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Recent studies show that current trends in yield improvement will not be sufficient to meet projected global food demand in 2050, and suggest that a further expansion of agricultural area will be required. However, agriculture is the main driver of losses of biodiversity and a major contributor to climate change and pollution, and so further expansion is undesirable. The usual proposed alternative—intensification with increased resource use—also has negative effects. It is therefore imperative to find ways to achieve global food security without expanding crop or pastureland and without increasing greenhouse gas emissions. Some authors have emphasized a role for sustainable intensification in closing global ‘yield gaps’ between the currently realized and potentially achievable yields. However, in this paper we use a transparent, data-driven model, to show that even if yield gaps are closed, the projected demand will drive further agricultural expansion. There are, however, options for reduction on the demand side that are rarely considered. In the second part of this paper we quantify the potential for demand-side mitigation options, and show that improved diets and decreases in food waste are essential to deliver emissions reductions, and to provide global food security in 2050.

Neurotensin (NT) is a tridecapeptide hormone in the periphery and neurotransmitter in the brain that principally activates three receptor subtypes, named NTS1, NTS2, and NTS3. Since little is known about its structure in the presence of its principal receptor NTS1, we determined it using the key domain of the receptor, i.e. the third extracellular loop. We conclude the following: (i) for the receptor fragment, NT binding modifies its central part, underlying the great flexibility and adaptability of this region; (ii) for bound NT, the extended conformation of its C-terminus is confirmed for the first time in experimental conditions and in the presence of a part of the receptor; and (iii) despite some substitutions, the human receptor residues that are involved in the interaction with NT could be similar to those of the rat receptor which play an important role in NT binding.

Polyamines are essential for mammalian cell growth and proliferation, and their synthesis in cells or transport from the extracellular environment has attracted much attention, especially in cancer research. Here it is shown for the first time that polyamines can be transferred from cell to cell via gap junctions to coordinate cell growth.

Mutations in SPG4, encoding the microtubule-severing protein spastin, are responsible for the most frequent form of hereditary spastic paraplegia (HSP), a heterogeneous group of genetic diseases characterized by degeneration of the corticospinal tracts. We previously reported that mice harboring a deletion in Spg4, generating a premature stop codon, develop progressive axonal degeneration characterized by focal axonal swellings associated with impaired axonal transport. To further characterize the molecular and cellular mechanisms underlying this mutant phenotype, we have assessed microtubule dynamics and axonal transport in primary cultures of cortical neurons from spastin-mutant mice. We show an early and marked impairment of microtubule dynamics all along the axons of spastin-deficient cortical neurons, which is likely to be responsible for the occurrence of axonal swellings and cargo stalling. Our analysis also reveals that a modulation of microtubule dynamics by microtubule-targeting drugs rescues the mutant phenotype of cortical neurons. Together, these results contribute to a better understanding of the pathogenesis of SPG4-linked HSP and ascertain the influence of microtubule-targeted drugs on the early axonal phenotype in a mouse model of the disease.

We report a detailed description of the energy migration dynamics in the phycocyanin 645 (PC645) antenna complex from the photosynthetic alga ChroomonasCCMP270. Many of the cryptophyceae are known to populate greater depths than most other algal families, having developed a 99.5% efficient light-harvesting system. In this study, we used femtosecond time-resolved spectroscopy and global analysis to characterize the excited-state dynamics of PC645. Several different pump colors were selected to excite different fractions of the four phycobiliprotein pairs present in the complex. Measurements were also performed at cryogenic temperature to enhance spectral resolution and selectively promote downhill energy transfers. Upon excitation of the highest-energy bilins (dihydrobiliverdins), energy is transferred from the core of the complex to the periphery within 0.82 ps. Four bilins (mesobiliverdin (MBV) A/B and phycocyanobilins (PCB) 158C/D), which are responsible for the central band of the absorption spectrum, show concerted spectral dynamics. These chromophores show a biphasic decay with lifetimes of 0.6 ps (MBV) and 5–7 ps (PCB 158) to the lowest bilin pair (PCB 82C/D) absorbing around 650–657 nm. Within this lifetime of several picoseconds, the excitations reach the PCB 82 bilins on the two poles at the smaller sides of PC645. A slow 44–46 ps energy transfer step to the lowest-energy PCB 82 bilin concludes the dynamics.

