Ferrous Campylobacter jejuni truncated hemoglobin P displays an extremely high reactivity for cyanide - a comparative study

Campylobacter jejuni hosts two hemoglobins (Hbs). The Camplylobacter jejuni single-domain Hb (called Cgb) is homologous to the globin domain of flavohemoglobin, and it has been proposed to protect the bacterium against nitrosative stress. The second Hb is called Ctb (hereafter Cj-trHbP), belongs to truncated Hb group III, and has been hypothesized to be involved in O(2) chemistry. Here, the kinetics and thermodynamics of cyanide binding to ferric and ferrous Cj-trHbP [Cj-trHbP(III) and Cj-trHbP(II), respectively] are reported and analyzed in parallel with those of related heme proteins, with particular reference to those from Mycobacterium tuberculosis. The affinity of cyanide for Cj-trHbP(II) is higher than that reported for any known (in)vertebrate globin by more than three orders of magnitude (K = 1.2 x 10(-6) m). This can be fully attributed to the highest (ever observed for a ferrous Hb) cyanide-binding association rate constant (k(on) = 3.3 x 10(3) m(-1).s(-1)), even though the binding process displays a rate-limiting step (k(max) = 9.1 s(-1)). Cj-trHbP(III) shows a very high affinity for cyanide (L = 5.8 x 10(-9) m); however, cyanide association kinetics are independent of cyanide concentration, displaying a rate-limiting step (l(max) = 2.0 x 10(-3) s(-1)). Values of the first-order rate constant for cyanide dissociation from Cj-trHbP(II)-cyanide and Cj-trHbP(III)-cyanide (k(off) =5.0 x 10(-3) s(-1) and l(off) > or = 1 x 10(-4) s(-1), respectively) are similar to those reported for (in)vertebrate globins. The very high affinity of cyanide for Cj-trHbP(II), reminiscent of that of horseradish peroxidase(II), suggests that this globin may participate in cyanide detoxification.     

Archaeal protoglobin structure indicates new ligand diffusion paths and modulation of haem-reactivity

The structural adaptability of the globin fold has been highlighted by the recent discovery of the 2-on-2 haemoglobins, of neuroglobin and cytoglobin. Protoglobin from Methanosarcina acetivorans C2A-a strictly anaerobic methanogenic Archaea-is, to the best of our knowledge, the latest entry adding new variability and functional complexity to the haemoglobin (Hb) superfamily. Here, we report the 1.3 A crystal structure of oxygenated M. acetivorans protoglobin, together with the first insight into its ligand-binding properties. We show that, contrary to all known globins, protoglobin-specific loops and an amino-terminal extension completely bury the haem within the protein matrix. Access of O(2), CO and NO to the haem is granted by the protoglobin-specific apolar tunnels reaching the haem distal site from locations at the B/G and B/E helix interfaces. Functionally, M. acetivorans dimeric protoglobin shows a selectivity ratio for O(2)/CO binding to the haem that favours O(2) ligation and anticooperativity in ligand binding. Both properties are exceptional within the Hb superfamily.     

Messaging on Gigabit Ethernet: Some Experiments with GAMMA and Other Systems

The Genoa Active Message MAchine (GAMMA) is a lightweight communication system based on the Active Ports paradigm, originally designed for efficient implementation over low-cost Fast Ethernet interconnects. In this paper we report about the recently completed porting of GAMMA to the Packet Engines GNIC-II and the Netgear GA620 Gigabit Ethernet adapters, and provide a comparison among GAMMA, MPI/GAMMA, TCP/IP, and MPICH, on such commodity interconnects, using different performance metrics. With a combination of low end-to-end latency (9.5 s with GNIC-II, 32 s with GA620) and high transmission throughput (almost 97 MByte/s with GNIC-II and 125 MByte/s with GA620, the latter obtained without changing the firmware of the adapter), GAMMA demonstrates the potential for Gigabit Ethernet lightweight protocols to yield messaging performance comparable to the best Myrinet-based messaging systems. This result is of interest, given the envisaged drop in cost of Gigabit Ethernet due to the transition from fiber optic to UTP cabling and ever increasing mass market production of such standard interconnect. We also reports about a technique for message fragmentation that is commonly exploited to increase the throughput with short message. When a different, though more widely used, performance metrics is considered, such a technique results into a performance loss rather than improvement.     

Effect of axial coordination on the kinetics of assembly and folding of the two halves of horse heart cytochrome C

The kinetics of the assembly of two complementary fragments of oxidized horse heart cytochrome c (cyt c), namely the heme-containing fragment-(1-56) and the fragment-(57-104), have been characterized at different pH values. At neutral pH the fragment-(1-56) is hexacoordinated and has two histidines axially ligated to the heme-Fe(III) (Santucci, R., Fiorucci, L., Sinibaldi, F., Polizio, F., Desideri, A., and Ascoli, F. (2000) Arch. Biochem. Biophys. 379, 331-336), thus mimicking what occurs in the folding intermediate of cyt c. The kinetics of the formation of the complex between the two fragments are characterized at pH 7.0 by a slow rate constant that is independent of the concentration of the reactants; conversely, at a low pH the kinetics are much faster and depend on the concentration of the fragments. This behavior suggests that the rate-limiting step observed in the recombination process of the fragments at neutral pH (that leads to the final coordination of Met-80) has to be ascribed to the detachment of the "misligated" histidine. Thus, the faster recombination rate at a low pH can be related to the fact that histidine is protonated and not able to coordinate to the metal. Furthermore, the independence of the rate constant on the concentration of the reactants observed at pH 7.0 can be accounted for by the occurrence of a conformational transition, which takes place immediately after the two fragments collapse together, likely simulating what induces the detachment of the misligated histidine during cytochrome folding.     

