Buried water molecules in helical transmembrane proteins

Buried water molecules (having no contact with bulk solvent) in 30 helical transmembrane (TM) protein structures were identified. The average amount of buried water in helical TM proteins is about the same as for all water-soluble (WS) proteins, but it is greater than the average for helical WS proteins. Buried waters in TM proteins make more polar contacts, and are more frequently found contacting helices than in WS proteins. The distribution of the buried water binding sites across the membrane profile shows that the sites to some extent reflect protein function. There is also evidence for asymmetry of the sites, with more in the extracellular half of the membrane. Many of the buried water contact sites are conserved across families of proteins, including family members having different functions. This suggests that at least some buried waters play a role in structural stabilization. Disease-causing mutations, which are known to result in misfolded TM proteins, occur at buried water contact sites at a higher than random frequency, which also supports a stabilizing role for buried water molecules.     

Poly(adenosine diphosphate ribose) synthetase activity in nuclei of dividing and of non-dividing but differentiating intestinal epithelial cells.

Poly(ADP-ribose) synthetase activity is found in nuclei of regenerating epithelial cells in the lower half of the crypts of guinea-pig small intestine. Nuclei from non-dividing but differentiating and maturing cells in the upper crypts and on the villi contain no more than about 10% of the synthetase activity of lower-crypt cell nuclei. The product in the active nuclei is shown to be 80% poly(ADP-ribosylated) protein and 20% mono(ADP-ribosylated) protein; 60% of thetotal labelled product was attached to acid-soluble proteins (including histones), and 40% to acid-insoluble (non-histone) proteins. The average number of ADP-ribosyl units in the oligomeric chains of the poly(ADP-ribosylated) proteins was 15 but the range of sizes of (ADP-ribose) oligomers attached to nuclear proteins was smaller in villus than in crypt cell nuclei.     

Genomic organization of the fungus Phycomyces

The fungus Phycomyces blakesleeanus has a relatively small genome, 30 megabases (Mb), with a low guanine and cytosine (G+C) content, 35%; the coding sequences cloned to date all have a G+C content of about 50%. In order to investigate the organization of the genome of this fungus, we have cloned and sequenced 251 DNA fragments. One hundred and twenty-six clones were obtained by digestion with MspI (target sequence 5′-CCGG-3′) and 125 random clones were obtained by sonication. The average length of sequence obtained was about 200 base pairs (bp) and the total length was about 50 kilobases (kb). The G + C content is not homogeneous throughout the genome: sequences obtained after digestion with MspI have an average of 5% more G + C content than the random fragments, and are enriched in coding sequences. Fourteen MspI fragments show similarities to known proteins and 21 encode ribosomal RNA (rRNA). By contrast, only three of the random fragments are similar to known proteins and only one to a rRNA. We conclude that the Phycomyces genome is composed of G+C-rich genes surrounded by G+C-poor areas. Two clones have similarities to the transposase of the transposon Tcl from Caenorhabditis elegans. This result suggests the presence of a high copy number of a Tcl-like transposable element in the Phycomyces genome. Another clone was similar to the transposon Txl from Xenopus laevis. A novel repetitive nt sequence has been characterized; about 5% of the total genome is a repetition of any of two consensus sequences of 31 by named PrAI and PrA2.     

