabilizer giving over 4-fold improvement in protein stability compared to without the stabilizer. PEG 200 above 10% (w/v) in the array solution prevented the evaporation of water in the sample and thereby improved protein stability in the microarray. When the streptavidin-biotin binding reaction was performed under optimized conditions, biotin-BSA-fluorescein isothiocyanate (FITC) was detected from 1 ng ml^–1 to 5 g ml^–1 by fluorescence analysis.     

C-reactive protein reduces protein S-nitrosylation in endothelial cells

Glycodelin A (GdA) is a dimeric glycoprotein synthesized by the human endometrium under progesterone regulation. Based on the high sequence similarity with β-lactoglobulin, it is placed under the lipocalin superfamily. The protein is one of the local immunomodulators present at the feto-maternal interface which affects both the innate as well as the acquired arms of the immune system, thereby bringing about successful establishment and progression of pregnancy. Our previous studies revealed that the domain responsible for the immunosuppressive activity of glycodelin lies on its protein backbone and the glycans modulate the same. This study attempts to further delineate the apoptosis inducing region of GdA. Our results demonstrate that the stretch of amino acid sequence between Met24 to Leu105 is necessary and sufficient to inhibit proliferation of T cells and induce apoptosis in them. Further, within this region the key residues involved in harboring the activity were shown to be present between Asp52 and Ser65.     

药物靶标蛋白在蛋白网络中的分布

药物靶标发现是目前生物学研究领域的热点和难点问题。从已有药物靶标中寻找规律可以为新靶标的发现总结规律,提供依据。随着功能基因组学的发展,这种组学数据的积累为这一问题的研究提供了契机。本文研究了已有靶标在蛋白网络中的分布,并分析了它们的蛋白功能域组成情况。结果显示靶标基因倾向位于网络的核心区域,并且集中在一些特定蛋白家族中。这些规律的总结将对药物研发过程中药物靶点的选择提供一定的帮助。     

The protein as a variable in protein crystallization

Strategies for growing protein crystals have for many years been essentially empirical, the protein, once purified to a certain homogeneity, being mixed with a selection of crystallization agents selected in a more or less trial-and-error fashion. Screening for the correct conditions has been made easier through automation and by the introduction of commercially available crystallization kits. Many parameters can be changed in these experiments, such as temperature, pH, and ionic strength, but perhaps the most important variable has been ignored, namely the protein. The crystallization properties of a protein vary greatly: some crystallize readily, whereas others have proven extremely difficult or even impossible to obtain in a crystalline state. The possibility of altering the intrinsic characteristics of a protein for crystallization has become a feasible strategy. Some historical perspectives and advances in this area will be reviewed.     

Flexible ligand-flexible protein docking in protein kinase systems

Quite a few reviews on molecular docking have already appeared. This mini-review focuses on methods that incorporate protein flexibility in docking rather than those that treat protein targets as rigid molecules. This is still a challenging problem but there are encouraging recent advances. These methods will be reviewed particularly in light of their applications to protein kinases and phosphatases. In addition to obtaining correct docking pose, recent developments on exploring docking pathways are also highlighted.     

Patterns of protein protein interactions in salt solutions and implications for protein crystallization

The second osmotic virial coefficients of seven proteins-ovalbumin, ribonuclease A, bovine serum albumin, alpha-lactalbumin, myoglobin, cytochrome c, and catalase-were measured in salt solutions. Comparison of the interaction trends in terms of the dimensionless second virial coefficient b(2) shows that, at low salt concentrations, protein-protein interactions can be either attractive or repulsive, possibly due to the anisotropy of the protein charge distribution. At high salt concentrations, the behavior depends on the salt: In sodium chloride, protein interactions generally show little salt dependence up to very high salt concentrations, whereas in ammonium sulfate, proteins show a sharp drop in b(2) with increasing salt concentration beyond a particular threshold. The experimental phase behavior of the proteins corroborates these observations in that precipitation always follows the drop in b(2). When the proteins crystallize, they do so at slightly lower salt concentrations than seen for precipitation. The b(2) measurements were extended to other salts for ovalbumin and catalase. The trends follow the Hofmeister series, and the effect of the salt can be interpreted as a water-mediated effect between the protein and salt molecules. The b(2) trends quantify protein-protein interactions and provide some understanding of the corresponding phase behavior. The results explain both why ammonium sulfate is among the best crystallization agents, as well as some of the difficulties that can be encountered in protein crystallization.     

