Conformational changes in arginine kinase upon ligand binding seen by small-angle X-ray scattering

Small-angle X-ray scattering is used to study the effects of substrate binding to lobster arginine kinase in solution. We measure the radius of gyration of the enzyme in the absence and in the presence of ligands. We find that the radius of gyration decreases by 1.20 ± 0.25 Å upon binding ADP-Mg and L-arginine to form the ternary complex. The same decrease is also observed upon binding ADP-Mg alone or ATP-Mg. These results indicate a large conformational change consistent with the hinge motion of domains observed in other phosphokinases.     

The kinetics of protein-protein recognition

We examine a simple kinetic model for association that incorporates the basic features of protein-protein recognition within the rigid body approximation, that is, when no large conformation change occurs. Association starts with random collision at the rate k_coll predicted by the Einstein-Smoluchowski equation. This creates an encounter pair that can evolve into a stable complex if and only if the two molecules are correctly oriented and positioned, which has a probability p_r. In the absence of long-range interactions, the bimolecular rate of association is p_r k_coll. Long-range electrostatic interactions affect both k_coll and p_r. The collision rate is multiplied by q_t, a factor larger than 1 when the molecules carry net charges of opposite sign as coulombic attraction makes collisions more frequent, and less than 1 in the opposite case. The probability p_r is multiplied by a factor q_r that represents the steering effect of electric dipoles, which preorient the molecules before they collide. The model is applied to experimental data obtained by Schreiber and Fersht (Nat. Struct. Biol. 3:427–431, 1996) on the kinetics of barnase-barstar association. When long-range electrostatic interactions are fully screened or mutated away, q_tq_r ≈1, and the observed rate of productive collision is p_r k_coll ≈10⁵ M⁻¹ · s⁻¹. Under these conditions, p_r ≈1.5 · 10⁻⁵ is determined by geometric constraints corresponding to a loss of rotational freedom. Its value is compatible with computer docking simulations and implies a rotational entropy loss ΔS_rot ≈ 22 e.u. in the transition state. At low ionic strength, long-range electrostatic interactions accelerate barnase-barstar association by a factor q_tq_rof up to 10⁵ as favorable charge-charge and charge-dipole interactions work together to make it much faster than free diffusion would allow. Proteins 28:153–161, 1997. © 1997 Wiley-Liss Inc.     

Peptide aMptD-mediated capture PCR for detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples

A peptide-mediated capture PCR for the detection of Mycobacterium avium subsp. paratuberculosis in bulk milk samples was developed and characterized. Capture of the organism was performed using peptide aMptD, which had been shown to bind to the M. avium subsp. paratuberculosis MptD protein (J. Stratmann, B. Strommenger, R. Goethe, K. Dohmann, G. F. Gerlach, K. Stevenson, L. L. Li, Q. Zhang, V. Kapur, and T. J. Bull, Infect. Immun. 72:1265-1274, 2004). Consistent expression of the MptD receptor protein and binding of the aMptD ligand were demonstrated by capturing different Mycobacterium avium subsp. paratuberculosis type I and type II strains and subsequent PCR analysis using ISMav2-based primers. The analytical sensitivity of the method was determined to be 5 x 10(2) CFU ml(-1) for artificially contaminated milk. The specificity of aMptD binding was confirmed by culture and competitive capture assays, showing selective enrichment of M. avium subsp. paratuberculosis (at a concentration of 5 x 10(2) CFU ml(-1)) from samples containing 100- and 1,000-fold excesses of other mycobacterial species, including M. avium subsp. avium and M. avium subsp. hominissuis. The aMptD-mediated capture of M. avium subsp. paratuberculosis using paramagnetic beads, followed by culture, demonstrated the ability of this approach to capture viable target cells present in artificially contaminated milk. Surface plasmon resonance experiments revealed that the aMptD peptide is a high-affinity ligand with a calculated association rate constant of 9.28 x 10(3) and an association constant of 1.33 x 10(9). The potential use of the method on untreated raw milk in the field was investigated by testing 423 bulk milk samples obtained from different dairy farms in Germany, 23 of which tested positive. Taken together, the results imply that the peptide-mediated capture PCR might present a suitable test for paratuberculosis screening of dairy herds, as it has an analytical sensitivity sufficient for detection of M. avium subsp. paratuberculosis in bulk milk samples under field conditions, relies on a defined and validated ligand-receptor interaction, and is adaptable to routine diagnostic laboratory automation.     

Biochronologie et corrélation des bassins néogènes du Couloir sud-rifain (Maroc) fondées sur les évènements de foraminifères planctoniques et de nannofossiles calcaires

Qualitative and quantitative analyses of planktonic foraminifera (completed by calcareous nannofossils in some localities) from 8 sections and boreholes located in 7 Neogene basins of the South Rifian Corridor (Morocco) enable us to identify 16 bioevents calibrated with the geomagnetic polarity time scale. These events are useful for (1) assigning the sediments to the Tortonian (bioevents 1 to 7), the Messinian (bioevents 9 to 14, the event 8 indicates the Tortonian/Messinian boundary), the Early Pliocene (bioevent 15) and the Middle Pliocene (bioevent 16), (2) establishing high resolution correlations between the sections and boreholes studied herein, (3) locating the South Rifian Corridor sections and boreholes within the framework of biostratigraphic events recognized during the latest ten years in time-equivalent Atlantic and Mediterranean sequences, and (4) estimating the variation of sedimentation rates in the studied basins. With respect to Morocco, previous detailed studies concerned only the Rabat area (Bou Regreg valley) and Guercif basin. Our results extend correlations to the other basins of the South Rifian Corridor, a key area to understand connections between the Atlantic Ocean and the Mediterranean Sea at the end of the Miocene.     

