Blood glucose minimum predicts maximal lactate steady state on running.

This study analyzed if the running speed corresponding to glucose minimum (GM) could predict the maximal lactate steady state (MLSS). Thirteen physically active men (25.2+/-4.2 years, 73.4+/-8.0 kg, 180.0+/-1.0 cm) completed three running tests on different days: 1) a 1 600-m time trial to calculate the average speed; 2) after 10-min of recovery from a 150-m sprint to elevate [lac], participants performed 6 series of 800-m respectively at 78, 81, 84, 87, 90 and 93% of the 1 600-m speed to identify the lactate minimum (LM) and GM speeds and 3) 2-4 constant intensity exercise sessions for the MLSS. Repeated measures ANOVA showed no differences between running speeds associated to the GM (201.7+/-23.8 m.min (-1)), LM (200.0+/-23.9 m.min (-1)) and MLSS (201.5+/-23.1 m.min (-1)), with high correlation between GM vs. LM (r=0.984), GM vs. MLSS (r=0.947) and LM vs. MLSS (r=0.961) (P<0.01). Bland and Altman plots showed good agreement [Bias (+/-95% CI)] for MLSS and GM [0.2(15.3) m.min (-1)], MLSS and LM [-1.4(13.2) m.min (-1)], as well as for LM and GM [1.7(8.5) m.min (-1)]. These running speeds occurred at approximately 84.4% of 1 600-m speed, which would have practical applications for exercise prescription. We concluded that GM running speed is a good predictor of the MLSS for physically active individuals.

Detection of activation phases and quantification of coupling in NREM sleep EEG by pointwise transinformation.

BACKGROUND: The coupling dynamics of two time series can be assessed by pointwise transinformation (PTI). Due to its high temporal resolution, this algorithm is ideal for analysis of sleep microstructure. Different types of electroencephalographic (EEG) activation phases, like single K-complexes, K-complexes associated with spindle or alpha activity, K-complexes mixed with delta waves, and arousals, can be detected and changes in EEG coupling can be quantified. METHODS: Nine hundred ninety-one one-minute EEG segments (C3-A2/C4-A1) containing the described types of activation phases were selected from the sleep EEGs of 12 healthy persons. PTI was calculated with 250 Hz resolution and an embedding dimension of 20. An averaged PTI curve was assessed for single K-complexes and K-complexes followed by spindle and alpha activity, respectively. RESULTS: During background activity, PTI was nearly 0. With the onset of a K-complex, PTI increased significantly in a sequence of distinct phases (rising - peak - decay). For single K-complexes, the PTI curve had a nearly symmetric dome-shaped form. The decay phase was prolonged by subsequent spindle or alpha activity. In K-complexes mixed with delta activity and in arousals, repetitive maxima of PTI were obtained. The durations of arousals and their coupling phases were correlated (r=0.83). CONCLUSIONS: PTI displays the coupling dynamics of the sleep EEG with high resolution. It detects phases of activation represented by single K-complexes and various types of arousals. These induce a specific run of the PTI curve clearly distinguishable from background activity. PTI might, therefore, prove useful in the analysis of sleep microstructure.

The laminin-binding domain of agrin is structurally related to N-TIMP-1.

Agrin is the key organizer of postsynaptic differentiation at the neuromuscular junction. This organization activity requires the binding of agrin to the synaptic basal lamina. Binding is conferred by the N-terminal agrin (NtA) domain, which mediates a high-affinity interaction with the coiled coil domain of laminins. Here, we report the crystal structure of chicken NtA at 1.6 A resolution. The structure reveals that NtA harbors an oligosaccharide/oligonucleotide-binding fold with several possible sites for the interaction with different ligands. A high structural similarity of NtA with the protease inhibition domain in tissue inhibitor of metalloproteinases-1 (TIMP-1) supports the idea of additional functions of agrin besides synaptogenic activity.

Stabilization of short collagen-like triple helices by protein engineering.

