Comparison of the predicted structure for the activated form of the P21 protein with the X-ray crystal structure

The predicted conformation and position of the central transforming region (residues 55–67) of the p21 protein are compared with the conformation and position of this segment in a recently determined X-ray crystal structure of residues 1–166 of this protein in the activated state bound to a nonhydrolyzable GTP derivative. We previously predicted that this segment of the protein would adopt a roughly extended conformation from Ile 55-Thr 58, a reverse turn at Ala 59-Gln 61, followed by an -helix from Glu 62-Met 67. We further predicted that this region of the activated protein occupies a position that is virtually identical to corresponding regions in the homologous purine nucleotide-binding proteins, bacterial elongation factor (EF-tu), and adenylate kinase (ADK). We find that there is a close correspondence between the conformation and position of our predicted structure and those found in the X-ray crystal structure. A mechanism for activation of the protein is proposed and is corroborated by X-ray crystallographic data.     

Protein phosphorylation in rat liver mitochondria

Summary Incubation of rat liver mitochondria in the presence of either [³²P] Pi or ₃₂ ^y -P] ATP resulted in a phosphorylation of four proteins with Mr 50, 47, 44 and 36 kDa, respectively. The endogenous phosphorylation of these proteins in the presence of [³²P] Pi was markedly influenced by the osmolarity of the incubation medium and differentially affected by various effectors of mitochondrial functions, such as Ca²⁺, oligomycin, FCCP, arsenite and dichloroacetate. In particular, the 36 kDa protein, unlike the other proteins, appears to be phosphorylated also by direct incorporation of [³²P], independently of respiratory chain-linked ATP synthesis. The four proteins, located in the mitoplasts, seem to be phosphorylated by diiferent protein kinases, as suggested by the observation that the endogenous phosphorylation of 36 kDa protein resulted selectively increased by addition of exogenous protein kinases, such as casein kinases S and TS. A tentative identification of these phosphorylatable protein is discussed.     

Calmodulin binds to a tubulin binding site of the microtubule-associated protein tau

Previous studies have demonstrated that the microtubule - associated proteins MAP-2 and tau interact selectively with common binding domains on tubulin defined by the low-homology segments a (430–441) and (422–434). It has been also indicated that the synthetic peptide VRSKIGSTENLKHQPGGG corresponding to the first tau repetitive sequence represents a tubulin binding domain on tau. The present studies show that the calcium-binding protein calmodulin interacts with a tubulin binding site on tau defined by the second repetitive sequence VTSKCGSLGNIHHKPGGG. It was shown that both tubulin and calmodulin bind to tau peptide-Sepharose affinity column. Binding of calmodulin occurs in the presence of 1 mM Ca 2+ and it can be eluted from the column with 4 mM EGTA. These findings provide new insights into the regulation of microtubule assembly, since Ca 2+/calmodulin inhibition of tubulin polymerization into microtubules could be mediated by the direct binding of calmodulin to tau, thus preventing the interaction of this latter protein with tubulin.     

Non-histone chromatin proteins in beef thyroid: distinct phosphorylation patterns of several protein kinases

Summary Non-histone chromatin protein (NHCP) fractions were extracted from purified beef thyroid nuclear preparations and tested for the presence of protein kinase activities using several known mediators of thyroid regulation, as well as potential phosphotransferase substrates using purified or partially purified protein kinase activities. The addition of cAMP/3-isobutyl-l-methylxanthine had no effect on NHCP historic kinase activity; the addition of 10 g of the heat-stable cAMP-dependent protein kinase A inhibitor, however, resulted in a 47% reduction in histone H₂ kinase activity. Nuclear casein kinase II activity was present in the NHCP fractions as evidenced by the capacity of spermine to stimulate (ED₅₀ = 0.19 mM) and heparin to inhibit (ID₅₀ = 0.09 g/ml) the phosphorylation of casein; further, the phosphotransferase activity could be purified by sequential casein-agarose and spermine-agarose affinity chromatography. Neither calcium-calmodulin nor calcium/phosphatidylserine/diolein had an effect on NHCP casein kinase or histone kinase activities, respectively. The addition of cAMP-dependent protein kinase A catalytic subunit, nuclear casein kinase II, calcium-activated calmodulin-dependent protein kinase and diacylglycerol-activated calcium/phospholipid-dependent protein kinase C activities exhibited distinct phosphorylation patterns when NHCP were used as substrates and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography. We conclude that NHCP fraction from beef thyroid: 1) contains both cAMP-dependent protein kinase A catalytic subunit and nuclear casein kinase II and 2) substrates for cAMP-dependent protein kinase A, calcium-activited calmodulin-dependent protein kinase, protein kinase C, and nuclear casein kinase II.Abbreviations NHCP Non-Histone Chromatin Proteins - PK-A cAMP-Dependent Protein Kinase - CAMPK Calcium-Activated Calmodulin-Dependent Protein Kinase - PK-C Diacylglycerol-Activated Calcium/phospholipid-dependent Protein Kinase - NK-11 Nuclear Casein Kinase 11 - CK-G Cytosolic Casein Kinase G or 11 - PMSF Phenylmethyl Sulfonyl Fluoride - PKI the Heat Stable PK-A Inhibitor (Walsh inhibitor) - SDS-PAGE Sodium Dodecylsulfate Polyacrylamide Gel Electrophoresis - EDTA Ethylenediamine Tetraacetic Acid - EGTA Ethyleneglycol bis- (B-aminoethyl ether) N,N,N,N,-Tetraacetic Acid - PS Phosphatidylserine - DO 1,2-Diolein     