Phospholipid metabolism and protein kinase C mediated protein phosphorylation in dietary protein deficiency in rat lung

Nutritional deprivation of proteins decreases the protein kinase C (PKC) activity in rat lung. The activity of (PKC) is influenced by lipid metabolism. Changes in PKC activity may influence phosphorylation of its substrate proteins in the tissues. Therefore, alterations in phospholipid metabolism and PKC mediated protein phosphorylation in dietary protein deficiency in rat lung were envisaged. The study was conducted on rats fed on three different types of diet viz., casein (20% protein), deficient (4% protein, rice flour as source of protein) and supplemented (deficient diet supplemented with L-lysine and DL-threoning). Feeding of protein deficient diet caused reduction in incorporation of [3H] myo-inositol in the total phosphoinositides in lungs and an increase in total inositol phosphate pool. There was a significant reduction in the contents and turnover rate of phosphatidyl inositol and phosphatidyl inositol monophosphate. Supplementation of diet with L-lysine and DL-threonine had a reversing effect on total pool of phosphoinositides and, the metabolism of phosphatidyl inositol bisphosphate and phosphatidyl inositol. In phosphatidyl choline metabolism, the dietary protein deficiency led to a decrease in incorporation of [14C-methyl] choline-chloride in total phospholipids. In contrast, its incorporation increased in phosphatidyl choline pool. The contents of phosphatidyl choline and residue, incorporation of [14C-methyl] choline-chloride in them and their turnover rate also increased. Supplementation of diet had a reversal effect on most of these parameters. Phosphorylation of proteins of 84, 47, 35 and 16 kDa was identified to be mediated by PKC. In dietary protein deficiency, phosphorylation of all these proteins, except that of 47 kDa, increased. Supplementation of diet reversed the pattern except that of 84 kDa. The findings suggest that changes in phospholipid metabolism in dietary protein deficiency may effect the activity of PKC thereby influencing the phosphorylation of its substrate proteins and hence associated functions that may lead to pathophysiology of lung.     

Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions

We have previously described a strategy for detecting protein protein interactions based on protein interaction assisted folding of rationally designed fragments of enzymes. We call this strategy the protein fragment complementation assay (PCA). Here we describe PCAs based on the enzyme TEM-1 beta-lactamase (EC: 3.5.2.6), which include simple colorimetric in vitro assays using the cephalosporin nitrocefin and assays in intact cells using the fluorescent substrate CCF2/AM (ref. 6). Constitutive protein protein interactions of the GCN4 leucine zippers and of apoptotic proteins Bcl2 and Bad, and the homodimerization of Smad3, were tested in an in vitro assay using cell lysates. With the same in vitro assay, we also demonstrate interactions of protein kinase PKB with substrate Bad. The in vitro assay is facile and amenable to high-throughput modes of screening with signal-to-background ratios in the range of 10:1 to 250:1, which is superior to other PCAs developed to date. Furthermore, we show that the in vitro assay can be used for quantitative analysis of a small molecule induced protein interaction, the rapamycin-induced interaction of FKBP and yeast FRB (the FKBP-rapamycin binding domain of TOR (target of rapamycin)). The assay reproduces the known dissociation constant and number of sites for this interaction. The combination of in vitro colorimetric and in vivo fluorescence assays of beta-lactamase in mammalian cells suggests a wide variety of sensitive and high-throughput large-scale applications, including in vitro protein array analysis of protein protein or enzyme protein interactions and in vivo applications such as clonal selection for cells expressing interacting protein partners.     