State-of-the-art biomolecular core facilities: a comprehensive survey

A survey of 124 protein and/or nucleic acid chemistry facilities has provided a basis for estimating the resources needed to establish a facility, the financial support needed to keep it operating, and the technical capabilities it might reasonably be expected to achieve. Based on these data, an average core facility occupied 870 ft2, was staffed by three full-time personnel, and was equipped with 4-5 major instrument systems. Because user fees generated an average of about $101,000/year in income compared with an average operating budget of about $197,000/year, even a facility that charged user fees would, on average, still require an annual subsidy of about $96,000. Although most government and industrial core facilities did not assess user fees, at least 83 of the 124 respondents did have a preestablished schedule of service charges that enabled a compilation to be made of the average cost of providing a number of typical facility analyses and syntheses. The greater than 100-fold range in charges assessed in core facilities for seemingly identical services was shown to result from the equally large range in the degree of subsidization of these laboratories. Although an average facility might be expected to offer four or five of the following six major services--amino acid sequencing, amino acid analysis, HPLC peptide isolation, peptide synthesis, fragmentation of proteins and DNA synthesis--less than 10% of the responding laboratories provided mass spectrometry, capillary zone electrophoresis, or RNA synthesis. With the exception of peptide synthesis, which had an average turn-around time of about 24 days, all other major services had turn-around times that averaged in the range of 4-9 days. Additional data are summarized regarding average sample throughput in core laboratories and the amount of protein that is needed for hydrolysis/amino acid analysis and sequencing.     

Genetic polymorphism in normal human fibroblasts as analyzed by two-dimensional polyacrylamide gel electrophoresis.

Two dimensional gel electrophoresis has been used to measure the degree of genetic polymorphism among the proteins of normal human fibroblasts. Autoradiographic analysis of the gel protein profiles from radioactively labeled cells allowed comparison of as many as 300 discrete polypeptides at a time. In addition, a newly developed technique for double label autoradiography was used to increase the sensitivity of the system for detection of small differences in the protein profiles of different cell lines. Only about 1.2% of the proteins of different cell lines were found to differ in their electrophoretic mobility. This corresponds to an average heterozygosity of approximately 0.6%. Previous studies of genetic polymorphism using different methods of one-dimensional electrophoretic analysis have estimated the average heterozygosity of the human population at about 6.7%. Detailed mathematical analysis shows the variation of the observed from the expected number of differences to be statistically highly significant. While the reasons for this difference are not clear, the observation of low levels of genetic polymorphism on two-dimensional gels should enhance the usefulness of this technique for detection of altered proteins in inherited disease.     

不同营养方式真菌中分泌蛋白数量及其功能对比研究

【目的】分泌蛋白在植物病原真菌致病过程中发挥着重要的作用,前人多以单种菌株分泌蛋白预测分析,尚未见多种类型真菌中分泌蛋白的预测及对比研究报道。【方法】本研究基于多种不同营养方式真菌的全基因组序列,根据分泌蛋白所具有的基本特征,采用在线分析程序(主要包括SignalP v5.0、ProtComp v9.0等)对模式生物、死体营养型真菌、半活体营养型真菌及活体营养型真菌共19种菌株的分泌蛋白开展预测及功能分析。【结果】在上述真菌约13万条蛋白序列中,分泌蛋白所占比例为0.74%–4.83%,其中,活体营养型真菌所具有的分泌蛋白数量占比最高,平均为3.51%,而死体营养型真菌和模式生物平均比例最低,平均为1.36%。同时,活体营养型真菌具有的功能种类最多,为433种,其次为半活体营养型真菌,为266种,而模式生物具有的功能种类最少,为100种,其中,功能为假设蛋白和非特征蛋白的蛋白数量最多。【结论】该研究为深入解析分泌蛋白在实现不同营养方式真菌的致病机制方面提供理论依据。     

Measurement of protein turnover in rat liver. Analysis of the complex curve for decay of label in a mixture of proteins.

The curve for decay of 14C in rat liver protein labelled by injection of NaH14CO3 was analysed to obtain the average turnover rate of mixed liver protein. Three different methods of analysis were used. (1) Unlike decay curves from homogeneous proteins, the curve did not fit a single exponential, but a good fit was obtained with three exponentials. By assuming that the mixture contained three major components with different turnover rates, the calculated value for the average turnover rate (k) was close to 40% per day. (2) k was also calculated from the area under the decay curve, a method which makes no assumptions about the number of proteins in the mixture. This method also gave a value close to 40% per day. (3) It was shown empirically, both by simulation of decay of label in model mixtures of protein and with the decay curve measured in vivo, that k can be calculated from the time taken for the specific radioactivity to fall to 10% of its maximum value. This is an advantage, since the other two methods require the decay curve to be measured over a much longer period of time.     

