Design of a functional calcium channel protein: inferences about an ion channel-forming motif derived from the primary structure of voltage-gated calcium channels.

To identify sequence-specific motifs associated with the formation of an ionic pore, we systematically evaluated the channel-forming activity of synthetic peptides with sequence of predicted transmembrane segments of the voltage-gated calcium channel. The amino acid sequence of voltage-gated, dihydropyridine (DHP)-sensitive calcium channels suggests the presence in each of four homologous repeats (I-IV) of six segments (S1-S6) predicted to form membrane-spanning, alpha-helical structures. Only peptides representing amphipathic segments S2 or S3 form channels in lipid bilayers. To generate a functional calcium channel based on a four-helix bundle motif, four-helix bundle proteins representing IVS2 (T4CaIVS2) or IVS3 (T4CaIVS3) were synthesized. Both proteins form cation-selective channels, but with distinct characteristics: the single-channel conductance in 50 mM BaCl2 is 3 pS and 10 pS. For T4CaIVS3, the conductance saturates with increasing concentration of divalent cation. The dissociation constants for Ba2+, Ca2+, and Sr2+ are 13.6 mM, 17.7 mM, and 15.0 mM, respectively. The conductance of T4CaIVS2 does not saturate up to 150 mM salt. Whereas T4CaIVS3 is blocked by microM Ca2+ and Cd2+, T4CaIVS2 is not blocked by divalent cations. Only T4CaIVS3 is modulated by enantiomers of the DHP derivative BayK 8644, demonstrating sequence requirement for specific drug action. Thus, only T4CaIVS3 exhibits pore properties characteristic also of authentic calcium channels. The designed functional calcium channel may provide insights into fundamental mechanisms of ionic permeation and drug action, information that may in turn further our understanding of molecular determinants underlying authentic pore structures.     

Structural analysis based on state-space modeling.

A new method has been developed to compute the probability that each amino acid in a protein sequence is in a particular secondary structural element. Each of these probabilities is computed using the entire sequence and a set of predefined structural class models. This set of structural classes is patterned after Jane Richardson''s taxonomy for the domains of globular proteins. For each structural class considered, a mathematical model is constructed to represent constraints on the pattern of secondary structural elements characteristic of that class. These are stochastic models having discrete state spaces (referred to as hidden Markov models by researchers in signal processing and automatic speech recognition). Each model is a mathematical generator of amino acid sequences; the sequence under consideration is modeled as having been generated by one model in the set of candidates. The probability that each model generated the given sequence is computed using a filtering algorithm. The protein is then classified as belonging to the structural class having the most probable model. The secondary structure of the sequence is then analyzed using a "smoothing" algorithm that is optimal for that structural class model. For each residue position in the sequence, the smoother computes the probability that the residue is contained within each of the defined secondary structural elements of the model. This method has two important advantages: (1) the probability of each residue being in each of the modeled secondary structural elements is computed using the totality of the amino acid sequence, and (2) these probabilities are consistent with prior knowledge of realizable domain folds as encoded in each model. As an example of the method''s utility, we present its application to flavodoxin, a prototypical alpha/beta protein having a central beta-sheet, and to thioredoxin, which belongs to a similar structural class but shares no significant sequence similarity.     

ARK2 stabilizes the plus-end of microtubules and promotes microtubule bundling in Arabidopsis

Microtubule dynamics and organization are important for plant cell morphogenesis and development. The microtubule-based motor protein kinesins are mainly responsible for the transport of some organelles and vesicles, although several have also been shown to regulate microtubule organization. The ARMADILLO REPEAT KINESIN (ARK) family is a plant-specific motor protein subfamily that consists of three members (ARK1, ARK2, and ARK3) in Arabidopsis thaliana. ARK2 has been shown to participate in root epidermal cell morphogenesis. However, whether and how ARK2 associates with microtubules needs further elucidation. Here, we demonstrated that ARK2 co-localizes with microtubules and facilitates microtubule bundling in vitro and in vivo. Pharmacological assays and microtubule dynamics analyses indicated that ARK2 stabilizes cortical microtubules. Live-cell imaging revealed that ARK2 moves along cortical microtubules in a processive mode and localizes both at the plus-end and the sidewall of microtubules. ARK2 therefore tracks and stabilizes the growing plus-ends of microtubules, which facilitates the formation of parallel microtubule bundles.     

