Role of conserved residues in structure and stability: tryptophans of human serum retinol-binding protein, a model for the lipocalin superfamily

Serum retinol binding protein (RBP) is a member of the lipocalin family, proteins with up-and-down beta-barrel folds, low levels of sequence identity, and diverse functions. Although tryptophan 24 of RBP is highly conserved among lipocalins, it does not play a direct role in activity. To determine if Trp24 and other conserved residues have roles in stability and/or folding, we investigated the effects of conservative substitutions for the four tryptophans and some adjacent residues on the structure, stability, and spectroscopic properties of apo-RBP. Crystal structures of recombinant human apo-RBP and of a mutant with substitutions for tryptophans 67 and 91 at 1.7 A and 2.0 A resolution, respectively, as well as stability measurements, indicate that these relatively exposed tryptophans have little influence on structure or stability. Although Trp105 is largely buried in the wall of the beta-barrel, it can be replaced with minor effects on stability to thermal and chemical unfolding. In contrast, substitutions of three different amino acids for Trp24 or replacement of Arg139, a conserved residue that interacts with Trp24, lead to similar large losses in stability and lower yields of native protein generated by in vitro folding. The results and the coordinated nature of natural substitutions at these sites support the idea that conserved residues in functionally divergent homologs have roles in stabilizing the native relative to misfolded structures. They also establish conditions for studies of the kinetics of folding and unfolding by identifying spectroscopic signals for monitoring the formation of different substructures.     

MPtopo: A database of membrane protein topology

The reliability of the transmembrane (TM) sequence assignments for membrane proteins (MPs) in standard sequence databases is uncertain because the vast majority are based on hydropathy plots. A database of MPs with dependable assignments is necessary for developing new computational tools for the prediction of MP structure. We have therefore created MPtopo, a database of MPs whose topologies have been verified experimentally by means of crystallography, gene fusion, and other methods. Tests using MPtopo strongly validated four existing MP topology-prediction algorithms. MPtopo is freely available over the internet and can be queried by means of an SQL-based search engine.     

Comparing function and structure between entire proteomes

More than 30 organisms have been sequenced entirely. Here, we applied a variety of simple bioinformatics tools to analyze 29 proteomes for representatives from all three kingdoms: eukaryotes, prokaryotes, and archaebacteria. We confirmed that eukaryotes have relatively more long proteins than prokaryotes and archaes, and that the overall amino acid composition is similar among the three. We predicted that approximately 15%-30% of all proteins contained transmembrane helices. We could not find a correlation between the content of membrane proteins and the complexity of the organism. In particular, we did not find significantly higher percentages of helical membrane proteins in eukaryotes than in prokaryotes or archae. However, we found more proteins with seven transmembrane helices in eukaryotes and more with six and 12 transmembrane helices in prokaryotes. We found twice as many coiled-coil proteins in eukaryotes (10%) as in prokaryotes and archaes (4%-5%), and we predicted approximately 15%-25% of all proteins to be secreted by most eukaryotes and prokaryotes. Every tenth protein had no known homolog in current databases, and 30%-40% of the proteins fell into structural families with >100 members. A classification by cellular function verified that eukaryotes have a higher proportion of proteins for communication with the environment. Finally, we found at least one homolog of experimentally known structure for approximately 20%-45% of all proteins; the regions with structural homology covered 20%-30% of all residues. These numbers may or may not suggest that there are 1200-2600 folds in the universe of protein structures. All predictions are available at http://cubic.bioc.columbia.edu/genomes.     

Utilization ofDrosophila eye to probe the functions of two mammalian serine/threonine kinases, Snk and HsHPK

Here we report a quick functional analysis of two mammalian serine/threonine kinases, a serum inducible kinase (Snk) and Homo sapiens hepatoma protein kinase (HsHPK), using Drosophila eye as a model system. We generated transgenic fly lines carrying constructs of both kinases under control of the GAL upstream activating sequence (UAS). Each UAS line was then crossed to a line in which GAL4 expression was driven by one of the following promoters, eyeless (ey), glass or decapentaplegic. Thus, different kinase mutants can be ectopically expressed in a promoter-dependent manner. We observed that the ectopic expression of either the wild-type or active form of Snk driven by the glass promoter resulted in a rough-eye phenotype. Nevertheless, the ectopic expression of HsHPK under the control of the ey promoter resulted in a small-eye phenotype. The results of this study demonstrated that ectopic expression of these two mammalian genes could be achieved by the regulation of Drosophila promoters. In addition, the effects of these ectopically expressed genes on eye development could be an implication of their functions with respect to cell proliferation and differentiation. Thus, Drosophila eye, with the powerful genetic tools and vast information on eye development available, can be a useful system to probe the functions of mammalian genes in the postgenome era.     