Recombinant expression of collagens and fragments of collagens is often difficult, as their biosynthesis requires specific post-translational enzymes, in particular prolyl 4-hydroxylase. Although the use of hydroxyproline-deficient variants offers one possibility to overcome this difficulty, these proteins usually differ markedly in stability when compared with the hydroxyproline-containing analogs. Here, we report a method to stabilize collagen-like peptides by fusing them to the N terminus of the bacteriophage T4 fibritin foldon domain. The isolated foldon domain and the chimeric protein (GlyProPro)(10)foldon were expressed in a soluble form in Escherichia coli. The recombinant proteins and the synthetic (ProProGly)(10) peptide were characterized by circular dichroism (CD) spectroscopy, differential scanning calorimetry, and analytical ultracentrifugation. We show that the foldon domain, which comprises only 27 amino acid residues, forms an obligatory trimer with a high degree of thermal stability. The CD thermal unfolding profiles recorded from foldon are monophasic and completely reversible upon cooling. Similar Van't Hoff and calorimertic enthalpy values of trimer formation indicated a cooperative all-or-none transition. As reported previously, (ProProGly)(10) peptides form collagen triple helices of only moderate stability. When fused to the foldon domain, however, triple helix formation of (GlyProPro)(10) is concentration independent, and the midpoint temperature of the triple helix unfolding is significantly increased. The stabilizing function of the trimeric foldon domain is explained by the close vicinity of its N termini, which induce a high local concentration in the range of 1 M for the C termini of the collagen-like-peptide. Collagen-foldon fusion proteins should be potentially useful to study receptor-collagen interactions.

An intrahelical salt bridge within the trigger site stabilizes the GCN4 leucine zipper.

We previously reported that a helical trigger segment within the GCN4 leucine zipper monomer is indispensable for the formation of its parallel two-stranded coiled coil. Here, we demonstrate that the intrinsic secondary structure of the trigger site is largely stabilized by an intrahelical salt bridge. Removal of this surface salt bridge by a single amino acid mutation induced only minor changes in the backbone structure of the GCN4 leucine zipper dimer as verified by nuclear magnetic resonance. The mutation, however, substantially destabilized the dimeric structure. These findings support the proposed hierarchic folding mechanism of the GCN4 coiled coil in which local helix formation within the trigger segment precedes dimerization.

Crystal structure of a naturally occurring parallel right-handed coiled coil tetramer.

The crystal structure of a polypeptide chain fragment from the surface layer protein tetrabrachion from Staphylothermus marinus has been determined at 1.8 A resolution. As proposed on the basis of the presence of 11-residue repeats, the polypeptide chain fragment forms a parallel right-handed coiled coil structure. Complementary hydrophobic interactions and complex networks of surface salt bridges result in an extremely thermostable tetrameric structure with remarkable properties. In marked contrast to left-handed coiled coil tetramers, the right-handed coiled coil reveals large hydrophobic cavities that are filled with water molecules. As a consequence, the packing of the hydrophobic core differs markedly from that of a right-handed parallel coiled coil tetramer that was designed on the basis of left-handed coiled coil structures.

Interaction of agrin with laminin requires a coiled-coil conformation of the agrin-binding site within the laminin gamma1 chain.

Coiled-coil domains are found in a wide variety of proteins, where they typically specify subunit oligomerization. Recently, we have demonstrated that agrin, a multidomain heparan sulfate proteoglycan with a crucial role in the development of the nerve-muscle synapse, binds to the three-stranded coiled-coil domain of laminin-1. The interaction with laminin mediates the integration of agrin into basement membranes. Here we characterize the binding site within the laminin-1 coiled coil in detail. Binding assays with individual laminin-1 full-length chains and fragments revealed that agrin specifically interacts with the gamma1 subunit of laminin-1, whereas no binding to alpha1 and beta1 chains was detected. By using recombinant gamma1 chain fragments, we mapped the binding site to a sequence of 20 residues. Furthermore, we demonstrate that a coiled-coil conformation of this binding site is required for its interaction with agrin. The finding that recombinant gamma1 fragments bound at least 10-fold less than native laminin-1 indicates that the structure of the three-stranded coiled-coil domain of laminin is required for high-affinity agrin binding. Interestingly, no binding to a chimeric gamma2 fragment was observed, indicating that the interaction of agrin with laminin is isoform specific.

Heterodimerization of a functional GABAB receptor is mediated by parallel coiled-coil alpha-helices.

A detailed understanding of GABAB receptor assembly is an important issue in view of its role as attractive target for treatment of epilepsy, anxiety, depression, cognitive defects, and nociceptive disorders. Heteromerization of GABAB-R1 and GABAB-R2 subunits is a prerequisite for the formation of a functional GABAB receptor. Each individual subunit contains one stretch of approximately 30 amino acid residues within its intracellular C-terminal domain that mediates heteromer formation. To investigate the mechanism of the GABAB-R1/GABAB-R2 interaction and to assess the subunit stoichiometry of the complex, recombinant polypeptide chain fragments containing the heteromerization site were produced by heterologous gene expression in Escherichia coli. When mixed in equimolar amounts, these peptides preferentially formed parallel coiled-coil heterodimers under physiological buffer conditions. This demonstrates that the short C-terminal regions are sufficient to determine the specificity of interaction between GABAB receptor subunits. In contrast, isolated GABAB-R1 peptides folded into relatively unstable homodimers, whereas GABAB-R2 peptides were largely unstructured. Together with the data reported in the literature, the results presented here indicate that the functional GABAB receptor is a heterodimer assembled by parallel coiled-coil alpha-helices.