Soluble and membrane-bound ferrisiderophore reductases of Escherichia coli K-12

After uptake of microbial ferrisiderophores, iron is assumed to be released by reduction. Two ferrisiderophore-reductase activities were identified in Escherichia coli K-12. They differed in cellular location, susceptibility to amytal, and competition between oxygen and ferrichrome-iron(III) reduction. The ferrisiderophore reductase associated with the 40,000×g sediment (membrane-bound enzyme) was inhibited by 10 mM amytal in contrast to the ferrisiderophore reductase present in the 100,000×g supernatant (soluble enzyme). Reduction by the membrane-bound enzyme followed sigmoid kinetics, but was biphasic in the case of the soluble enzyme. The soluble reductase could be assigned to a protein consisting of a single polypeptide of M _r 26000. Reduction of iron(III) by the purified enzyme depended on the addition of NADH or NADPH which were equally active reductants. The cofactor FMN and to a lesser degree FAD stimulated the reaction. Substrate specificity of the soluble reductase was low. In addition to the hydroxamate siderophores arthrobactin, schizokinen, fusigen, aerobactin, ferrichrome, ferrioxamine B, coprogen, and ferrichrome A, the iron(III) complexes of synthetic catecholates, dihydroxy benzoic acid, and dicitrate, as well as carrier-free iron(III) were accepted as substrates. Both ferrisiderophore reductases were not controlled by the fur regulatory system and were not suppressed by anaerobic growth.Abbreviations DHB dihydroxybenzoic acid - MECAM 1,3,5-N,N,N-tris-(2,3-dihydroxybenzoyl)-triamino-methylbenzene - MECAMS 2,3-dihydroxy-5-sulfonyl-derivative of MECAM     

Organization and nucleotide sequence of the genes for ribosomal protein S2 and elongation factor Ts in Spirulina Platensis

A 6.5 kb region from the genome of the cyanobacterium Spirulina platensis was cloned using as a probe the Escherichia coli gene for ribosomal protein S2. Sequence analysis revealed, in this region, the presence of the gene for ribosomal protein S2 and part of the gene for the elongation factor Ts (EF-Ts). The arrangement rpsB-spacer-tsf resembles that reported for E. coli. The deduced amino acid sequences of the platensis S2 and EF-Ts show significant homology with the E. coli counterparts.     

Suppression of protein kinase C and the stimulation of glucocorticoid receptor synthesis by dexamethasone in human fibroblasts derived from tumor tissue

Exposure of fibroblasts derived from keloid tissues, desmoid and dermal tissue from individuals with Gardner's syndrome (GS) to dexamethasone resulted in the suppression of protein kinase C (PKC) activity and [3H]thymidine incorporation into DNA, and a 20-fold induction of glutamine synthetase activity. Treatment of GS and keloid fibroblasts with 0.1 microM dexamethasone for 36 h increased glucocorticoid receptor (GR) synthesis, as determined by [35S]methionine labeling and immunoprecipitation with a monoclonal antibody to the human GR. The suppression of PKC activity by dexamethasone was shown to result from a loss of protein mass as determined by immunoblotting using an antibody to PKC type III. In contrast to these results, exposure of fibroblasts isolated from normal tissues to dexamethasone did not result in the suppression PKC and [3H]thymidine incorporation, there was only a sixfold induction of glutamine synthetase, and a decrease of GR synthesis. As no primary receptor binding defect could be detected, the altered response of tumor cells to steroid-occupied receptor indicates a partial post-receptor binding defect in GS and keloid cells.     