Lincomycin-induced alteration in the contents of chlorophyll-protein complexes of dimorphic maize chloroplasts and its effect on the temperature-induced spectral changes

The difference spectroscopy technique has been utilized to investigate the temperature-induced spectral changes in mesophyll and bundle sheath chloroplasts of maize ( Zea mays L. cv. Ganga-5) in order to assess the role of different pigment-protein complexes in the manifestation of temperature effect on the chloroplast membranes. Cooling and heating of both mesophyll and bundle sheath chloroplasts resulted in absorbance difference (AA) bands at similar wavelengths but the degree of absorb-ance changes were significantly higher in bundle sheath chloroplasts. For example, upon cooling to 7-8°C, positive AA bands were observed at 440, 490 and 680 nm in mesophyll chloroplasts and at 440, 495–500 and 680 nm in bundle sheath chloroplasts but the absorbance change at 680 nm was ca 2% in mesophyll chloroplasts, whereas it was ca 5% in bundle sheath chloroplasts, which have a lower content of light-harvesting pigment-protein complex. The role of chlorophyll-protein complexes was further investigated by monitoring the temperature-induced spectral changes of mesophyll and bundle sheath chloroplasts isolated from lincomycin-treated maize plants where lincomycin selectively inhibits the biosynthesis of specific chlorophyll-protein complexes. Results indicated that depletion of certain pigment-protein complexes in mesophyll chloroplasts made them more susceptible (a ca 4% vs ca 2% absorbance change upon cooling and a ca 6% vs ca 4% absorbance change upon heating) and less tolerant to temperature variation (a 76% vs 39% reversibility during ambient→Cooling→ambient temperature cycle). The data indicate that pigment-protein complexes play a significant role in protecting the chloroplast membranes against temperature variation.     

Introduction of a [4Fe-4S (S-cys)4]+1,+2 iron-sulfur center into a four-alpha helix protein using design parameters from the domain of the Fx cluster in the Photosystem I reaction center.

We describe the insertion of an iron-sulfur center into a designed four alpha-helix model protein. The model protein was re-engineered by introducing four cysteine ligands required for the coordination of the mulinucleate cluster into positions in the main-chain directly analogous to the domain predicted to ligand the interpeptide [4Fe-4S (S-cys)4] cluster, Fx, from PsaA and PsaB of the Photosystem I reaction center. This was achieved by inserting the sequence, CDGPGRGGTC, which is conserved in PsaA and PsaB, into interhelical loops 1 and 3 of the four alpha-helix model. The holoprotein was characterized spectroscopically after insertion of the iron-sulfur center in vitro. EPR spectra confirmed the cluster is a [4Fe-4S] type, indicating that the cysteine thiolate ligands were positioned as designed. The midpoint potential of the iron-sulfur center in the model holoprotein was determined via redox titration and shown to be -422 mV (pH 8.3, n = 1). The results support proposals advanced for the structure of the domain of the [4Fe-4S] Fx cluster in Photosystem I based upon sequence predictions and molecular modeling. We suggest that the lower potential of the Fx cluster is most likely due to factors in the protein environment of Fx rather than the identity of the residues proximal to the coordinating ligands.     

Protein conformational landscapes: Energy minimization and clustering of a long molecular dynamics trajectory

Using energy minimization and cluster analysis, we have analyzed a 1020 ps molecular dynamics trajectory of solvated bovine pancreatic trypsin inhibitor. Elucidation of conformational sub states in this way both illustrates the degree of conformational convergence in the simulation and reduces the structural data to a tractable subset. The relative movement of structures upon energy minimization was used to estimate the sizes of features on the protein potential energy surface. The structures were analyzed using their pairwise root-mean-square C_α deviations, which gave a global measure of conformational changes that would not be apparent by monitoring single degrees of freedom. At time scales of 0.1 ps, energy minimization detected sharp transitions between energy minima separated by 0.1 Å rms deviation. Larger conformational clusters containing these smaller minima and separated by 0.25 Å were seen at 1 ps time scales. Both of these small features of the conformational landscape were characterized by movements in loop regions associated with small, correlated backbone dihedral angle shifts. On a nanosecond time scale, the main features of the protein energy landscape were clusters separated by over 0.7 Å rms deviation, with only seven of these sub states visited over the 1 ns trajectory. These substates, discernible both before and after energy minimization, differ mainly in a monotonic pivot of the loop residues 11–18 over the course of the simulation. This loop contains lysine 17, which specifically binds to trypsin in the active site. The trajectory did not return to previously visited clusters, indicating that this trajectory has not been shown to have completely sampled the conformational substates available to it. Because the apparent convergence to a single region of conformation space depends on both the time scale of observation and the size of the conformational features examined, convergence must be operationally defined within the context of the simulation. © 1995 Wiley-Liss, Inc.     

Leaf vasculature in sugarcane (Saccharum officinarum L.)