Synaptic pattern of sex complements and sperm head malformation in X-autosome translocation carrier bulls

Testicular activity and semen characteristics of bulls carrying an X-autosome translocation t(Xp +;23q-) revealed all stages of spermatogenesis although their semen consisted of few and, exclusively, of malformed spermatozoa. Chromosome painting on metaphase spreads of their mother and synaptonemal complex analysis on these and normal bulls were carried out to test whether the location and meiotic pairing behaviour of the rearranged segments could have contributed to the sperm head malformation and oligospermia in our X-autosome translocation (X-AT) carrier bulls. Spermatocytes of X-AT carriers displayed the rearranged chromosomes in a univalent-trivalent association, with 23q- always remaining as a univalent and Xp + in synapsis with normal chromosome 23 and the Y chromosome. Chromosome painting studies to test whether the total absence of meiocytes showing a quadrivalent is due to the non-reciprocal nature of this translocation, identified Xp sequence homology with the distal end of 23q- confirming its relocation to the terminal segment of 23q-. Our synaptonemal complex analyses also confirmed that the bovine pseudo-autosomal region (PAR) is at the distal ends of Xq and Yp and further revealed that over 85% of spermatocytes of X-AT carriers (and up to 13% of spermatocytes of normal bulls) sustain a Y-axis break adjacent to the PAR. Although the exact cause of a Y-axis break in bovine spermatocytes is not known at present, we believe that the break and possible loss of Yq in such high proportions of spermatocytes of X-AT carriers could have contributed to the sperm head malformation and oligospermia in our X-AT carrier bulls.     

Fluorescence correlation spectroscopy and its potential for intracellular applications

Fluorescence correlation spectroscopy (FCS) is a time-averaging fluctuation analysis of small molecular ensembles, combining maximum sensitivity with high statistical confidence. Among a multitude of physical parameters that are, in principle, accessible by FCS, it most conveniently allows to determine local concentrations, mobility coefficients, and characteristic rate constants of fast-reversible and slow-irreversible reactions of fluorescently labeled biomolecules at very low (nanomolar) concentrations, under equilibrium conditions and without physical separation. Its presently most popular instrumentation by confocal-microscope setups allows for a spatial resolution of fractions of femtoliters for the measurement volumes, containing sparse or even single molecules at any time, and encourages the adaptation of the solution-based technique for cellular applications. The scope of this review is thus, to introduce the FCS technique in particular to the reader with biological background, searching for new methods for a precise quantification of physical parameters governing cellular mechanisms and dynamics, especially if high sensitivity and fast dynamic resolution are required. After a short theoretical introduction, examples are given for the so far most important experimental applications, with respect to their implementation in cellular systems. As an interesting alternative to the confocal instrumentation, two-photon excitation will be introduced, offering a number of important advantages especially in cellular systems with high-noise and low-signal levels.     

Down-regulation of particulate protein kinase Cepsilon and up-regulation of nuclear activator protein-1 DNA binding in liver following in vivo exposure of B6C3F1 male mice to heptachlor epoxide

The effects of in vivo administration of the cyclodiene tumor promoter heptachlor epoxide on mouse liver protein kinase C were studied in male B6C3F1 mice by protein kinase C activity assays and Western blotting under conditions known to increase the incidence of hepatocellular carcinoma because protein kinase C is thought to be critical in phorbol ester-induced tumor promotion. Under these test conditions, 20 ppm dietary heptachlor epoxide for 1-20 days increased cytosolic and decreased particulate total protein kinase C activities, while 10 ppm had no effect. Further, total cytosolic and particulate protein kinase C activities were decreased within 1 hour by 10 mg/kg intraperitoneal (i.p.) heptachlor epoxide. Western blotting showed that conventional protein kinase Calpha and beta isoforms were unaffected by heptachlor epoxide. Particulate novel protein kinase Cepsilon, however, was selectively down-regulated by 1, 10, and 20 ppm dietary heptachlor epoxide, whereas the cytosolic isoform was decreased by 1 and 10 ppm heptachlor epoxide for 10 days. The high-dose treatment for 24 hours also decreased particulate novel protein kinase Cepsilon but increased the cytosolic titer. These results demonstrate that this isoform is unique in its sensitivity to heptachlor epoxide. Activator protein-1 DNA binding, a critical factor in tumor promotion, was substantially increased at 3 and 6 hours with 3.7 mg/kg (i.p.) heptachlor epoxide and at 3 and 10 days with 20 ppm dietary heptachlor epoxide. The effects of heptachlor epoxide on protein kinase C and activator protein-1 are similar to those caused by phorbol ester treatments and correlate well to heptachlor levels found to induce tumors in mice. However, heptachlor epoxide did not initially activate protein kinase C with in vivo treatments or with in vitro treatments of a plasma membrane fraction aimed at demonstrating direct activation, as has been shown for phorbol esters. The ability of heptachlor epoxide to down-regulate particulate novel protein kinase Cepsilon correlates to dosages used in in vivo tumor promotion studies. However, this may represent a negative feedback response rather than a causative effect.     