Large scale protein separations: engineering aspects of chromatography

The engineering considerations common to large scale chromatographic purification of proteins are reviewed. A discussion of the industrial chromatography fundamentals is followed by aspects which affect the scale of separation. The separation column geometry, the effect of the main operational parameters on separation performance, and the physical characteristics of column packing are treated. Throughout, the emphasis is on ion exchange and size exclusion techniques which together constitute the major portion of commercial chromatographic protein purifications. In all cases, the state of current technology is examined and areas in need of further development are noted.

The physico-chemical advances now underway in chromatographic separation of biopolymers would ensure a substantially enhanced role for these techniques in industrial production of products of new biotechnology.     

The Cellular Mechanism of Orcadian Rhythms-A View on Evidence, Hypotheses and Problems

A stable period length is a characteristic property of circadian oscillations. The question about whether higher frequency oscillators (0.5-8 hr) contribute to or establish the stable circadian periodicity cannot be answered at present. A sequential coupling of quantal subcycles appears possible on the basis of known “ultradian” oscillations. There is, however, no supporting evidence for such a concept. Phase response curves of the circadian clock derived from various perturbing pulses allow qualitative conclusions concerning the perturbed clock process. Deductions from computer simulations also allow conclusions about the phase of this oscillatory process.

The distinction between processes (a) essential to the clock mechanism, (b) maintaining and controlling the clock (inputs) and (c) depending on the clock (outputs) on the basis of “oscillatory” and “change of φ or τ after perturbation” seems to be useful but not stringent. Protein synthesis may be an essential or input process. Oscillatory changes of this process may be due to periodic translational control or RNA-supply. Circadian changes in protein concentration and/or activity may depend on periodic synthesis, proteolysis, covalent modifications or aggregations. Specific essential proteins have not been identified conclusively. The large overlap between the group of agents and treatments that phase shift the clock and the group that induces stress proteins suggest that the latter may play a role in the controlling (input) or essential domain.

The role of membranes in the clock mechanism is not clear: concepts assuming an essential function are based on circumstantial evidence. The membrane potential as well as Ca²⁺ may be involved in either input or essential function. Ca²⁺ -calmodulin may also be important as concluded from inhibitor experiments. It is tempting to assume that a calmodulin-dependent kinase is part of a periodic protein phosphorylation process, yet it is not clear whether the periodic protein phosphorylation that has been observed is essential or is just another output process.     

Conformational and helicoidal analysis of the molecular dynamics of proteins: "curves," dials and windows for a 50 psec dynamic trajectory of BPTI

A new procedure for the graphic analysis of molecular dynamics (MD) simulations on proteins is introduced, in which comprehensive visualization of results and pattern recognition is greatly facilitated. The method involves determining the conformational and helicoidal parameters for each structure entering the analysis via the method "Curves," developed for proteins by Sklenar, Etchebest, and Lavery (Proteins: Structure, Function Genet. 6:46-60, 1989) followed by a novel computer graphic display of the results. The graphic display is organized systematically using conformation wheels ("dials") for each torsional parameter and "windows" on the range values assumed by the linear and angular helicoidal parameters, and is present in a form isomorphous with the primary structure per se. The complete time evolution of dynamic structure can then be depicted in a set of four composite figures. Dynamic aspects of secondary and tertiary structure are also provided. The procedure is illustrated with an analysis of a 50 psec in vacuo simulation on the 58 residue protein, bovine pancreatic trypsin inhibitor (BPTI), in the vicinity of the local minimum on the energy surface corresponding to a high resolution crystal structure. The time evolution of 272 conformational and 788 helicoidal parameters for BPTI is analyzed. A number of interesting features can be discerned in the analysis, including the dynamic range of conformational and helicoidal motions, the dynamic extent of 2 degrees structure motifs, and the calculated fluctuations in the helix axis. This approach is expected to be useful for a critical analysis of the effects of various assumptions about force field parameters, truncation of potentials, solvation, and electrostatic effects, and can thus contribute to the development of more reliable simulation protocols for proteins. Extensions of the analysis to present differential changes in conformational and helicoidal parameters is expected to be valuable in MD studies of protein complexes with substrates, inhibitors, and effectors and in determining the nature of structural changes in protein-protein interactions.