An autonomous folding unit mediates the assembly of two-stranded coiled coils.

Subunit oligomerization of many proteins is mediated by coiled-coil domains. Although the basic features contributing to the thermodynamic stability of coiled coils are well understood, the mechanistic details of their assembly have not yet been dissected. Here we report a 13-residue sequence pattern that occurs with limited sequence variations in many two-stranded coiled coils and that is absolutely required for the assembly of the Dictyostelium discoideum actin-bundling protein cortexillin I and the yeast transcriptional activator GCN4. The functional relationship between coiled-coil "trigger" sequences was manifested by replacing the intrinsic trigger motif of GCN4 with the related sequence from cortexillin I. We demonstrate that these trigger sequences represent autonomous helical folding units that, in contrast to arbitrarily chosen heptad repeats, can mediate coiled-coil formation. Aside from being of general interest for protein folding, trigger motifs should be of particular importance in the protein de novo design.

Crystallization and preliminary X-Ray diffraction analysis of the 190-A-long coiled-coil dimerization domain of the actin-bundling protein cortexillin I from dictyostelium discoideum

We have crystallized the approximately 190-A-long parallel two-stranded coiled-coil oligomerization domain of the actin-bundling protein cortexillin I from Dictyostelium discoideum. The orthorhombic crystals belong to the space group C2221 with unit cell dimensions of a = 71.3 A, b = 127.8 A, and c = 91.6 A. As both native and selenomethionine-substituted protein crystals diffract to 3.0 and 2.85 A resolution, respectively, using synchrotron radiation, they are suitable for the first high-resolution structural analysis of a two-stranded coiled coil comprising more than six heptad repeats. Moreover, because the polypeptide chain fragment contains a recently identified two-heptad-repeat long sequence that is indispensable for the assembly of the cortexillin I coiled-coil oligomerization domain, its high-resolution structure should enable us to extend our knowledge on the molecular mechanisms underlaying coiled-coil formation and to establish the precise manner in which the two "trigger" sequences interact with one another in the dimer. Copyright 1998 Academic Press.

A distinct 14 residue site triggers coiled-coil formation in cortexillin I.

We have investigated the process of the assembly of the Dictyostelium discoideum cortexillin I oligomerization domain (Ir) into a tightly packed, two-stranded, parallel coiled-coil structure using a variety of recombinant polypeptide chain fragments. The structures of these Ir fragments were analyzed by circular dichroism spectroscopy, analytical ultracentrifugation and electron microscopy. Deletion mapping identified a distinct 14 residue site within the Ir coiled coil, Arg311-Asp324, which was absolutely necessary for dimer formation, indicating that heptad repeats alone are not sufficient for stable coiled-coil formation. Moreover, deletion of the six N-terminal heptad repeats of Ir led to the formation of a four- rather than a two-helix structure, suggesting that the full-length cortexillin I coiled-coil domain behaves as a cooperative folding unit. Most interestingly, a 16 residue peptide containing the distinct coiled-coil 'trigger' site Arg311-Asp324 yielded approximately 30% helix formation as monomer, in aqueous solution. pH titration and NaCl screening experiments revealed that the peptide's helicity depends strongly on pH and ionic strength, indicating that electrostatic interactions by charged side chains within the peptide are critical in stabilizing its monomer helix. Taken together, these findings demonstrate that Arg311-Asp324 behaves as an autonomous helical folding unit and that this distinct Ir segment controls the process of coiled-coil formation of cortexillin I.

Tenascin-C hexabrachion assembly is a sequential two-step process initiated by coiled-coil alpha-helices.

We have investigated the oligomerization process of tenascin-C using a variety of recombinant wild-type and mutant polypeptide chain fragments produced by heterologous gene expression in Escherichia coli. Biochemical and biophysical analyses of the structures and assemblies of these fragments indicated a sequential two-step oligomerization mechanism of tenascin-C involving the concerted interaction of two distinct domains and cysteines 64, 111, and 113. First, the sequence between alanine 114 and glutamine 139 initiates hexabrachion formation via a parallel three-stranded coiled coil. Subsequently, the tenascin assembly domain, which is unique to the tenascins, is responsible for the connection of two triplets to a hexamer. The oligomerization of the tenascin assembly domains by the three-stranded coiled coil increases their homophilic binding affinity and is an important prerequisite for tenascin-C hexamerization. Although formation of the characteristic hexabrachion structure involves the covalent linkage of the six subunits by cysteine residues, mutational analysis indicates that hexamer formation is not dependent on intermolecular disulfide bonds. Most interestingly, substitution of glutamate 130 within the coiled-coil domain by leucine or alanine resulted in the formation of parallel four-stranded helix structures, which further associated to dodecamers. Aside from supporting a sequential process of tenascin-C assembly, this finding provides experimental evidence that non-core residues can have profound effects on the oligomerization states of coiled coils.