Activities of the Sex-lethal protein in RNA binding and protein:protein interactions.

The Drosophila sex determination gene Sex-lethal (Sxl) controls its own expression, and the expression of downstream target genes such as transformer , by regulating pre-mRNA splicing and mRNA translation. Sxl codes an RNA-binding protein that consists of an N-terminus of approximately 100 amino acids, two 90 amino acid RRM domains, R1 and R2, and an 80 amino acid C-terminus. In the studies reported here we have examined the functional properties of the different Sxl protein domains in RNA binding and in protein:protein interactions. The two RRM domains are responsible for RNA binding. Specificity in the recognition of target RNAs requires both RRM domains, and proteins which consist of the single domains or duplicated domains have anomalous RNA recognition properties. Moreover, the length of the linker between domains can affect RNA recognition properties. Our results indicate that the two RRM domains mediate Sxl:Sxl protein interactions, and that these interactions probably occur both in cis and trans. We speculate that cis interactions between R1 and R2 play a role in RNA recognition by the Sxl protein, while trans interactions stabilize complex formation on target RNAs that contain two or more closely spaced binding sites. Finally, we show that the interaction of Sxl with the snRNP protein Snf is mediated by the R1 RRM domain.     

C-reactive protein decreases protein phosphorylation in stimulated human neutrophils

Treatment of human neutrophils with C-reactive protein (CRP) causes a concentration-dependent in the extent of activation of superoxide production and of granule secretion, induced by phorbol-12-myristate-13-acetate (PMA) or N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF). The same treatment also causes a significant reduction in the degree of PMA- and fMLF-stimulated phosphorylation of several cell proteins. These include the proteins of 43-47 kDa, whose extent of phosphorylation correlates with the activation of superoxide production and of secretion. Contrary to the effects exerted on protein phosphorylation, CRP does not affect the fMLF-elicited increase in neutrophil cytosolic Ca2+.     

Two protein/protein interaction assays in one go

Discovering and characterizing protein–protein interactions (PPIs) that contribute to cellular homeostasis, development, and disease is a key priority in proteomics. Numerous assays for protein–protein interactions have been developed, but each one comes with its own strengths, weaknesses, and false‐positive/false‐negative rates. Therefore, it seems rather intuitive that combining multiple assays is beneficial for robust and reliable discovery of interactions. Along those lines, in their recent study, Wanker and colleagues (Trepte et al, 2018 ) combined two complementary and quantitative interaction assays in one pot. One assay is luminescence‐based and depends on protein proximity in living cells, while the other relies on formation of more stable complexes detected by co‐precipitation with a luminescence‐based readout, which facilitates confident identification and quantitation of interactions in high throughput.     

Thermal motion of whole protein molecules in protein solids

A preliminary analysis is presented of whether and to what extent thermal motion of protein molecules as rigid bodies contributes to the mobility found in X-ray and M?ssbauer studies. A simple theory is advanced enabling the estimation of mean-square amplitudes of translational and librational motion of the protein molecules in crystals and amorphous glasses from the experimental data on the elastic properties of these solids. The values calculated and their dependence on the crystal packing, temperature and hydration level were found to be in good accord with the data of X-ray analysis and M?ssbauer spectroscopy. The external modes were concluded to contribute significantly to the values of mean value of chi 2 measured by the last two methods meaning that the conventional amplitudes of internal motion in proteins were overestimated. The real average amplitude of thermal motion of atoms in the protein interior should be close to that in molecular crystals, in accordance with the crystal-like packing of atoms inside the protein globule and some other "crystal-like" physical characteristics such as Young's modulus, adiabatic compressibility and thermal expansion coefficient. Factors are discussed which determine the temperature dependence of the amplitudes of external and internal modes of protein motion.     

