NMR determination of the major solution conformation of a peptoid pentamer with chiral side chains

Polymers of N-substituted glycines (“peptoids”) containing chiral centers at the α position of their side chains can form stable structures in solution. We studied a prototypical peptoid, consisting of five para-substituted (S)-N-(1-phenylethyl)glycine residues, by NMR spectroscopy. Multiple configurational isomers were observed, but because of extensive signal overlap, only the major isomer containing all cis-amide bonds was examined in detail. The NMR data for this molecule, in conjunction with previous CD spectroscopic results, indicate that the major species in methanol is a right-handed helix with cis-amide bonds. The periodicity of the helix is three residues per turn, with a pitch of ≈6 Å. This conformation is similar to that anticipated by computational studies of a chiral peptoid octamer. The helical repeat orients the amide bond chromophores in a manner consistent with the intensity of the CD signal exhibited by this molecule. Many other chiral polypeptoids have similar CD spectra, suggesting that a whole family of peptoids containing chiral side chains is capable of adopting this secondary structure motif. Taken together, our experimental and theoretical studies of the structural properties of chiral peptoids lay the groundwork for the rational design of more complex polypeptoid molecules, with a variety of applications, ranging from nanostructures to nonviral gene delivery systems.

Cleavage motifs of the yeast 20S proteasome β subunits deduced from digests of enolase 1

The 436-amino acid protein enolase 1 from yeast was degraded in vitro by purified wild-type and mutant yeast 20S proteasome particles. Analysis of the cleavage products at different times revealed a processive degradation mechanism and a length distribution of fragments ranging from 3 to 25 amino acids with an average length of 7 to 8 amino acids. Surprisingly, the average fragment length was very similar between wild-type and mutant 20S proteasomes with reduced numbers of active sites. This implies that the fragment length is not influenced by the distance between the active sites, as previously postulated. A detailed analysis of the cleavages also allowed the identification of certain amino acid characteristics in positions flanking the cleavage site that guide the selection of the P1 residues by the three active β subunits. Because yeast and mammalian proteasomes are highly homologous, similar cleavage motifs might be used by mammalian proteasomes. Therefore, our data provide a basis for predicting proteasomal degradation products from which peptides are sampled by major histocompatibility complex class I molecules for presentation to cytotoxic T cells.

Two-dimensional IR correlation spectroscopy: Sequential events in the unfolding process of the λ Cro-V55C repressor protein

A question often posed in protein folding/unfolding studies is whether the process is fully cooperative or whether it contains sequential elements. To address this question, one needs tools capable of resolving different events. It seems that, at least in certain cases, two-dimensional (2D) IR correlation spectroscopy can provide answers to this question. To illustrate this point, we have turned to the Cro-V55C dimer of the λ Cro repressor, a protein known to undergo thermal unfolding in two discrete steps through a stable equilibrium intermediate. The secondary structure of this intermediate is compatible with that of a partially unfolded protein and involves a reorganization of the N terminus, whereas the antiparallel β-ribbon formed by the C-terminal part of each subunit remains largely intact. To establish whether the unfolding process involves sequential events, we have performed a 2D correlation analysis of IR spectra recorded over the temperature range of 20–95°C. The 2D IR correlation analysis indeed provides evidence for a sequential formation of the stable intermediate, which is created in three (closely related) steps. A first step entails the unfolding of the short N-terminal β-strand, followed by the unfolding of the α-helices in a second step, and the third step comprises the reorganization of the remaining β-sheet and of some unordered segments in the protein. The complete unfolding of the stable intermediate at higher temperatures also undergoes sequential events that ultimately end with the breaking of the H bonds between the two β-strands at the dimer interface.

From calcium blips to calcium puffs: Theoretical analysis of the requirements for interchannel communication

In the cytoplasm of cells of different types, discrete clusters of inositol 1,4,5-trisphosphate-sensitive Ca2+ channels generate Ca2+ signals of graded size, ranging from blips, which involve the opening of only one channel, to moderately larger puffs, which result from the concerted opening of a few channels in the same cluster. These channel clusters are of unknown size or geometrical characteristics. The aim of this study was to estimate the number of channels and the interchannel distance within such a cluster. Because these characteristics are not attainable experimentally, we performed computer stochastic simulations of Ca2+ release events. We conclude that, to ensure efficient interchannel communication, as experimentally observed, a typical cluster should contain two or three tens of inositol 1,4,5-trisphosphate-sensitive Ca2+ channels in close contact.

A numerical strategy for efficient modeling of the equatorial wave guide

Convection in the tropics is observed to involve a wide-ranging hierarchy of scales from a few kilometers to the planetary scales and also has a profound impact on short-term climate. The mechanisms responsible for this behavior present a major unsolved problem. A promising emerging approach to address these issues is cloud-resolving modeling. Here a family of numerical models is introduced specifically to model the feedback of small-scale deep convection on tropical planetary waves and tropical circulation in a highly efficient manner compatible with the approach through cloud-resolving modeling. Such a procedure is also useful for theoretical purposes. The basic idea in the approach is to use low-order truncation in the meriodonal direction through Gauss–Hermite quadrature projected onto a simple discrete radiation condition. In this fashion, the cloud-resolving modeling of equatorially trapped planetary waves reduces to the solution of a small number of purely zonal two-dimensional wave systems along a few judiciously chosen meriodonal layers that are coupled only by some additional source terms. The approach is analyzed in detail with full mathematical rigor for linearized equatorial primitive equations with source terms.