Microtubules (MTs) are cylindrical cytoskeleton polymers composed of α-βtubulin heterodimers whose dynamic properties are essential to fulfill their numerous cellular functions. In response to spatial confinement, dynamic MTs, even in the absence of protein partners, were shown to self-organize into higher order structures (spindle or striped structures) which lead to interesting dynamical properties (MT oscillations). In this study, we considered the assembly and sensitivity of dynamic MTs when in bundles. To perform this study, spermine, a natural tetravalent polyamine present at high concentrations in all eukaryote cells, was used to trigger MT bundling while preserving MT dynamics. Interestingly, we first show that, near physiological ionic strengths, spermine promotes the bundling of MTs whereas it does not lead to aggregation of free tubulin, which would have been detrimental to MT polymerization. Experimental and theoretical results also indicate that, to obtain a high rate of bundle assembly, bundling should take place at the beginning of assembly when rapid rotational movements of short and newly nucleated MTs are still possible. On the other hand, the bundling process is significantly slowed down for long MTs. Finally, we found that short MT bundles exhibit a higher sensitivity to cold exposure than do isolated MTs. To account for this phenomenon, we suggest that a collective behavior takes place within MT bundles because an MT entering into a phase of shortening could increase the probability of the other MTs in the same bundle to enter into shortening phase due to their close proximity. We then elaborate on some putative applications of our findings to in vivo conditions including neurons.

This article reports stable photoluminescence and high-contrast optically detected electron spin resonance (ODESR) from single nitrogen-vacancy (NV) defect centers created within ultrasmall, disperse nanodiamonds of radius less than 4 nm. Unexpectedly, the efficiency for the production of NV fluorescent defects by electron irradiation is found to be independent of the size of the nanocrystals. Fluorescence lifetime imaging shows lifetimes with a mean value of around 17 ns, only slightly longer than the bulk value of the defects. After proper surface cleaning, the dephasing times of the electron spin resonance in the nanocrystals approach values of some microseconds, which is typical for the type Ib diamond from which the nanoparticle is made. We conclude that despite the tiny size of these nanodiamonds the photoactive nitrogen-vacancy color centers retain their bulk properties to the benefit of numerous exciting potential applications in photonics, biomedical labeling, and imaging.

Biomolecular motors have inspired the design and construction of artificial nanoscale motors and machines based on nucleic acids, small molecules, and inorganic nanostructures. However, the high degree of sophistication and efficiency of biomolecular motors, as well as their specific biological function, derives from the complexity afforded by protein building blocks. Here, we discuss a novel bottom‐up approach to understanding biological motors by considering the construction of synthetic protein motors. Specifically, we present a design for a synthetic protein motor that moves along a linear track, dubbed the “Tumbleweed.” This concept uses three discrete ligand‐dependent DNA‐binding domains to perform cyclically ligand‐gated, rectified diffusion along a synthesized DNA molecule. Here we describe how de novopeptide design and molecular biology could be used to produce the Tumbleweed, and we explore the fundamental motor operation of such a design using numerical simulations. The construction of this and more sophisticated protein motors is an exciting challenge that is likely to enhance our understanding of the structure‐function relationship in biological motors.

This article presents an investigation of the energy migration dynamics in intact cells of the unicellular photosynthetic cryptophyte Chroomonas CCMP270 by steady-state and time-resolved fluorescence measurements. By kinetic modeling of the fluorescence data on chlorophyll and phycocyanin 645 excitation (at 400 and 582nm respectively), it has been possible to show the excited state energy distribution in the photosynthetic antenna of this alga. Excitation energy from phycocyanin 645 is distributed nearly equally between photosystem I and photosystem II with very high efficiency on a 100-ps timescale. The excitation energy trapping times for both photosystem I (∼30ps) and photosystem I (200 and ∼540ps) correspond well to those obtained from experiments on isolated photosystems. The results are compared with previous results for another cryptophyte species, Rhodomonas CS24, and suggest a similar membrane organization for the cryptophytes with the phycobiliproteins tightly packed in the thylakoid lumen around the periphery of the photosystems.