Compartments of protein metabolism in the developing brain.

We investigated whether the higher rate of amino acid incorporation into immature than into mature brain protein is due to (a) rapid growth, (b) a small rapidly metabolized protein pool, or (c) a higher turnover rate of most of the protein. We measured net growth and the incorporation of [14C]tyrosine or [14C]valine into brain proteins in young rats and mice. The specific activity of the free amino acid pool was kept constant in the tyrosine experiments. Incorporation of tyrosine into protein was continued for up to 30 h by which time the specific activity of protein-bound amino acid reached 1/3 of that of the free (precursor) amino acid. The growth (accretion) of brain proteins was approx. 0.635% per h in mice and rats in the 1-4 day period after birth. In previous studies we found that the turnover rate of the bulk (about 96%) of adult brain proteins is below 0.3% per h. Because of the presence of a small (about 4%) active pool the average turnover rate is 0.6% per h. The present experiments show a degradation rate of 0.7-1.1% per h in the brain proteins of the young. This high metabolic rate is not due to a small rapidly degraded fraction of protein. The very rapid protein fraction previously seen in adult rats is either very small (below 1%) or absent in the young. Thus most of the proteins in the immature brain during the rapid growth phase are formed and broken down at a rate that is approximately three times higher than that of the bulk of proteins in the adult brain. The small active protein pool in the adult on the other hand has a metabolic rate higher than that of the immature brain proteins.     

Synthesis, Transport, and Morphogenesis of Type 5 Adenovirus Capsid Proteins

During the period between 20 and 24 hr after infection of KB cells with type 5 adenovirus, at a time when approximately 85% of the proteins made were virus-specific, viral proteins were synthesized on polyribosomes with an average sedimentation coefficient of 200S. The polypeptide chains synthesized during a 1-min period of labeling with (14)C-amino acids had an average sedimentation coefficient of 3.4S in sucrose gradients containing 1% sodium dodecyl sulfate. Within 1 min after completion, the newly made polypeptide chains were released from polyribosomes, and the majority were transported into the nuclei within 6 min. Meanwhile, the immunological reactivity of the newly synthesized proteins also increased rapidly. During the same 6-min interval after synthesis, the single polypeptide chains assembled into multimeric proteins with average sedimentation coefficients of 6S, 9S, and 12S. The 6S and 12S proteins were identified immunologically as the fiber and hexon capsid proteins, respectively. The 9S protein was trypsin-sensitive and appeared to be the precursor of the penton; it was tentatively identified as the penton base. The penton had a sedimentation coefficient of about 10.5S and sedimented with the hexon in sucrose gradients. The concomitant migration of nascent proteins into the nuclei, development of the capsid proteins' immunological reactivity, and morphogenesis of the multimeric capsid proteins suggest that the single polypeptide chains or small complexes were transported into the nuclei where they assembled into mature structural proteins of the virion.     

Prediction of protein secondary structure by an enhanced neural network.

Computational model of neural network is used for prediction of secondary structure of globular proteins of known sequence. In contrast to earlier works some information about expected tertiary interactions were built in into the neural network. As a result the prediction accuracy was improved by 3% to 5%. Possible applications of this new approach are briefly discussed.     

Highly efficient production of soluble proteins from insoluble inclusion bodies by a two-step-denaturing and refolding method

The production of recombinant proteins in a large scale is important for protein functional and structural studies, particularly by using Escherichia coli over-expression systems; however, approximate 70% of recombinant proteins are over-expressed as insoluble inclusion bodies. Here we presented an efficient method for generating soluble proteins from inclusion bodies by using two steps of denaturation and one step of refolding. We first demonstrated the advantages of this method over a conventional procedure with one denaturation step and one refolding step using three proteins with different folding properties. The refolded proteins were found to be active using in vitro tests and a bioassay. We then tested the general applicability of this method by analyzing 88 proteins from human and other organisms, all of which were expressed as inclusion bodies. We found that about 76% of these proteins were refolded with an average of >75% yield of soluble proteins. This "two-step-denaturing and refolding" (2DR) method is simple, highly efficient and generally applicable; it can be utilized to obtain active recombinant proteins for both basic research and industrial purposes.     