The targets of CAPRI rounds 6-12

Six protein-protein complexes and two homodimeric proteins involved in a variety of biological processes were offered as targets to CAPRI by crystallographers in Rounds 6-12. CAPRI predictor groups had to predict their structure by docking the free proteins, which they did with a degree of success that depended largely on the amplitude of the conformation changes. In one case at least, the prediction pointed to alternative possibilities of interactions in the crystal of a complex, showing that docking methods have value even when there is an experimental structure.     

Systemin and jasmonic acid regulate constitutive and herbivore-induced systemic volatile emissions in tomato, Solanum lycopersicum

Transgenic tomato (Solanum lycopersicum) plants that overexpress the Prosystemin gene (35S::PS) and plants with a mutation in the JA biosynthetic pathway (def1) are known to exhibit a constitutive or reduced wound response, respectively. Here it is demonstrated that several independent 35S::PS lines emit high levels of specific volatiles in addition to increased accumulation of proteinase inhibitors (PIs). Furthermore, the temporal dynamics of systemically induced volatile compounds including green-leaf volatiles, terpenes, and shikimic acid-derivatives from 35S::PS and def1 plants in response to herbivore wounding and treatment with jasmonic acid (JA) are described. Application of JA induced defense protein accumulation and volatile emissions in wild type plants, but did not further increase systemic volatile emissions from 35S::PS plants. Wounding by Manduca sexta larvae induced synthesis of defense proteins and emission of volatiles in wild type plants, but not in def1 plants. Application of jasmonic acid restored the local and systemic accumulation of defense proteins in def1, as well as enhanced herbivore-induced volatile emissions. These results provide strong support for the role of prosystemin- and JA-signaling in the regulation of volatile emissions in tomato plants.     

DnaB helicase dynamics in bacterial DNA replication resolved by single-molecule studies

In Escherichia coli, the DnaB helicase forms the basis for the assembly of the DNA replication complex. The stability of DnaB at the replication fork is likely important for successful replication initiation and progression. Single-molecule experiments have significantly changed the classical model of highly stable replication machines by showing that components exchange with free molecules from the environment. However, due to technical limitations, accurate assessments of DnaB stability in the context of replication are lacking. Using in vitro fluorescence single-molecule imaging, we visualise DnaB loaded on forked DNA templates. That these helicases are highly stable at replication forks, indicated by their observed dwell time of ∼30 min. Addition of the remaining replication factors results in a single DnaB helicase integrated as part of an active replisome. In contrast to the dynamic behaviour of other replisome components, DnaB is maintained within the replisome for the entirety of the replication process. Interestingly, we observe a transient interaction of additional helicases with the replication fork. This interaction is dependent on the τ subunit of the clamp-loader complex. Collectively, our single-molecule observations solidify the role of the DnaB helicase as the stable anchor of the replisome, but also reveal its capacity for dynamic interactions.     

Determination of Angptl4 mRNA as a Diagnostic Marker of Primary and Metastatic Clear Cell Renal-Cell Carcinoma

Background

We have previously shown that angiopoietin-like 4 (angptl4) mRNA, a hypoxia-inducible gene, is highly expressed in clear cell renal-cell carcinoma (ccRCC), the most common subtype of RCC for which no specific marker is available. We here investigated whether angptl4 mRNA 1) could be a useful diagnostic and/or prognostic marker of ccRCC in a large and comprehensive retrospective series, 2) induction is dependent on the VHL status of tumors.

Methodology/Principal Findings

Using in situ hybridization, we report that angptl4 mRNA is expressed in 100% of both sporadic (n = 102) and inherited (n = 6) primary ccRCCs, without any statistical association with nuclear grade (p = 0.39), tumor size (p = 0.09), stage grouping (p = 0.17), progression-free survival (p = 0.94), and overall survival (p = 0.80). Angptl4 mRNA was also expressed in 26 (87%) of 30 secondary ccRCCs but neither in any other secondary RCCs (n = 7). In contrast, angptl4 mRNA was neither expressed in 94% non-ccRCC renal tumors (papillary RCCs (n = 46), chromophobe RCCs (n = 28), and oncocytomas (n = 9)), nor in non-renal clear cell carcinomas (n = 39). Angptl4 expression was also examined in tumors associated (n = 23) or not associated (n = 66) with VHL disease. 40 (98%) hemangioblastomas expressed angptl4 whereas all pheochromocytomas (n = 23) and pancreatic tumors (n = 25) were angptl4-negative, whatever their VHL status.

Conclusions/Significance

Angptl4 mRNA expression was highly associated with ccRCC (p = 1.5 10⁻⁴⁹, Chi square test) allowing to define its expression as a diagnosis marker for primary ccRCC. Moreover, angptl4 mRNA allows to discriminate the renal origin of metastases of clear-cell carcinomas arising from various organs. Finally, inactivation of VHL gene is neither necessary nor sufficient for angptl4 mRNA induction.