Homophilic adhesion of E-cadherin occurs by a co-operative two-step interaction of N-terminal domains.

Cluster formation of E-cadherin on the cell surface is believed to be of major importance for cell-cell adhesion. To mimic this process the extracellular part of mouse E-cadherin (ECAD) was recombinantly fused to the assembly domain of rat cartilage oligomeric matrix protein (COMP), resulting in the chimeric protein ECAD-COMP. The COMP domain formed a five-stranded alpha-helical coiled-coil. This enabled the formation of a pentameric ECAD with bundled C-termini and free N-termini. The pentameric protein construct ECAD-COMP and the monomeric ECAD were expressed in human embryonal kidney 293 cells. Electron microscopy, analytical ultracentrifugation, solid phase binding and cell attachment assays revealed that pentamers showed strong self-association and cell attachment, whereas monomers exhibited no activity. At the high internal concentration in the pentamer the N-terminal EC1 domains of two E-cadherin arms interact to form a ring-like structure. Then the paired domains interact with a corresponding pair from another pentamer. None of the four other extracellular domains of E-cadherin is involved in this interaction. Based on these results, an in vivo mechanism is proposed whereby two N-terminal domains of neighbouring E-cadherins at the cell surface first form a pair, which binds with high affinity to a similar complex on another cell. The strong dependence of homophilic interactions on C-terminal clustering points towards a regulation of E-cadherin mediated cell-cell adhesion via lateral association.

Effect of high-dose tyrosine supplementation on brain function in adults with phenylketonuria.

OBJECTIVES: To characterize abnormalities of brain function in patients with phenylketonuria (PKU) who had relaxed or stopped the dietary regimen and to test whether oral high-dose tyrosine (Tyr) supplementation has a beneficial effect. DESIGN: Comparison with a control group; double-blind, placebo-controlled study comprising six test times; crossover treatment groups; oral high-dose Tyr therapy (100 mg/kg body weight per day) or placebo administration for 4 weeks. SUBJECTS: Twenty-four early-treated patients with PKU aged 20.8 (16 to 25) years; 24 control subjects. METHODS: Plasma concentrations of phenylalanine and Tyr were monitored. Neuropsychologic tasks, visual evoked potentials, and spectral analysis of electroencephalographic activity were used to evaluate brain function. RESULTS: When patients with PKU were compared with control subjects, deficits in certain aspects of brain function were confirmed (i.e., a decreased ability to sustain attention, prolonged latencies of visual evoked potential peaks N1 and P2, and a reduced amount of fast-wave activity on the electroencephalogram). Baseline plasma phenylalanine and Tyr concentrations were in the typical range of adult patients with PKU. The plasma Tyr concentration increased approximately 200% during Tyr supplementation, but no beneficial effects were observed. CONCLUSIONS: High-dose Tyr supplementation cannot be recommended as an "alternative" treatment for patients with PKU after relaxation or termination of strict dietary adherence.

High-affinity and low-affinity calcium binding and stability of the multidomain extracellular 40-kDa basement membrane glycoprotein (BM-40/SPARC/osteonectin).

Reversible binding of calcium ions to a single high-affinity binding site in the 40-kDa basement membrane protein (BM-40) caused a 33% increase of alpha-helicity, an about 60% change in intrinsic fluorescence and a dramatic increase of the rate of cleavage by alpha-chymotrypsin. All these effects exhibited identical dependencies on calcium concentration from which a dissociation constant Kd = 0.6 microM was determined. Calcium release was accompanied by an increase of the frictional ratio in solution but not by denaturation which occurred at about equal guanidine.HCl concentration for both calcium-saturated and calcium-depleted protein (midpoint 1.5 M). The cleavage sites for alpha-chymotrypsin are located in or near to the EF-hand domain IV of calcium-depleted BM-40 (also known as SPARC, i.e. secreted protein acidic and rich in cysteine, and osteonectin). These and other data indicate that binding occurs in the EF-hand domain from which a large conformational change is transmitted. Low-affinity calcium-binding sites in the N-terminal glutamic-acid-rich domain I of BM-40 were identified by human leukocyte elastase which was found to cleave very specifically in the middle of this domain. From the increase of cleavage rate with increasing calcium concentration a Kd greater than or equal to 10 mM was estimated. It is suggested that variations of calcium levels in the extracellular space in this range may regulate functions of BM-40 such as collagen binding and that high-affinity binding is important for stabilization, folding and secretion during biosynthesis.