A quadratic discriminant analysis of protein structure classification based on the Helix/Strand content

Based on the 210 non-homologous proteins (domains) classified manually by Michie et al. (J. Mol. Biol. 262, 168-185, 1996), a new structure classification criterion of globular proteins relying on the content of helix/strand has been proposed, using a quadratic discriminant method. Each protein is classified into one of the three classes, i.e. those of alpha class, beta class and alphabeta class (including alpha/beta and alpha+beta classes). According to the new structure classification criterion, of the 210 proteins in the training set, 207 are correctly classified and thus the accuracy is 207/210=98.57%. Multiple cross-validation tests are performed. The jackknife test shows that of the 210 proteins 207 are correctly classified with an accuracy of 98.57%. To test the method further, of 3577 proteins (domains) extracted from SCOP, 91.39% of them are correctly reclassified by the new classification criterion. On average, the accuracy of the new criterion is about 8 percentage points higher than that of the criterion proposed by Nakashima et al. (J. Biochem. 99, 153-162, 1986). Our result shows that the classification based solely on structures is basically consistent with that combining both structural and evolutionary information. Further complete automated classification scheme should consider both structures and evolutionary relationship. The methodology presented provides an appropriate mathematical format to reach this goal.     

Synthesis of cytoskeletal and contractile proteins by cultured IMR-90 fibroblasts

Models of the assembly of cytoskeletal and contractile proteins of eukaryotic cells require quantitative information about the rates of synthesis of individual component proteins. We applied the dual isotope technique of Clark and Zak (1981, J. Biol. Chem., 256:4863-4870) to measure the synthesis rates of cytoskeletal and contractile proteins in stationary and growing cultures of IMR-90 fibroblasts. Fibroblast proteins were labeled to equilibrium with [14C]leucine over several days, at the end of which there was a 4-h pulse with [3H]leucine. Fractional synthesis rates (percent per hour) were calculated from the 3H/14C ratio of cell protein extracts or protein purified by one- or two-dimensional polyacrylamide gel electrophoresis and the 3H/14C ratio of medium-free leucine. The average fractional synthesis rate for total, SDS- or urea-soluble; Triton-soluble; and cytoskeletal protein extracts in stationary cells each was approximately 4.0%/h. The range of values for the synthesis of individual proteins from total cell extracts or cytoskeletal extracts sliced from one-dimensional gels was similar, though this range was greater than that for major proteins of Triton-soluble protein extracts. Three specific cytoskeletal proteins--actin, vimentin, and tubulin--were synthesized at similar rates that were significantly slower than the average fractional synthesis rate for total protein. Myosin, on the other hand, was synthesized faster than average. Synthesis rates were the same for beta-and gamma-actin and polymerized (cytoskeletal extract) vs. Triton-soluble actin. The same was true for alpha- and beta-tubulin and two different forms of vimentin. Synthesis rates were uniformly higher in growing cells, though the same pattern of differential rates was observed as for stationary cells. Synthesis rates in growing cells were higher than the rate necessary to maintain the growth rate, even for those cytoskeletal proteins being synthesized slowly. Therefore, there appears to be some turnover of these cytoskeletal elements even during growth. We conclude that proteins in cytoskeletal extracts may have nonuniform rates of synthesis, but at least one important subclass of cytoskeletal proteins that comprise filament subunits have the same synthesis rates.     