Reactivity and endogenous modification by nitrite and hydrogen peroxide: does human neuroglobin act only as a scavenger?

NGB (human neuroglobin), a recently discovered haem protein of the globin family containing a six-co-ordinated haem, is expressed in nervous tissue, but the physiological function of NGB is currently unknown. As well as playing a role in neuronal O2 homoeostasis, NGB is thought to act as a scavenger of reactive species. In the present study, we report on the reactivity of metNGB (ferric-NGB), which accumulates in vivo as a result of the reaction of oxyNGB (oxygenated NGB) with NO, towards NO2- and H2O2. NO2- co-ordination of the haem group accounts for the activity of metNGB in the nitration of phenolic substrates. The two different metNGB forms, with and without the internal disulfide bond between Cys46 (seventh residue on the inter-helix region between helices C and D) and Cys55 (fifth residue on helix D), exhibit different reactivity, the former being more efficient in activating NO2-. The kinetics of the reactions, the NO2--binding studies and the analysis of the nitrated products from different substrates all support the hypothesis that metNGB is able to generate an active species with the chemical properties of peroxynitrite, at pathophysiological concentrations of NO2- and H2O2. Without external substrates, the targets of the reactive species generated by the metNGB/NO2-/H2O2 system are endogenous tyrosine (resulting in the production of 3-nitrotyrosine) and cysteine (oxidized to sulfinic acid and sulfonic acid) residues. These endogenous modifications were characterized by HPLC-MS/MS (tandem MS) analysis of metNGB after reaction with NO2- and H2O2 under various conditions. The internal S-S bond affects the functional properties of the protein. Therefore metNGB acts not only as scavenger of toxic species, but also as a target of the self-generated reactive species. Self-modification of the protein may be related to or inhibit its postulated neuroprotective activity.     

Dynamic Conduction and Repolarisation Changes in Early Arrhythmogenic Right Ventricular Cardiomyopathy versus Benign Outflow Tract Ectopy Demonstrated by High Density Mapping & Paced Surface ECG Analysis

Aims

The concealed phase of arrhythmogenic right ventricular cardiomyopathy (ARVC) may initially manifest electrophysiologically. No studies have examined dynamic conduction/repolarization kinetics to distinguish benign right ventricular outflow tract ectopy (RVOT ectopy) from ARVC''s early phase. We investigated dynamic endocardial electrophysiological changes that differentiate early ARVC disease expression from RVOT ectopy.

Methods

22 ARVC (12 definite based upon family history and mutation carrier status, 10 probable) patients without right ventricular structural anomalies underwent high-density non-contact mapping of the right ventricle. These were compared to data from 14 RVOT ectopy and 12 patients with supraventricular tachycardias and normal hearts. Endocardial & surface ECG conduction and repolarization parameters were assessed during a standard S₁-S₂ restitution protocol.

Results

Definite ARVC without RV structural disease could not be clearly distinguished from RVOT ectopy during sinus rhythm or during steady state pacing. Delay in Activation Times at coupling intervals just above the ventricular effective refractory period (VERP) increased in definite ARVC (43±20 ms) more than RVOT ectopy patients (36±14 ms, p = 0.03) or Normals (25±16 ms, p = 0.008) and a progressive separation of the repolarisation time curves between groups existed. Repolarization time increases in the RVOT were also greatest in ARVC (definite ARVC: 18±20 ms; RVOT ectopy: 5±14, Normal: 1±18, p<0.05). Surface ECG correlates of these intracardiac measurements demonstrated an increase of greater than 48 ms in stimulus to surface ECG J-point pre-ERP versus steady state, with an 88% specificity and 68% sensitivity in distinguishing definite ARVC from the other groups. This technique could not distinguish patients with genetic predisposition to ARVC only (probable ARVC) from controls.

Conclusions

Significant changes in dynamic conduction and repolarization are apparent in early ARVC before detectable RV structural abnormalities, and were present to a lesser degree in probable ARVC patients. Investigation of dynamic electrophysiological parameters may be useful to identify concealed ARVC in patients without disease pedigrees by using endocardial electrogram or paced ECG parameters.     

FRASS: the web-server for RNA structural comparison

Background  

The impressive increase of novel RNA structures, during the past few years, demands automated methods for structure comparison. While many algorithms handle only small motifs, few techniques, developed in recent years, (ARTS, DIAL, SARA, SARSA, and LaJolla) are available for the structural comparison of large and intact RNA molecules.     

Improving the quality of protein structure models by selecting from alignment alternatives

Background  

In the area of protein structure prediction, recently a lot of effort has gone into the development of Model Quality Assessment Programs (MQAPs). MQAPs distinguish high quality protein structure models from inferior models. Here, we propose a new method to use an MQAP to improve the quality of models. With a given target sequence and template structure, we construct a number of different alignments and corresponding models for the sequence. The quality of these models is scored with an MQAP and used to choose the most promising model. An SVM-based selection scheme is suggested for combining MQAP partial potentials, in order to optimize for improved model selection.