Dissection of laminin by cathepsin G into its long-arm and short-arm structures and localization of regions involved in calcium dependent stabilization and self-association.

Native laminin-nidogen complex isolated from mouse Engelbreth-Holm-Swarm tumor was treated with purified cathepsin G or leucocyte elastase, two neutral serine proteases which play a role in inflammatory processes accompanied by degradation of basement membranes. Both enzymes were found to be more active than porcine pancreatic elastase. In the absence of Ca2+, laminin fragments produced by leucocyte elastase resembled those formed by the pancreatic enzyme but at physiological concentrations of Ca2+ cleavage by cathepsin G was much more selective. Initially laminin (900 kDa) was cleaved at two major sites only with similar rates leading to three fragments. Fragment C1-4 (about 550 kDa) comprises the intact three short arms of the molecule and fragment C8-9 (about 350 kDa) contains the entire triple-coiled region by which its three chains are assembled and the major part of the terminal globular domain of the long arm. The remaining C-terminal region of this domain was recovered as fragment C3 of about 50 kDa. Stabilization against proteolytic attack was restricted to the region of fragment C1-4 and only this fragment exhibited strong Ca2+ dependent self-association similar to that of intact laminin or of its complex with nidogen. The associative properties of fragment C1-4 were dramatically diminished upon removal of the tip of one of the short arms comprising fragment 4. In addition, this provides a clear assignment of the important laminin function to a distinct domain in one of its short arms. The new fragment C8-9 may be employed for exploring the properties and possible functions of the upper long-arm region which so far has not been available as a fragment.

Binding of Ca2+ influences susceptibility of laminin to proteolytic digestion and interactions between domain-specific laminin fragments.

Ca2+ was found to influence the patterns of limit digests of laminin obtained with various neutral proteases. In the presence of Ca2+, larger fragments were obtained from the central part of laminin than in its absence. This was interpreted as being due to a stabilization of the central short-arm domains of laminin by bound Ca2+. When proteolytic fragments were tested for their ability to aggregate, only large fragments containing intact short arms were active, indicating an important role for these domains in laminin self-aggregation.

A comparison of three predictors of an individual's memory performance: the individual's feeling of knowing versus the normative feeling of knowing versus base-rate item difficulty.

Distinctions are drawn between different predictors of an individual's memory performance, with emphasis on the notion of privileged access to idiosyncratic knowledge. Research is reported in which undergraduates attempted to recall the answers to general-information questions, then made feeling-of-knowing judgments on nonrecalled items, and subsequently had a criterion test (relearning, perceptual identification, or one of two versions of recognition). For predicting an individual's criterion performance, the individual's own feeling-of-knowing predictions were intermediate between two kinds of normative predictions: The individual's feeling-of-knowing predictions were more accurate than predictions derived from normative feeling-of-knowing ratings but were less accurate than predictions derived from base-rate item difficulty (normative probabilities of correct recall). Subsidiary analyses showed that factors other than unreliability are responsible for the partial inaccuracy of the individual's feeling of knowing. Ramifications are discussed for possible ways to improve the accuracy of an individual's feeling-of-knowing predictions.

Interaction of fibronectin with C1q and collagen. Effects of ionic strength and denaturation of the collagenous component.

By attaching native collagen and C1q to Sepharose, it was possible to test the binding of fibronectin (Fn) to the native and heat-denatured forms of these proteins without complications due to aggregation, precipitation, or fibril formation. Binding to the native proteins occurred only at low (sub-physiological) ionic strength whereas binding to the denatured proteins occurred even in 1 M NaCl. Thus both of these proteins possess one or more strong sites which are masked in the native state and become exposed during thermal denaturation. Fn did not bind to albumin-Sepharose or IgG-Sepharose either before or after heat-denaturation. C1q bound readily to native IgG-Sepharose but did not mediate the binding of Fn. Nor did Fn inhibit the reconstitution of C1 on antibody-coated erythrocytes. The fluorescence polarization of fluorescein-labeled collagen in 1 M NaCl displayed a downward transition at 38-40 degrees C consistent with unfolding of the triple helix. In the presence of Fn, the same material displayed an upward transition at slightly lower temperature suggesting that gross unfolding is not required to expose the strong binding site(s).