Role of electrostatic interactions for the stability and folding behavior of cold shock protein

The impacts of three charged‐residue‐involved mutations, E46A, R3E, and R3E/L66E, on the thermostability and folding behavior of the cold shock protein from the themophile Bacillus caldolyticus (Bc‐Csp) were investigated by using a modified Gō‐like model, in which the nonspecific electrostatic interactions of charged residues were taken into account. Our simulation results show that the wild‐type Bc‐Csp and its three mutants are all two‐sate folders, which is consistent with the experimental observations. It is found that these three mutations all lead to a decrease of protein thermodynamical stability, and the effect of R3E mutation is the strongest. The lower stability of these three mutants is due to the increase of the enthalpy of the folded state and the entropy of the unfolded state. Using this model, we also studied the folding kinetics and the folding/unfolding pathway of the wild‐type Bc‐Csp as well as its three mutants and then discussed the effects of electrostatic interactions on the folding kinetics. The results indicate that the substitutions at positions 3 and 46 largely decrease the folding kinetics, whereas the mutation of residue 66 only slightly decreases the folding rate. This result agrees well with the experimental observations. It is also found that these mutations have little effects on the folding transition state and the folding pathway, in which the N‐terminal β sheet folds earlier than the C‐terminal region. We also investigated the detailed unfolding pathway and found that it is really the reverse of the folding pathway, providing the validity of our simulation results. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Grading the commercial optical biosensor literature—Class of 2008: ‘The Mighty Binders’

Optical biosensor technology continues to be the method of choice for label‐free, real‐time interaction analysis. But when it comes to improving the quality of the biosensor literature, education should be fundamental. Of the 1413 articles published in 2008, less than 30% would pass the requirements for high‐school chemistry. To teach by example, we spotlight 10 papers that illustrate how to implement the technology properly. Then we grade every paper published in 2008 on a scale from A to F and outline what features make a biosensor article fabulous, middling or abysmal. To help improve the quality of published data, we focus on a few experimental, analysis and presentation mistakes that are alarmingly common. With the literature as a guide, we want to ensure that no user is left behind. Copyright © 2009 John Wiley & Sons, Ltd.     

Kinetic mechanism and catalysis of Trypanosoma cruzi dihydroorotate dehydrogenase enzyme evaluated by isothermal titration calorimetry

Trypanosoma cruzi dihydroorotate dehydrogenase (TcDHODH) catalyzes the oxidation of l-dihydroorotate to orotate with concomitant reduction of fumarate to succinate in the de novo pyrimidine biosynthetic pathway. Based on the important need to characterize catalytic mechanism of TcDHODH, we have tailored a protocol to measure TcDHODH kinetic parameters based on isothermal titration calorimetry. Enzymatic assays lead to Michaelis-Menten curves that enable the Michaelis constant (K_M) and maximum velocity (V_max) for both of the TcDHODH substrates: dihydroorotate (K_M = 8.6 ± 2.6 μM and V_max = 4.1 ± 0.7 μM s^-1) and fumarate (K_M = 120 ± 9 μM and V_max = 6.71 ± 0.15 μM s^-1). TcDHODH activity was investigated using dimethyl sulfoxide (10%, v/v) and Triton X-100 (0.5%, v/v), which seem to facilitate the substrate binding process with a small decrease in K_M. Arrhenius plot analysis allowed the determination of thermodynamic parameters of activation for substrates and gave some insights into the enzyme mechanism. Activation entropy was the main contributor to the Gibbs free energy in the formation of the transition state. A factor that might contribute to the unfavorable entropy is the hindered access of substrates to the TcDHODH active site where a loop at its entrance regulates the open-close channel for substrate access.     

Disruption of quaternary structure in Escherichia coli dihydrodipicolinate synthase (DHDPS) generates a functional monomer that is no longer inhibited by lysine

Escherichia coli dihydrodipicolinate synthase (DHDPS, E.C. 4.2.1.52), a natively homotetrameric enzyme was converted to a monomeric species through the introduction of destabilising interactions at two different subunit interfaces allowing exploration of the roles of the quaternary structure in affecting catalytic competency. The double mutant DHDPS-L197D/Y107W displays gel filtration characteristics consistent with a single non-interacting monomeric species, which was confirmed by sedimentary velocity experiments. This monomer was shown to be catalytically active, but with reduced catalytic efficiency (k_cat = 9.8 ± 0.5 s⁻¹), displaying 8% of the specific activity of the wild-type enzyme. The Michaelis constants for the substrates pyruvate and for (S)-aspartate semialdehyde increased by an order of magnitude, indicating that quaternary structure plays a significant role in substrate specificity. This monomeric species exhibited an enhanced propensity for aggregation and inactivation, indicating that whilst the oligomerization is not an intrinsic criterion for catalysis, higher oligomeric forms may benefit from both increased catalytic efficiency and diminished aggregation propensity. Furthermore, allosteric inhibition by (S)-lysine was abolished for DHDPS-L197D/Y107W, confirming the importance of the dimeric unit as the minimal functional assembly for efficient (S)-lysine binding.     