The vascular system of the leaves of Saccharum officinarum L. is composed in part of a system of longitudinal strands that in any given transverse section may be divided into three types of bundle according to size and structure: small, intermediate, and large. Virtually all of the longitudinal strands intergrade, however, from one type bundle to another. For example, virutually all of the strands having large bundle anatomy appear distally in the blade as small bundles, which intergrade into intermediates and then large bundles as they descend the leaf. These large bundles, together with the intermediates that arise midway between them, extend basipetally into the sheath and stem. Most of the remaining longitudinal strands of the blade do not enter the sheath but fuse with other strands above and in the region of the blade joint. Despite the marked decrease in number of bundles at the base of the blade, both the total and mean cross-sectional areas (measured with a digitizer from electron micrographs) of sieve tubes and tracheary elements increase as the bundles continuing into the sheath increase in size. Linear relationships exist between leaf width and total bundle number, and between cross-sectional area of vascular bundles and both total and mean cross-sectional areas of sieve tubes and tracheary elements.     

Chemical induction of β-carotene biosynthesis

Marsh white seedless grapefruit were treated with the 2-diethylaminoethanol esters of the following acids: benzoic, phenylacetic, hydrocinnamic, 4-phenylbutyric, 5-phenylvaleric, valeric, hexanoic, heptanoic, octanoic, nonanoic, 5-chlorovaleric, cyclohexanecarboxylic, phenoxyacetic, p-chlorophenoxyacetic, 3-phenoxypropionic, cinnamic and p-chlorocinnamic. Several of these esters, in particular the hexanoate, 4-phenylbutyrate and cinnamate, caused the accumulation of large amounts of β-carotene. The effects of the hexanoate and of 2-phenoxytriethylamine, which causes only lycopene accumulation, were studied as functions of time. The hexanoate caused the rapid accumulation of lycopene during the first day. The amount of lycopene then began to decrease and that of β-carotene increased until, after 14 days, β-carotene was the major pigment. 2-Phenoxytriethylamine caused rapid lycopene accumulation during the first day and a slow steady increase afterwards. Thus, the mode of action of the β-carotene inducers may be similar to that of the lycopene inducers except that the former are probably rapidly hydrolysed by the esterase(s) in the flavedo, so that they no longer inhibit the cyclase(s), and β-carotene is accumulated at the expanse of lycopene.     

The effect of nitrogen and phosphorus on starch accumulation and net photosynthesis in two variants of Panicum maximum Jacq.

Abstract. Two anatomical variants of Panicum maximum Jacq. were observed to accumulate an unusually large number of starch grains in the bundle sheath chloroplasts when grown under controlled environmental conditions in a nutrient medium containing a low level of nitrate nitrogen (20 mg N dm⁻³ as KNO₃). When these plants were placed under dark conditions the chloroplasts were destarched, but exhibited a marked distortion of the thylakoid membranes. Under a higher level of nitrate nitrogen supply (200 mg N dm⁻³ as KNO₃) the number of starch grains was markedly reduced compared to that observed above in both plant variants. When the nitrogen was supplied as ammonium nitrogen (200 mg N dm⁻³ as NH₄Cl) there was again a high level of starch in the bundle sheath chloroplasts, the level being only slightly lower than that observed at the low KNO₃ supply. An unusually large number of starch grains accumulated in the bundle sheath chloroplasts in the absence of added phosphorus in the nutrient medium, in the presence of the higher nitrate nitrogen level. It is suggested that the increased starch accumulation results from a reduced trans-location of Calvin cycle intermediates out of the chloroplasts into the cytoplasm and that both nitrate nitrogen and phosphorus may play an important role in this process. A good correlation between high net photosynthetic activity and low bundle sheath starch content was observed. Nutrient medium requirements favouring low starch content in chloroplasts also favoured high net photosynthetic rates.     

Effects of Frequency and Pattern of Medial Forebrain Bundle Stimulation on Caudate Dialysate Dopamine and Serotonin

In vivo microdialysis was employed to detect changes in extracellular dopamine and serotonin in the rat caudate in response to electrical stimulation of the medial forebrain bundle. Extracellular dopamine concentrations increased linearly as a function of the frequency (4-33 Hz) of evenly spaced stimuli in both the presence and absence of cocaine added to the dialysate. Because dopamine neurons are known to fire in single-spike and burst patterns, stimulation pulses were also delivered in a bursting pattern. The response of extracellular dopamine was augmented in both the presence and absence of cocaine when the same number of stimuli were delivered in bursts as compared to an evenly spaced pattern. Serotonin, which was only assessed in the presence of cocaine, similarly increased linearly with frequency, but, in contrast to the dopamine response, levels of serotonin were not augmented by stimuli presented in bursts. These results suggest that microdialysis can be used to detect physiological changes in synaptic transmitter concentrations.