Extracellular fluid proteins of goldfish brain: Studies of concentration and labeling patterns

An extraction procedure for the isolation of proteins from the extracellular fluid (ECF) of goldfish brain was developed and applied in an investigation of the time course and pattern of labeling of ECF proteins. The results indicate that two out of the many protein bands present, which migrated at 32,000 and 26,000 daltons on SDS-polyacrylamide electrophoretic gels, could incorporate as much as 50% of the label of the ECF fraction, even though their concentration was only 14%. Measurements of the protein content of the ECF and its volume (24% of the brain) by the inulin method were used to calculate the protein concentration in the extracellular space of goldfish brain. This gave a value of 1.6–2%, i.e., about 50% of the value obtained for the protein concentration of the cytoplasmic fraction devoid of particulate matter. Such a result suggests that the goldfish brain intracellular and extracellular fluids, separated by the neural membranes, contain relatively comparable levels of proteins.     

Labial Gland and Its Protein Patterns of Hydropsychid Caddisfly (Hydropsyche kozhantschikovi Martynov: Trichoptera)

ABSTRACT There is a pair of labial gland of hydropsychid caddisfly ( Hydropsyche kozhantschikovi Martynov) larva. It is in 'Z' formation in the body and the total length is about 20 mm. Hydropsychid caddishfly larvae that were raised under the lab conditions were able to form a nest-spining by connecting the small grains of sand provided. By repeatedly treating the extracted labial gland with methanol/D.W., the cell layer was removed. Accordingly, only the matrix within the labial gland that did not dissolve in water was obtained. The matrix inside the methanol/D.W. treated labial gland was dissolved with 5% acetic acid. Then the results of an acidic electrophoresis with a number of conditions indicated that 5% acetic acid/ 5 M urea/ 8% PAGE was the most effective. Moreover, the result of 2-D PAGE on the labial gland of these hydropsychid caddisfly larva, the number of proteins in the labial gland including the cell layer was about 350 and the number of proteins in the labial gland treated with methanol/D.W. was about 80, showing a substantially small number of proteins.     

Effect of Ultraviolet Light on Mengovirus: Formation of Uracil Dimers, Instability and Degradation of Capsid, and Covalent Linkage of Protein to Viral RNA

UV irradiation of purified mengovirus resulted in a very rapid inactivation of the infectivity of the virions (D(37) [37% survival dose] = 700 ergs/mm(2)) which correlated in time with the formation of uracil dimers in the viral RNA. During the first 2 min of irradiation, an average of 1.7 uracil dimers were formed per PFU of virus inactivated. Hemagglutination activity of the virions began to decrease only after a lag period of about 5 min and at a much lower rate (D(37) = 84,000 ergs/mm(2)). This decrease coincided in time with the appearance of altered proteins in the capsid and a structural change in the capsid. Although 10- to 20-min irradiated virions appeared intact in the electron microscope and sedimented at 150S in sucrose density gradients, the RNA of the virions became accessible to RNase and extractable by low concentrations of sodium dodecyl sulfate, and the virions broke down upon equilibrium centrifugation in CsCl gradients. During longer periods of irradiation (30 to 60 min), a progressively greater proportion of the virions were converted to 14S protein particles and 80S ribonucleoprotein particles composed of intact viral RNA and about 30% of the capsid proteins, alpha, beta, and gamma. Empty capsids were not detectable at any time during 60 min of irradiation, by which time disruption of the virions was complete. Irradiation of complete virions also resulted in an increased sedimentation rate of the viral RNA and in the covalent linkage to the viral RNA of about 1% of the total capsid protein in the form of heterogeneous low-molecular-weight polypeptides. The two observations seem to be causally related, since irradiation of isolated viral RNA did not result in an increase in sedimentation rate of the RNA, even though uracil dimer formation in viral RNA occurred at about the same rate and to the same extent whether intact virions or viral RNA were irradiated.     