Using liquid chromatography−mass spectrometry, 528 proteins were identified that are expressed during growth at 4 °C in the cold adapted archaeon, Methanococcoides burtonii. Of those, 135 were annotated previously as unique or conserved hypothetical proteins. We have performed a comprehensive, integrated analysis of the latter proteins using threading, InterProScan, predicted subcellular localization and visualization of conserved gene context across multiple prokaryotic genomes. Functional information was obtained for 55 proteins, providing new insight into the physiology of M. burtonii. Many of the proteins were predicted to be involved in DNA/RNA binding or modification and cell signaling, suggesting a complex, uncharacterized regulatory network controlling cellular processes during growth at low-temperature. Novel enzymatic functions were predicted for several proteins, including a putative candidate gene for the posttranslational modification of the key methanogenesis enzyme coenzyme M methyl reductase. A bacterial-like CRISPR locus was identified as a strong candidate for archaeal-bacterial lateral gene transfer. Gene context analysis proved a valuable augmentation to the other predictive methods in several cases, by revealing conserved gene associations and annotations in other microbial genomes. Our results underscore the importance of addressing the “hypothetical protein problem” for a complete understanding of cell physiology. Keywords: proteome • LC/LC−MS/MS • archaea • methanogenesis • psychrophile • CRISPR locus • hypothetical proteins • conserved gene context

ABSTRACTThe muscle protein myosin binding protein C (MyBPC) is a large multi-domain protein whose role in the sarcomere is complex and not yet fully understood. Mutations in MyBPC are strongly associated with the heart disease familial hypertrophic cardiomyopathy (FHC) and these experiments of nature have provided some insight into the intricate workings of this protein in the heart. While some regions of the MyBPC molecule have been assigned a function in the regulation of muscle contraction, the interaction of other regions with various parts of the myosin molecule and the sarcomeric proteins, actin and titin, remain obscure. In addition, several intra-domain interactions between adjacent MyBPC molecules have been identified. Although the basic structure of the molecule (a series of immunoglobulin and fibronectin domains) has been elucidated, the assembly of MyBPC in the sarcomere is a topic for debate. By analysing the MyBPC sequence with respect to FHC-causing mutations it is possible to identify individual residues or regions of each domain that may be important either for binding or regulation. This review looks at the current literature, in concert with alignments and the structural models of MyBPC, in an attempt to understand how FHC mutations may lead to the disease state.

Stathmin is a ubiquitous 17 kDa cytosolic phosphoprotein proposed to play a general role in the integration and relay of intracellular signalling pathways. It is believed to regulate microtubule dynamics by sequestering tubulin in a complex made of two tubulin heterodimers per stathmin molecule (T2S complex). The other proteins of the stathmin family can also bind two tubulin heterodimers through their SLD (stathmin-like domain), but the different tubulin:SLD complexes display varying stabilities. In this study, we analysed the relative influence of three regions of SLDs on the interaction with tubulin and the mechanistic processes that lead to its sequestration. Tubulin-binding properties of fragments and chimaeras of stathmin and RB3SLD were studied in vitro by tubulin polymerization, size-exclusion chromatography and surface plasmon resonance assays. Our results show that the N-terminal region of SLDs favours the binding of the first tubulin heterodimer and that the second C-terminal tubulinbinding site confers the specific stability of a given tubulin:SLD complex. Our results highlight the molecular processes by which tubulin co-operatively interacts with the SLDs. This knowledge may contribute to drug development aimed at disturbing microtubules that could be used for the treatment of cancer.

