Spectroscopic characterization and structural modeling of prolamin from maize and pearl millet

Biophysical methods and structural modeling techniques have been used to characterize the prolamins from maize (Zea mays) and pearl millet (Pennisetum americanum). The alcohol-soluble prolamin from maize, called zein, was extracted using a simple protocol and purified by gel filtration in a 70% ethanol solution. Two protein fractions were purified from seed extracts of pearl millet with molecular weights of 25.5 and 7 kDa, as estimated by SDS-PAGE. The high molecular weight protein corresponds to pennisetin, which has a high -helical content both in solution and the solid state, as demonstrated by circular dichroism and Fourier transform infrared spectra. Fluorescence spectroscopy of both fractions indicated changes in the tryptophan microenvironments with increasing water content of the buffer. Low-resolution envelopes of both fractions were retrieved by ab initio procedures from small-angle X-ray scattering data, which yielded maximum molecular dimensions of about 14 nm and 1 nm for pennisetin and the low molecular weight protein, respectively, and similar values were observed by dynamic light scattering experiments. Furthermore, ¹H nuclear magnetic resonance spectra of zein and pennisetin do not show any signal below 0.9 ppm, which is compatible with more extended solution structures. The molecular models for zein and pennisetin in solution suggest that both proteins have an elongated molecular structure which is approximately a prolate ellipsoid composed of ribbons of folded -helical segments with a length of about 14 nm, resulting in a structure that permits efficient packing within the seed endosperm.     

Gravitropism in Phycomyces: threshold determination on a clinostat centrifuge

The absolute sensitivity of sporangiophores of Phycomyces blakesleeanus to centrifugal acceleration was determined on a clinostat centrifuge. The centrifuge provides centrifugal accelerations ranging from 10(-4) to 6 x g. The rotor of the centrifuge, which accommodates 96 culture vials with single sporangiophores, is clinostatted, that is, turning "head over", at slow speed (1 rev min(-1)) while it is running. The negative gravitropism of sporangiophores is characterized by two components: a polar angle, which is measured in the plane of bending, and an aiming-error angle, which indicates the deviation of the plane of bending from the vector of the centrifugal acceleration. Dose-response curves were generated for both angles with centrifugations lasting 3, 5, and 8 h. The threshold for the polar angle depends on the presence of statoliths, so-called octahedral protein crystals in the vacuoles. The albino strain C171 carAcarR (with crystals) has a threshold near 10(-2) x g while the albino strain C2 carAgeo-3 (without crystals) has a threshold of about 2 x 10(-1) x g. The threshold for the aiming error angle is ill defined and is between 10(-2) and 10(-1) x g. The threshold for the polar angle of the wild type NRRL 1555 (with crystals) is near 8 x 10(-2) x g.     

Evolving nature of the AP2 alpha-appendage hub during clathrin-coated vesicle endocytosis

Clathrin-mediated endocytosis involves the assembly of a network of proteins that select cargo, modify membrane shape and drive invagination, vesicle scission and uncoating. This network is initially assembled around adaptor protein (AP) appendage domains, which are protein interaction hubs. Using crystallography, we show that FxDxF and WVxF peptide motifs from synaptojanin bind to distinct subdomains on alpha-appendages, called 'top' and 'side' sites. Appendages use both these sites to interact with their binding partners in vitro and in vivo. Occupation of both sites simultaneously results in high-affinity reversible interactions with lone appendages (e.g. eps15 and epsin1). Proteins with multiple copies of only one type of motif bind multiple appendages and so will aid adaptor clustering. These clustered alpha(appendage)-hubs have altered properties where they can sample many different binding partners, which in turn can interact with each other and indirectly with clathrin. In the final coated vesicle, most appendage binding partners are absent and thus the functional status of the appendage domain as an interaction hub is temporal and transitory giving directionality to vesicle assembly.     

Development of a polyvalent assay system for lead identification

In an effort to identify new approaches to lead discovery a polyvalent assay was developed to allow identification of weak inhibitors. This approach involves the polyvalent display of a protein binder off a Tenta-gel scaffold and the generation of a polyvalent display of protein by biotinylation followed by complexation with fluorescently labeled streptavidin. Subsequent exposure of the streptavidin complexed protein to Tenta-gel beads with active protein binders results in fluorescent beads, which are easily viewed under a fluorescent microscope.     