Ethanol- and nicotine-induced membrane changes in embryonic and neonatal chick brains

In order to study the effects of EtOH and/or nicotine on brain membrane fatty acid composition, various concentrations of EtOH and/or nicotine were injected into the air sac of chicken eggs at 0 days of incubation. Controls were injected with saline. Experimental groups were injected with either 200 micromol EtOH/kg egg, 100 micromol nicotine/kg egg, 200 micromol nicotine/kg egg, 200 micromol EtOH/kg and 100 micromol nicotine/kg egg, or 200 micromol EtOH/kg and 200 micromol nicotine/kg egg. In all experimental groups, EtOH- and nicotine-induced decreases in brain long-chain polyunsaturated membrane fatty acids were observed in stage 44 embryos, stage 45 embryos, and neonatal chicks. These EtOH- and nicotine-induced decreases in brain membrane polyunsaturated fatty acids correlated with elevated levels of brain lipid hydroperoxides and reduced brain acetylcholinesterase (AChE; EC. 3.1.1.7) activities.     

Region of heat-stable enterotoxin II of Escherichia coli involved in translocation across the outer membrane

Heat-stable enterotoxin II of Escherichia coli (STII) is synthesized as a precursor form consisting of pre- and mature regions. The pre-region is cleaved off from the mature region during translocation across the inner membrane, and the mature region emerges in the periplasm. The mature region, composed of 48 amino acid residues, is processed in the periplasm by DsbA to form an intramolecular disulfide bond between Cys-10 and Cys-48 and between Cys-21 and Cys-36. STII formed with these disulfide bonds is efficiently secreted out of the cell through the secretory system, including TolC. However, it remains unknown which regions of STII are involved in interaction with TolC. In this study, we mutated the STII gene and examined the secretion of these STIIs into the culture supernatant. A deletion of the part covering from amino acid residue 37 to the carboxy terminal end did not markedly reduce the efficiency of secretion of STII into the culture supernatant. On the other hand, the efficiency of secretion of the peptide covering from the amino terminal end to position 18 to the culture supernatant was significantly low. These observations indicated that the central region of STII from amino acid residue 19 to that at position 36 is involved in the secretion of STII into the milieu. The experiment using a dsbA-deficient strain of E. coli showed that the disulfide bond between Cys-21 and Cys-36 by DsbA is necessary for STII to adapt to the structure that can cross the outer membrane.     

Genomic analysis of protein kinases,protein phosphatases and two-component regulatory systems of the cyanobacterium Anabaena sp. strain PCC 7120

Anabaena sp. PCC 7120 is a cyanobacterium capable of performing several important biological functions: photosynthesis, nitrogen fixation, cell differentiation, cell-cell communication, etc. These activities require an extensive signaling capability in order to respond to the changing environment. Based on the genomic data, we have retrieved several gene families encoding signaling components. It is estimated that 211 genes encode two-component signaling elements, and 66 genes encode Ser/Thr kinases and phosphatases. These genes together represent 4.2% of the coding capacity of the whole genome, making Anabaena PCC 7120 a leading member among prokaryotes in terms of its signaling potential. It is known that two-component systems are composed of a few basic modules that can arrange into different structures best adapted for each signaling system. Many proteins in Anabaena PCC 7120 have incorporated both modules of two-component systems and catalytic domains of either Ser/Thr kinases or phosphatases. A family of 13 genes encode proteins with both a Ser/Thr kinase domain and a His kinase domain, and another four genes were also found whose products have both a response regulator domain and a Ser/Thr phosphatase domain. Of all the signaling proteins in Anabaena PCC 7120, about one third (35%) are conserved in the genome of the unicellular cyanobacterium strain Synechocystis sp. PCC 6803. Interestingly, one subfamily of His kinases and two subfamilies of response regulators are found in Anabaena PCC 7120 but are absent in Synechocystis PCC 6803. This study constitutes a basis for analyses of signal transduction in Anabaena PCC 7120 using functional genomic approaches.