SecA protein is directly involved in protein secretion in Escherichia coli

A high-expression plasmid for the secA gene was constructed. The SecA protein was then overproduced in E. coli and purified. The purified SecA stimulated the in vitro translocation of a model secretory protein into inverted membrane vesicles pretreated with 4 M urea. Membrane vesicles from a secAts mutant exhibited lower translocation activity, which was enhanced by SecA. These results indicate that SecA is directly involved in protein secretion across the cytoplasmic membrane.     

A fatty acid-binding protein and a protein disulphide isomerase-related protein expressed in urochordate gonad cytosol

Despite the evolutionary-tree data suggesting that gene duplication leading to the divergence of the three branches which heart, liver and intestinal fatty acid-binding proteins belong to must have occurred before the vertebrate/invertebrate split, only the heart fatty acid-binding protein has been reported for invertebrates. In an attempt to shed light on this apparent inconsistency the presence of the other two branch members was investigated in the Urochordata Molgula pedunculata, an ascidian species close to vertebrates. The mantle-, gonad- and digestive tube-cytosolic fractions, obtained by centrifugation at 106,000 g, were incubated separately with [1-(14)C]palmitic acid and then fractionated on a Sephadex G-75 column. In the case of gonads and digestive tube, radioactive peaks corresponding to a molecular mass of 14-16 kDa, characteristic of fatty acid-binding proteins, were detected. When the experiment was performed on the mantle, this peak showing fatty acid binding capacity was absent. Western Blot of the radioactive 14-16 kDa Sephadex fraction from the urochordate gonad cross-reacted with rat liver fatty acid-binding protein anti-serum but did not do so with anti-rat intestinal, adipocyte or heart fatty acid-binding protein antisera. The material from the digestive tube was not recognized by any of the antisera. The most abundant protein in said 14-16 kDa fraction was a protein disulphide isomerase-related protein. Its partial amino acid sequence was determined.     

Differential dynamics of neurofilament-H protein and neurofilament-L protein in neurons

Neurofilaments (NFs) are composed of triplet proteins, NF-H, NF-M, and NF-L. To understand the dynamics of NFs in vivo, we studied the dynamics of NF-H and compared them to those of NF-L, using the combination of microinjection technique and fluorescence recovery after photobleaching. In the case of NF-L protein, the bleached zone gradually restored its fluorescence intensity with a recovery half time of approximately 35 min. On the other hand, recovery of the bleached zone of NF-H was considerably faster, taking place in approximately 19 min. However, in both cases the bleached zone was stationary. Thus, it was suggested that NF-H is the dynamic component of the NF array and is interchangeable, but that it assembles with the other neurofilament triplet proteins in a more exchangeable way, implying that the location of NF-H is in the periphery of the core NF array mainly composed of NF- L subunits. Immunoelectron microscopy investigations of the incorporation sites of NF-H labeled with biotin compounds also revealed the lateral insertion of NF-H subunits into the preexisting NF array, taking after the pattern seen in the case of NF-L. In summary, our results demonstrate that the dynamics of the L and H subunit proteins in situ are quite different from each other, suggesting different and separated mechanisms or structural specialization underlying the behavior of the two proteins.     

Dual function of protein confinement in chaperonin-assisted protein folding.

The GroEL/GroES chaperonin system mediates the folding of a range of newly synthesized polypeptides in the bacterial cytosol. Using a rapid biotin-streptavidin-based inhibition of chaperonin function, we show that the cage formed by GroEL and its cofactor GroES can have a dual role in promoting folding. First, enclosure of nonnative protein in the GroEL:GroES complex is essential for folding to proceed unimpaired by aggregation. Second, folding inside the cage can be significantly faster than folding in free solution, independently of ATP-driven cycles of GroES binding and release. This suggests that confinement of unfolded protein in the narrow hydrophilic space of the chaperonin cage smoothes the energy landscape for the folding of some proteins, increasing the flux of folding intermediates toward the native state.