P3 cap modified Phe*-Ala series BACE inhibitors

With the aim of reducing molecular weight and adjusting log D value of BACE inhibitors to more favorable range for BBB penetration and better bioavailability, we synthesized and evaluated several series of P3 cap modified BACE inhibitors obtained via replacement of the P3NHBoc moiety as seen in 3 with other polar functional groups such as amino, hydroxyl and fluorine. Several promising inhibitors emerging from this P3 cap SAR study (e.g., 15 and 19) demonstrated good enzyme inhibitory potencies (BACE-1 IC(50) <50 nM) and whole cell activities (IC(50) approximately 1 microM).     

Disparate roles for the regulatory A subunit isoforms in Arabidopsis protein phosphatase 2A

The heterotrimeric protein phosphatase 2A (PP2A) complex comprises a catalytic subunit and regulatory A and B subunits that modulate enzyme activity and mediate interactions with other proteins. We report here the results of a systematic analysis of the Arabidopsis (Arabidopsis thaliana) regulatory A subunit gene family, which includes the ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 (RCN1), PP2AA2, and PP2AA3 genes. All three A subunit isoforms accumulate in the organs of seedlings and adult plants, suggesting extensive overlap in expression domains. We have isolated pp2aa2 and pp2aa3 mutants and found that their phenotypes are largely normal and do not resemble that of rcn1. Whereas rcn1 pp2aa2 and rcn1 pp2aa3 double mutants exhibit striking abnormalities in all stages of development, the pp2aa2 pp2aa3 double mutant shows only modest defects. Together, these data suggest that RCN1 performs a cardinal role in regulation of phosphatase activity and that PP2AA2 and PP2AA3 functions are unmasked only when RCN1 is absent.     

Do mitochondria make nitric oxide? no?

Several papers have claimed that mitochondria contain nitric oxide synthase (NOS) and make nitric oxide (NO*) in amounts sufficient to affect mitochondrial respiration. However, we found that the addition of L-arginine or the NOS inhibitor L-NMMA to intact rat liver mitochondria did not have any effect on the respiratory rate in both State 3 and State 4. We did not detect mitochondrial NO* production by the oxymyoglobin oxidation assay, or electrochemically using an NO* electrode. An apparent NO* production detected by the Griess assay was identified as an artifact. NO* generated by eNOS added to the mitochondria could easily be detected, although succinate-supplemented mitochondria appeared to consume NO*. Our data show that NO* production by normal rat liver mitochondria cannot be detected in our laboratory, even though the levels of production claimed in the literature should easily have been measured by the techniques used. The implications for the putative mitochondrial NOS are discussed.     

Increased phosphorylation of myosin light chain associated with slow-to-fast transition in rat soleus

In striated muscles myosin light chain (MLC)2 phosphorylation regulates calcium sensitivity and mediates sarcomere organization. Little is known about the changes in MLC2 phosphorylation in relation to skeletal muscle plasticity. We studied changes in MLC2 phosphorylation in rats receiving three treatment conditions causing slow-to-fast transitions: 1) atrophy induced by 14 days of hindlimb suspension (HS), 2) hypertrophy induced by 14 days of clenbuterol administration (CB), and 3) 14 days of combined treatment (CB-HS). Three variants of the slow (MLC2s) and two variants of the fast MLC2 (MLC2f) isoform were separated with two-dimensional electrophoresis and identified with monoclonal and polyclonal antibodies specific for MLC2; their relative proportions were densitometrically quantified. In control soleus muscle MLC2s predominated over MLC2f (91.4 ± 3.9% vs. 8.5 ± 3.9%) and was separated into two spots, the less acidic spot being 73.5 ± 4.3% of the total. All treatments caused a decrease of the less acidic unphosphorylated spot of MLC2s (CB: 64.1 ± 5.6%, HS: 62.4 ± 6.8%, CB-HS: 56.4 ± 4.4%), the appearance of a third more acidic variant of MLC2s (representing 3.9–5.9% of total MLC2s), an increase of MLC2f (CB: 30.9 ± 3.1%, HS: 23.9 ± 3.3%, CB-HS: 25.3 ± 3.9%), and the phosphorylation of a large fraction of MLC2f (CB: 30.4 ± 6.7%, HS: 28.7 ± 6.5%, CB-HS: 21.8 ± 2.1%). Treatment with alkaline phosphatase or with protein phosphatase 1 (PP1) removed the most acidic spots of both MLC2f and MLC2s. We conclude that in rat skeletal muscles an increase of MLC2 phosphorylation is associated with the slow-to-fast transition regardless of whether hypertrophy or atrophy develops. muscle atrophy; muscle hypertrophy; clenbuterol; hindlimb suspension