Soil spatial distribution, i.e. the spatial distribution of soils within landscapes, is difficult to predict because numerous processes operate simultaneously, but variably, over time. Quantifications of large areas with an acceptable degree of precision and low in cost require the development of specific methods making the best possible use of existing soil data and auxiliary information such as soil-forming factors. The quantification of the influence of soil-forming factors on soil spatial distribution is seldom performed over large areas such as regions. This study aimed to quantify the relationship between soil spatial distribution and the soil-forming factors of geology, topography, climate, and tectonic regime in order to predict soil spatial distribution over a wide region (30,000 km2). The Armorican Massif (western France), a complex basement of Proterozoic and Paleozoic rocks affected by recent tectonic activity and characterized by variations in topography and climate, was chosen as the study site. Detailed soil maps (125,000) were used to describe soil spatial distribution along transects. An ANOVA performed on 314 transects showed a high correlation between the occurrence of soils with particular features (namely redoximorphic, leached, glossic, and albic) and geological substrate, uplift ratio, mean slope gradient, and net rainfall. No such correlation was found with fluvic soils. These soil-forming factors seem to act through saprolite quality and erosion processes, which in turn control the development of soil features. A quantification of the relationship between soil features and soil-forming factors was performed by regression analysis in order to allow further prediction of the soil spatial distribution over the entire Armorican Massif. These results revealed and quantified the hitherto unrecognized role of tectonism on soil distribution and its relative importance in respect to other soil-forming factors. Finally, such an analysis, which is based on existing maps, can help to describe, quantify, and predict detailed soil spatial distribution at smaller scales.

Quantitative soil-landscape models, based on topographic attributes, make possible the characterization of large areas because of the widespread availability of digital elevation models (DEMs). However, these soil-landscapes models, which are usually generated and validated on the same detailed, single research site, such as a hillslope or an elementary catchment, may show high prediction errors when applied to other areas of a region. The effect of the regional variations of topography, climate, parent material, land-use, and/or soil has seldom been analyzed. The objective of this study was to test multiple-regression relationships between the soil hydromorphic index (HI) and topographic attributes on different catchments of the Armorican Massif (30,000 km2) in western France. Regression models were validated using 565 data points collected from four sites along hillslopes. These four sites, located throughout this region, were included in three catchments with surface areas of 78, 82, and 120 ha, respectively, and differing in topography (mean elevation from 39 to 202 m and slope gradient from 3.4 to 7.9), parent material (granite and schist), and precipitation (700 to 900 mm y−1). The existing models were multiple regression equations between the HI and the elevation above the stream bank, the compound topographic index (regression 1, r20.84), or the upslope drained area (regression 2, r20.86). At each validation point, systematic soil observation for HI estimation was compared with estimations from terrain attributes derived from DEMs at a 30-m resolution. Results showed small prediction errors for all study sites, with mean absolute errors between 5 and 15 of the HI range. Errors were not spatially correlated. Minimum prediction errors were encountered in the catchment for which the models were generated and also in one other that differed only in the parent material. In the other validation site, the models systematically overestimated HI. At the site of model generation, both regressions were accurate. However on the other sites, prediction errors using regression 2 were systematically higher than for regression 1, which uses a topographic index with a physical basis. These results revealed that soil-landscape models may be useful for predicting soil hydromorphy over a region but only when validated under several environmental conditions.

CLIC1 (NCC27) is an unusual, largely intracellular, ion channel that exists in both soluble and membrane-associated forms. The soluble recombinant protein can be expressed in Escherichia coli, a property that has made possible both detailed electrophysiological studies in lipid bilayers and an examination of the mechanism of membrane integration. SolubleE. coli-derived CLIC1 moves from solution into artificial bilayers and forms chloride-selective ion channels with essentially identical conductance, pharmacology, and opening and closing kinetics to those observed in CLIC1-transfected Chinese hamster ovary cells. The process of membrane integration of CLIC1 is pH-dependent. Following addition of protein to the trans solution, small conductance channels with slow kinetics (SCSK) appear in the bilayer. These SCSK modules then appear to undergo a transition to form a high conductance channel with fast kinetics. This has four times the conductance of the SCSK and fast kinetics that characterize the native channel. This suggests that the CLIC1 ion channel is likely to consist of a tetrameric assembly of subunits and indicates that despite its size and unusual properties, it is able to form a completely functional ion channel in the absence of any other ancillary proteins.