Regulation of the pattern of protein synthesis in Schizophyllum commune by the incompatibility genes

The pattern of protein synthesis in various coisogenic mycelial types of Schizophyllum commune, viz. monokaryon, dikaryon, and homokaryons carrying primary mutations in the A and the B factors, was studied by two-dimensional gel electrophoresis. After pulse-labeling with ³⁵S-methionine, approximately 650 of 710 proteins analyzed were common to all mycelial types. Coisogenic monokaryons differed by only 2%, whereas the largest difference was found between these monokaryons and the dikaryon derived from them (6.6 and 7.7%). The majority of these differences fell into two about equally sized categories, i.e., proteins which were either specifically absent (“switched-off” proteins) or present (“switched-on” proteins) in the dikaryon. “Switched-on” proteins were on the average larger and slightly more acidic than “switched-off” proteins. The double factor mutant which best mimicked the dikaryon in morphology also best resembled the dikaryon in types of proteins synthesized. Unexpected, however, was the large overlap in proteins apparently controlled by each of the two incompatibility factors individually, despite the distinct morphological sequences directed by each of them.     

Differential inhibitory effects of antibiotics on the biosynthesis of envelope proteins of Escherichia coli

Inhibitory effects of six antibiotics (kasugamycin, tetracycline, chloramphenicol, sparsomycin, puromycin and rifampicin) on the biosynthesis of envelope proteins of Escherichia coli were examined and compared with those on the biosynthesis of cytoplasmic proteins. Kasugamycin, puromycin and rifampicin were much more inhibitory to the over-all biosynthesis of cytoplasmic proteins than to that of envelope proteins. On the contrary, tetracycline and sparsomycin showed much stronger inhibitory effects on the biosynthesis of envelope proteins than on that of cytoplasmic proteins. Chloramphenicol showed little difference in its inhibitory effect on the biosynthesis of envelope proteins and cytoplasmic proteins.The envelope proteins were labeled with [³H]arginine in the presence of the antibiotics and separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The inhibitory effects of the antibiotics on the biosynthesis of individual envelope proteins were then examined. Inhibition patterns were found to be widely different from one envelope protein to the other. For example, the biosynthesis of one major envelope protein of molecular weight 38,000 was more resistant to kasugamycin, chloramphenicol and sparsomycin than that of the other envelope proteins. On the other hand, the biosynthesis of another major envelope protein (lipoprotein) of about 7500 molecular weight was much more resistant to puromycin and rifampicin than that of the other envelope proteins. In the case of tetracycline, little differential inhibitory effect on the biosynthesis of individual envelope proteins was observed.Stability of messenger RNAs for individual envelope proteins was also determined from the inhibitory effect of rifampicin on their biosynthesis. It was found that the average of half lives of mRNAs for major envelope proteins examined (5.5 minutes) is twice as long as the average of those of mRNAs for cytoplasmic proteins (2 minutes), except for the lipoprotein of about 7500 molecular weight which has extremely stable mRNA with a half life of 11.5 minutes. From these results the envelope proteins of E. coli appear to be biosynthesized in a somewhat different manner from that of the cytoplasmic proteins. Furthermore, at least some envelope proteins may have their own specific biosynthetic systems.     

Glutamine synthetase II inRhizobium: Reexamination of the proposed horizontal transfer of DNA from eukaryotes to prokaryotes

Summary We have determined the DNA sequence of aRhizobium meliloti gene that encodes glutamine synthetase II (GSII). The deduced amino acid sequence was compared to that ofBradyrhizobium japonicum GSII and those of various plant and mammalian glutamine synthetases (GS) in order to evaluate a proposal that the gene for this enzyme was recently transferred from plants to their symbiotic bacteria. There is 83.6% identity between theR. meliloti andB. japonicum proteins. The bacterial GSII proteins average 42.5% identity with the plant GS proteins and 41.8% identity with their mammalian counterparts. The plant proteins average 53.7% identity with the mammalian proteins. Thus, the GS proteins are highly conserved and the divergence of these proteins is proportional to the phylogenetic divergence of the organisms from which the sequences were determined. No transfer of genes across large taxonomic gaps is needed to explain the presence of GSII in these bacteria.