Tolerance of a protein to multiple polar-to-hydrophobic surface substitutions

Hydrophobic substitutions at solvent-exposed positions in two alpha-helical regions of the bacteriophage P22 Arc repressor were introduced by combinatorial mutagenesis. In helix A, hydrophobic residues were tolerated individually at each of the five positions examined, but multiple substitutions were poorly tolerated as shown by the finding that mutants with more than two additional hydrophobic residues were biologically inactive. Several inactive helix A variants were purified and found to have reduced thermal stability relative to wild-type Arc, with a rough correlation between the number of polar-to-hydrophobic substitutions and the magnitude of the stability defect. Quite different results were obtained in helix B, where variants with as many as five polar-to-hydrophobic substitutions were found to be biologically active and one variant with three hydrophobic substitutions had a t(m) 6 degrees C higher than wild-type. By contrast, a helix A mutant with three similar polar-to-hydrophobic substitutions was 23 degrees C less stable than wild-type. Also, one set of three polar-to-hydrophobic substitutions in helix B was tolerated when introduced into the wild-type background but not when introduced into an equally active mutant having a nearly identical structure. Context effects occur both when comparing different regions of the same protein and when comparing the same region in two different homologues.     

Synthesis and catalysis by molecularly imprinted materials

Molecularly imprinted materials have been demonstrated to possess a very high degree of selectivity towards targeted substrates. In addition to such tailor-made molecular recognition, progress has been made in introducing reactive groups into the recognition sites. Putting teeth into imprinted matrices is one method of making true enzyme mimics or plastizymes, which are plastic polymer enzyme mimics.     

Models of temporal variation in questing activity in individuals of Ixodes recinus (Acari: Ixodidae)

Intensive observations of the questing activity of Ixodes ricinus ticks in the field were made to provide data on the range of durations of periods of continuous questing activity, and of the variation in questing activity between individuals. Continuous periods of questing were observed to extend to a maximum of 28 hours. Substantial variation in questing activity between individuals was observed. Models fitted to the distribution of durations of bouts of questing activity provide insights into the questing ecology of I. ricinus. Results suggest that questing duration may not be solely dependent on the state of hydration of the tick. A function fitted to the frequency distribution of the proportions of active life that individuals spend on questing, provides an empirically-based model that can be used to generate a stochastic expression of variation of questing activity in individuals in a questing population.     

Inhibition of Photosynthesis by Shift in the Balance of Excitation Energy Distribution Between Photosystems in Dithiothreitol Treated Soybean Leaves

Chlorophyll fluorescence kinetics was used to investigate the effect of 1,4-dithiothreitol (DTT) on the distribution of excitation energy between photosystem 1 (PS1) and photosystem 2 (PS2) in soybean leaves under high irradiance (HI). The maximum PS2 quantum yield (F_v/F_m) was hardly affected by the presence of DTT, however, photon-saturated photosynthesis was depressed distinctly. Photochemical efficiency of open PS2 reaction centres during irradiation (F_v/F_m) was enhanced by about 30–40 % by DTT treatment, whereas photochemical quenching (q_P) was depressed by about 40 % under HI. DTT treatment caused a 30 % decrease in allocation of excitation energy to PS1 under HI and a 20 % increase to PS2. An obvious shift in the balance of excitation energy distribution between photosystems was observed in DTT-treated leaves. Though high excitation pressure (1 - q_P) resulted from DTT treatment, non-photochemical quenching (q_N) was lower. DTT completely inhibited the formation of zeaxanthin and also distinctly depressed the state transition (q_T). The shift in the balance of excitation distribution between the two photosystems induced by DTT was mainly due to the enhancement of excitation energy capture by PS2 antenna and the inhibition of state transition. It might be the shift in the balance between the two photosystems that mainly induced the depression of photosynthesis. Thus, to keep high utilization efficiency of absorbed photon energy, it is necessary to maintain the balance of excitation distribution between PS2 and PS1.     

Circular dichroism spectra of human hemoglobin reveal a reversible structural transition at body temperature

Previously we have shown that human red blood cells (RBCs) undergo a sudden change from blocking to passing through a 1.3±0.2-µm micropipette when applying an aspiration pressure of 2.3 kPa at a critical transition temperature (T_c=36.4±0.3 °C). Low-shear viscosity measurements suggested that changes in the molecular properties of hemoglobin might be responsible for this effect. To evaluate structural changes in hemoglobin at the critical temperature, we have used circular dichroism (CD) spectroscopy. The thermal denaturation curves of human hemoglobin A (HbA) and hemoglobin S (HbS) upon heating between 25 and 60 °C were non-linear and showed accelerated denaturation between 35 and 39 °C with a midpoint at 37.2±0.6 °C. The transition was reversible below 39 °C and independent of solution pH (pH 6.8–7.8). It was also independent of the oxygenation state of hemoglobin, since a sample that was extensively deoxygenated with N₂ showed a similar transition by CD. These findings suggest that a structural change in hemoglobin may enable the cellular passage phenomenon as well as the temperature-dependent decrease in viscosity of RBC solutions.     