Curvature Induced by Amyloplast Magnetophoresis in Protonemata of the Moss Ceratodon purpureus1

After gravistimulation of Ceratodon purpureus (Hedw.) Brid. protonemata in the dark, amyloplast sedimentation was followed by upward curvature in the wild-type (WT) and downward curvature in the wwr mutant (wrong way response). We used ponderomotive forces induced by high-gradient magnetic fields (HGMF) to simulate the effect of gravity and displace the presumptive statoliths. The field was applied by placing protonemata either between two permanent magnets at the edge of the gap, close to the edge of a magnetized ferromagnetic wedge, or close to a small (<1 mm) permanent magnet. Continuous application of an HGMF in all three configurations resulted in plastid displacement and induced curvature in tip cells of WT and wwr protonemata. WT cells curved toward the HGMF, and wwr cells curved away from the HGMF, comparable to gravitropism. Plastids isolated from protonemal cultures had densities ranging from 1.24 to 1.38 g cm−3. Plastid density was similar for both genotypes, but the mutant contained larger plastids than the WT. The size difference might explain the stronger response of the wwr protonemata to the HGMF. Our data support the plastid-based theory of gravitropic sensing and suggest that HGMF-induced ponderomotive forces can substitute for gravity.

Geometric constraints explain much of the species richness pattern in African birds

The world contains boundaries (e.g., continental edge for terrestrial taxa) that impose geometric constraints on the distribution of species ranges. Thus, contrary to traditional thinking, the expected species richness pattern in absence of ecological or physiographical factors is unlikely to be uniform. Species richness has been shown to peak in the middle of a bounded one-dimensional domain, even in the absence of ecological or physiographical factors. Because species ranges are not linear, an extension of the approach to two dimensions is necessary. Here we present a two-dimensional null model accounting for effects of geometric constraints. We use the model to examine the effects of continental edge on the distribution of terrestrial animals in Africa and compare the predictions with the observed pattern of species richness in birds endemic to the continent. Latitudinal, longitudinal, and two-dimensional patterns of species richness are predicted well from the modeled null effects alone. As expected, null effects are of high significance for wide ranging species only. Our results highlight the conceptual significance of an until recently neglected constraint from continental shape alone and support a more cautious analysis of species richness patterns at this scale.

Quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II: a systematic study of the effect of carotenoid structure.

The role of carotenoids in quenching of chlorophyll fluorescence in the major light-harvesting complex of photosystem II has been studied with a view to understanding the molecular basis of the control of photoprotective nonradiative energy dissipation by the xanthophyll cycle in vivo. The control of chlorophyll fluorescence quenching in the isolated complex has been investigated in terms of the number of the conjugated double bonds for a series of carotenoids ranging from n = 5-19, giving an estimated first excited singlet state energy from 20,700 cm-1 to 10,120 cm-1. At pH 7.8 the addition of exogenous carotenoids with >=10 conjugated double bonds (including zeaxanthin) stimulated fluorescence quenching relative to the control with no added carotenoid, whereas those with n the light-harvesting complex of photosystem II was induced by a lowering of pH to 5.5, carotenoids with n of fluorescence quenching relative to the control. Of the 10 carotenoids tested, quenching induced by the addition of the tertiary amine compound, dibucaine, to isolated light-harvesting complex of photosystem II could only be reversed by violaxanthin. These results are discussed in terms of the two theories developed to explain the role of zeaxanthin and violaxanthin in nonphotochemical quenching of chlorophyll fluorescence.

Structure and evolution of the compact radio source in NGC 1275.

Investigations of the fine-scale structure in the compact nucleus of the radio source 3C 84 in NGC 1275 (New General Catalogue number) are reported. Structural monitoring observations beginning as early as 1976, and continuing to the present, revealed subluminal motions in a jet-like relatively diffuse region extending away from a flat-spectrum core. A counterjet feature was discovered in 1993, and very recent nearly simultaneous studies have detected the same feature at five frequencies ranging from 5 to 43 GHz. The counterjet exhibits a strong low-frequency cutoff, giving this region of the source an inverted spectrum. The observations are consistent with a physical model in which the cutoff arises from free-free absorption in a volume that surrounds the core but obscures only the counterjet feature. If such a model is confirmed, very-long-baseline radio interferometry observations can then be used to probe the accretion region, outside the radio jet, on parsec scales.

Long-term in vivo expression of the human glucocerebrosidase gene in nonhuman primates after CD34+ hematopoietic cell transduction with cell-free retroviral vector preparations.

Successful gene transfer into stem cells would provide a potentially useful therapeutic modality for treatment of inherited and acquired disorders affecting hematopoietic tissues. Coculture of primate bone marrow cells with retroviral producer cells, autologous stroma, or an engineered stromal cell line expressing human stem cell factor has resulted in a low efficiency of gene transfer as reflected by the presence of 0.1-5% of genetically modified cells in the blood of reconstituted animals. Our experiments in a nonhuman primate model were designed to explore various transduction protocols that did not involve coculture in an effort to define clinically useful conditions and to enhance transduction efficiency of repopulating cells. We report the presence of genetically modified cells at levels ranging from 0.1% (granulocytes) to 14% (B lymphocytes) more than 1 year following reconstitution of myeloablated animals with CD34+ immunoselected cells transduced in suspension culture with cytokines for 4 days with a retrovirus containing the glucocerebrosidase gene. A period of prestimulation for 7 days in the presence of autologous stroma separated from the CD34+ cells by a porous membrane did not appear to enhance transduction efficiency. Infusion of transduced CD34+ cells into animals without myeloablation resulted in only transient appearance of genetically modified cells in peripheral blood. Our results document that retroviral transduction of primate repopulating cells can be achieved without coculture with stroma or producer cells and that the proportion of genetically modified cells may be highest in the B-lymphoid lineage under the given transduction conditions.