A synthetic Synechocystissp. PCC6803 DnaB split mini-intein gene was constructed for the in vivocyclization of recombinant proteins expressed in Escherichia coli. The system was used to cyclize the NH2-terminal domain of E. coliDnaB, the structure of which had been determined previously by NMR spectroscopy. Cyclization was found to proceed efficiently, with little accumulation of precursor, and the product was purified in high yield. The solution structure of cyclic DnaB-N is not significantly different from that of linear DnaB-N and it unfolds reversibly at temperatures ∼14 °C higher. Improved hydrogen bonding was observed in the first and last helices, and the length of the last helix was increased, while the 9-amino acid linker used to join the NH2and COOH termini was found to be highly mobile. The measured thermodynamic stabilization of the structure (ΔΔG≈ 2 kcal/mol) agrees well with the value estimated from the reduced conformational entropy in the unfolded form. Simple polymer theory can be used to predict likely free energy changes resulting from protein cyclization and how the stabilization depends on the size of the protein and the length of the linker used to connect the termini.

CLIC1 (NCC27) is a member of the highly conserved class of chloride ion channels that exists in both soluble and integral membrane forms. Purified CLIC1 can integrate into synthetic lipid bilayers forming a chloride channel with similar properties to those observed in vivo. The structure of the soluble form of CLIC1 has been determined at 1.4-A resolution. The protein is monomeric and structurally homologous to the glutathione S-transferase superfamily, and it has a redox-active site resembling glutaredoxin. The structure of the complex of CLIC1 with glutathione shows that glutathione occupies the redox-active site, which is adjacent to an open, elongated slot lined by basic residues. Integration of CLIC1 into the membrane is likely to require a major structural rearrangement, probably of the N-domain (residues 1-90), with the putative transmembrane helix arising from residues in the vicinity of the redox-active site. The structure indicates that CLIC1 is likely to be controlled by redox-dependent processes.

The molecular interactions driving reactive center loop (RCL) insertion are of considerable interest in gaining a better understanding of the serpin inhibitory mechanism. Previous studies have suggested that interactions in the proximal hinge/breach region may be critical determinants of RCL insertion in serpins. In this study, conformational and functional changes in plasminogen activator inhibitor-2 (PAI-2) following incubation with a panel of synthetic RCL peptides indicated that the P14 residue is critical for RCL insertion, and hence inhibitory activity, in PAI-2. Only RCL peptides with a P14 threonine were able to induce the stressed to relaxed transition and abolish inhibitory activity in PAI-2, indicating that RCL insertion into beta-sheet A of PAI-2 is dependent upon this residue. The recently solved crystal structure of relaxed PAI-2 (PAI-2.RCL peptide complex) allowed detailed analysis of molecular interactions involving P14 related to RCL insertion. Of most interest is the rearrangement of hydrogen bonding around the breach region that accompanies the stressed to relaxed transition, in particular the formation of a side chain hydrogen bond between the threonine at P14 and an adjacent tyrosine on strand 2 of beta-sheet B in relaxed PAI-2. Structural alignment of known serpin sequences showed that this pairing (or the equivalent serine/threonine pairing) is highly conserved ( approximately 87%) in inhibitory serpins and may represent a general structural basis for serpin inhibitory activity.