A \sqrt N Dynamic Load Distribution Algorithm Using Anti-Tasks and Load State Vectors

One of the fundamental issues to ensure maximal performance improvement in a cluster computing environment is load distribution, which is commonly achieved by using polling-based load distribution algorithms. Such algorithms suffer from two weaknesses: (1) Load information exchanged during a polling session is confined to the two negotiating nodes only. (2) Such algorithms are not scalable in that growth of the distributed system is accompanied with increasing amount of polling sessions.In this paper, we proposed a LD algorithm which is based on anti-tasks and load state vectors. Anti-tasks travel around the distributed system for pairing up task senders and receivers. As an anti-task travels, timed load information is collected and disseminated over the entire system via the load state vector bundled with the anti-task. Guided by load state vectors, anti-tasks are spontaneously directed towards processing nodes having high transient workload, thus allowing their surplus workload to be relocated soonest possible. No peer-to-peer negotiations between senders and receivers are needed.To reduce the network bandwidth consumption caused by the anti-task algorithm, the number of hosts that an anti-task needs to travel to must be carefully limited. The algorithm achieves this by employing the mathematical notion of Finite Projective Plane (FPP). By employing FPP, the number of nodes that each anti-task has to travel is at most , where N is the number of nodes in the system, without sacrifying the spread of load information.     

Correspondence between anomalous m- and DeltaCp-values in protein folding

Proteins folding according to a classical two-state system characteristically show V-shaped chevron plots. We have previously interpreted the symmetrically curved chevron plot of the protein U1A as denaturant-dependent movements in the position of the transition state ensemble (TSE). S6, a structural analog of U1A, shows a classical V-shaped chevron plot indicative of straightforward two-state kinetics, but the mutant LA30 has a curved unfolding limb, which is most consistent with TSE mobility. The kinetic m-values (derivatives of the rate constants with respect to denaturant concentration) in themselves depend on denaturant concentration. To obtain complementary information about putative mobile TSEs, we have carried out a thermodynamic analysis of the three proteins, based on data for refolding and unfolding over the range 10 degrees C to 70 degrees C. The data at all temperatures can be fitted to two-state model systems. Importantly, for all three proteins the activation heat capacities are, within error, identical to the heat capacities measured in independent experiments under equilibrium conditions. Although the equilibrium heat capacities are essentially invariant with regard to denaturant concentration, the activation heat capacities, similar to the structurally equivalent kinetic m-values, show marked denaturant dependence. Furthermore, the values of beta++ at different denaturant concentrations measured by m-values and by heat capacity values are very similar. These observations are consistent with significant transition state movements within the framework of two-state folding. The basis for TSE movement appears to be enthalpic rather than entropic, suggesting that the binding energy of denaturant-protein interactions is a major determinant of the response of energy landscape contours to changing environments.     

Atomic resolution analysis of the catalytic site of an aspartic proteinase and an unexpected mode of binding by short peptides

The X-ray structures of native endothiapepsin and a complex with a hydroxyethylene transition state analog inhibitor (H261) have been determined at atomic resolution. Unrestrained refinement of the carboxyl groups of the enzyme by using the atomic resolution data indicates that both catalytic aspartates in the native enzyme share a single negative charge equally; that is, in the crystal, one half of the active sites have Asp 32 ionized and the other half have Asp 215 ionized. The electron density map of the native enzyme refined at 0.9 A resolution demonstrates that there is a short peptide (probably Ser-Thr) bound noncovalently in the active site cleft. The N-terminal nitrogen of the dipeptide interacts with the aspartate diad of the enzyme by hydrogen bonds involving the carboxyl of Asp 215 and the catalytic water molecule. This is consistent with classical findings that the aspartic proteinases can be inhibited weakly by short peptides and that these enzymes can catalyze transpeptidation reactions. The dipeptide may originate from autolysis of the N-terminal Ser-Thr sequence of the enzyme during crystallization.     

Production of singlet oxygen on irradiation of a photodynamic therapy agent, zinc-coproporphyrin III, with low host toxicity

Zinc-coproporphyrin III (Zincphyrin) acts efficiently as a photodynamic therapy (PDT) agent in mice, while it shows no tumor cell-killing activity in vitro and has a high LD₅₀ (low toxicity) in mice. It appears to have advantages over other porphyrins as a practical PDT reagent. In order to examine the action mechanism of Zincphyrin in PDT, we evaluated the photochemical characteristics of Zincphyrin by measurement of the near-infrared emission at 1268 nm, which provides direct evidence for formation of ¹O₂. Intense emission was observed in the presence of Zincphyrin, and was completely inhibited by NaN₃, a ¹O₂ scavenger. Based on a quenching study, the rate constant of the reaction of ¹O₂ with NaN₃ was determined to be 1.5–3.5 M^–1 s^–1, which is close to the reported value (3.8×10⁸ M^–1 s^–1). The intensity of the ¹O₂-specific emission was proportional to both the laser power and the concentration of Zincphyrin. The fluorescence quantum yield of Zincphyrin was 0.004 in phosphate buffer (100 mM, pH 7.4), which indicates that the excited state decays via other pathway(s) faster than through the fluorescence emission pathway. The lifetime of the triplet state of Zincphyrin (210 s) was relatively long compared to that of other porphyrins, such as hematoporphyrin (Hp) (40 s), coproporphyrin I (50 s), or coproporphyrin III (36 s). These results demonstrate the photodynamic generation of ¹O₂ by Zincphyrin.