The structure of the serpin, plasminogen activator inhibitor type-2 (PAI-2), in a complex with a peptide mimicking its reactive center loop (RCL) has been determined at 1.6-A resolution. The structure shows the relaxed state serpin structure with a prominent six-stranded beta-sheet. Clear electron density is seen for all residues in the peptide. The P1 residue of the peptide binds to a well defined pocket at the base of PAI-2 that may be important in determining the specificity of protease inhibition. The stressed-to-relaxed state (S --> R) transition in PAI-2 can be modeled as the relative motion between a quasirigid core domain and a smaller segment comprising helix hF and beta-strands s1A, s2A, and s3A. A comparison of the Ramachandran plots of the stressed and relaxed state PAI-2 structures reveals the location of several hinge regions connecting these two domains. The hinge regions cluster in three locations on the structure, ensuring a cooperative S --> R transition. We hypothesize that the hinge formed by the conserved Gly(206) on beta-strand s3A in the breach region of PAI-2 effects the S --> R transition by altering its backbone torsion angles. This torsional change is due to the binding of the P14 threonine of the RCL to the open breach region of PAI-2.

The Sm/Lsm proteins associate with small nuclear RNA to form the core of small nuclear ribonucleoproteins, required for processes as diverse as pre-mRNA splicing, mRNA degradation and telomere formation. The Lsm proteins from archaea are likely to represent the ancestral Sm/Lsm domain. Here, we present the crystal structure of the Lsmα protein from the thermophilic archaeon Methanobacterium thermoautotrophicum at 2.0 A˚ resolution. The Lsmα protein crystallizes as a heptameric ring comprised of seven identical subunits interacting via β-strand pairing and hydrophobic interactions. The heptamer can be viewed as a propeller-like structure in which each blade consists of a seven-stranded antiparallel β-sheet formed from neighbouring subunits. There are seven slots on the inner surface of the heptamer ring, each of which is lined by Asp, Asn and Arg residues that are highly conserved in the Sm/Lsm sequences. These conserved slots are likely to form the RNA-binding site. In archaea, the gene encoding Lsmα is located next to the L37e ribosomal protein gene in a putative operon, suggesting a role for the Lsmα complex in ribosome function or biogenesis.

Stathmin family phosphoproteins (stathmin, SCG10, SCLIP, and RB3/RB3′/RB3“) are involved in signal transduction and regulation of microtubule dynamics. With the exception of stathmin, they are expressed exclusively in the nervous system, where they display different spatio-temporal and functional regulations and hence play at least partially distinct and possibly complementary roles in relation to the control of development, plasticity, and neuronal activities. At the molecular level, each possesses a specific “stathmin-like domain” and, with the exception of stathmin, various combinations of N-terminal extensions involved in their association with intracellular membrane compartments. We show here that each stathmin-like domain also displays specific biochemical and tubulin interaction properties. They are all able to sequester two α/β tubulin heterodimers as revealed by their inhibitory action on tubulin polymerization and by gel filtration. However, they differ in the stabilities of the complexes formed as well as in their interaction kinetics with tubulin followed by surface plasmon resonance as follows: strong stability and slow kinetics for RB3; medium for SCG10, SCLIP, and stathmin; and weak stability and rapid kinetics for RB3′. These results suggest that the fine-tuning of their stathmin-like domains contributes to the specific functional roles of stathmin family proteins in the regulation of microtubule dynamics within the various cell types and subcellular compartments of the developing or mature nervous system.

Because soils are both a source and a sink for atmospheric CO2, there is an increasing need to characterize the spatial distribution of soil C pools. Large amounts of organic carbon (OC) accumulate in hydric bottom-lands soils. In the Armorican Massif (Western France) where these soils represent 20% of the total surface area, the spatial characterization of OC pools is difficult to assess due to methodological problems such as high spatial variability. Soil color indexes, which combine various characteristics of soil horizons or profiles, are an alternative approach for quantifying the differences in OC storage. In addition, terrain attributes derived from Digital Elevation Models (DEM) may be useful in characterizing the distribution of soil color indexes over large areas. Thus, the overall goal of this work was the development and application of a model for use in predicting the organic carbon (OC) content of soil areas. To accomplish this, extensive examination of soil morphology combined with selected terrain attributes measured in the field and calculated from a digital elevation model (DEM) were used. Soil samples were collected in Western France from a 2-ha agricultural parcel that forms the major part of a hillslope. The results indicate that OC stocks of the entire profile were correlated highly to a soil hydromorphic index (HI) (r2= 0.80). HI is a function of the percent of the total soil profile depth constituted by horizons with some degree of hydromorphic feature development and the moist color of the surface A horizon. Using a stepwise regression technique, we constructed a prediction model of HI distribution by using the relations between HI and (i) the elevation above the stream bank (ES) (r2= 0.80); (ii) the downslope gradient (DG) (r2= 0.55); and (iii) the upslope contributing area (AMU) (r2= 0.60). Validation of this model on a second site showed that topographical attributes explained up to 75% of the profile OC stock variability. These results confirmed that the integration of a soil index and topographical information is a useful tool for prediction of OC distribution. In addition, the use of soil morphologic indexes could significantly improved the construction and the validation of soil-landscape models because it would minimize laboratory measurements of OC reservoirs.

The advantages of the organism Dictyostelium discoideumas an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [15N]NH4Cl and [13C]glycerol was used to generate isotopically labeled Escherichia coli,which was subsequently introduced to D. discoideumcells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly 13C,15N-labeled protein secreted by approximately 1010D. discoideumcells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional 1H-13C HSQC spectrum confirms 13C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination.

d-Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) catalyses the central CO2-fixing reaction of photosynthesis in a complex, multiple-step process. Several structures of rubisco complexed with substrate analogues, inhibitors and products have been determined by X-ray crystallography. The structures fall into two well-defined and distinct states. The active site is either “open” or “closed”. The timing and mechanism of the transition between these two states have been uncertain. We solved the crystal structure of unactivated (metal-free) rubisco from tobacco with only inorganic phosphate bound and conclude that phosphate binding per sedoes not trigger closure, as it does in the similarly structured enzyme, triosephosphate isomerase. Comparison of all available rubisco structures suggests that, instead, the distance between the terminal phosphates (P1 and P2) of the bisphosphate ligand is the trigger: if that distance is less than 9.1 Å, then the active site closes; if it is greater than 9.4 Å then the enzyme remains open. Shortening of the inter-phosphate distance results from the ligand binding in a more curved conformation when O atoms of the ligand’s sugar backbone interact either with the metal, if it is present, or with charged groups in the metal-binding site, if the metal is absent. This shortening brings the P1 phosphate into hydrogen bonding contact with Thr65. Thr65 exists in two discrete states related by a rotation of the backbone ψ torsion angle. This rotation is coupled to domain rotation and hence to active site closure. Rotation of the side-chain of Thr65 also affects the C-terminal strand of large subunit which packs against Loop 6 after closure. The position of the C-terminal strand in the closed state is stabilised by multiple polar interactions with a distinctive highly-charged latch site involving the side-chain of Asp473. In the open state, this latch site may be occupied instead by phosphorylated anions.

Stathmin is a cytosoluble phosphoprotein proposed to be a regulatory relay integrating diverse intracellular signaling pathway. Its interaction with tubulin modulates microtubule dynamics by destabilization of assembled microtubules or inhibition of their polymerization from free tubulin. The aim of this study was to probe the native structure of stathmin and to delineate its minimal region able to interact with tubulin. Limited proteolysis of stathmin revealed four structured domains within the native protein, corresponding to amino acid sequences 22-81 (I), 95-113 (II), 113-128 (III), and 128-149 (IV), which allows us to propose stathmin folding hypotheses. Furthermore, stathmin proteolytic fragments were mixed to interact with tubulin, and those that retained affinity for tubulin were isolated by size exclusion chromatography and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The results indicate that, to interact with tubulin, a stathmin fragment must span a minimal core region from residues 42 to 126, which interestingly corresponds to the predicted alpha-helical "interaction region" of stathmin. In addition, an interacting stathmin fragment must include a short N- or C-terminal extension. The functional significance of these interaction constrains is further validated by tubulin polymerization inhibition assays with fragments designed on the basis of the tubulin binding results. The present results will help to optimize further stathmin structural studies and to develop molecular tools to target its interaction with tubulin.