Physiologic gating properties of unitary cardiac L-type Ca channels

The contraction of adult mammalian ventricular cardiomyocytes is triggered by the influx of Ca²⁺ ions through sarcolemmal L-type Ca²⁺ channels (LCCs). However, the gating properties of unitary LCCs under physiologic conditions have remained elusive. Towards this end, we investigated the voltage-dependence of the gating kinetics of unitary LCCs, with a physiologic concentration of Ca²⁺ ions permeating the channel. Unitary LCC currents were recorded with 2 mM external Ca²⁺ ions (in the absence of LCC agonists), using cell-attached patches on K-depolarized adult rat ventricular myocytes. The voltage-dependence of the peak probability of channel opening (Po vs. Vm) displayed a maximum value of 0.3, a midpoint of −12 mV, and a slope factor of 8.5. The maximum value for Po of the unitary LCC was significantly higher than previously assumed, under physiologic conditions. We also found that the mean open dwell time of the unitary LCC increased twofold with depolarization, ranging from 0.53 ± 0.02 ms at −30 mV to 1.08 ± 0.03 ms at 0 mV. The increase in mean LCC open time with depolarization counterbalanced the decrease in the single LCC current amplitude; the latter due to the decrease in driving force for Ca²⁺ ion entry. Thus, the average amount of Ca²⁺ ions entering through an individual LCC opening (∼300-400 ions) remained relatively constant over this range of potentials. These novel results establish the voltage-dependence of unitary LCC gating kinetics using a physiologic Ca²⁺ ion concentration. Moreover, they provide insight into local Ca²⁺-induced Ca²⁺ release and a more accurate basis for mathematical modeling of excitation-contraction coupling in cardiac myocytes.     

Intracellular metabolite determination in the presence of extracellular abundance: Application to the penicillin biosynthesis pathway in Penicillium chrysogenum

Important steps in metabolic pathways are formed by the transport of substrates and products over the cell membrane. The study of in vivo transport kinetics requires accurate quantification of intra‐ and extracellular levels of the transported compounds. Especially in case of extracellular abundance, the proper determination of intracellular metabolite levels poses challenges. Efficient removal of extracellular substrates and products is therefore important not to overestimate the intracellular amounts. In this study we evaluated two different rapid sampling methods, one combined with cold filtration and the other with centrifugation, for their applicability to determine intracellular amounts of metabolites which are present in high concentrations in the extracellular medium. The filtration‐based method combines fast sampling and immediate quenching of cellular metabolism in cold methanol, with rapid and effective removal of all compounds present outside the cells by means of direct filtration and subsequent filtration‐based washing. In the centrifugation‐based method, removal of the extracellular metabolites from the cells was achieved by means of multiple centrifugation and resuspension steps with the cold quenching solution. The cold filtration method was found to be highly superior to the centrifugation method to determine intracellular amounts of metabolites related to penicillin‐G biosynthesis and allowed the quantification of compounds of which the extracellular amounts were 3–4 orders of magnitude higher than the intracellular amounts. Using this method for the first time allowed to measure the intracellular levels of the side chain precursor phenylacetic acid (PAA) and the product penicillin‐G of the penicillin biosynthesis pathway, compounds of which the transport mechanism in Penicillium chrysogenum is still far from being sufficiently understood. Biotechnol. Bioeng. 2010;107: 105–115. © 2010 Wiley Periodicals, Inc.     

The Impact of Viral RNA on Assembly Pathway Selection

Many single-stranded RNA viruses self-assemble their protein containers around their genomes. The roles that the RNA plays in this assembly process have mostly been ignored, resulting in a protein-centric view of assembly that is unable to explain adequately the fidelity and speed of assembly in such viruses. Using bacteriophage MS2, we demonstrate here via a combination of mass spectrometry and kinetic modelling how viral RNA can bias assembly towards only a small number of the many possible assembly pathways, thus increasing assembly efficiency. Assembly reactions have been studied in vitro using phage coat protein dimers, the known building block of the T = 3 shell, and short RNA stem-loops based on the translational operator of the replicase cistron, a 19 nt fragment (TR). Mass spectrometry has unambiguously identified two on-pathway intermediates in such reactions that have stoichiometry consistent with formation of either a particle 3-fold or 5-fold axis. These imply that there are at least two sub-pathways to the final capsid. The flux through each pathway is controlled by the length of the RNA stem-loop triggering the assembly reaction and this effect can be understood in structural terms. The kinetics of intermediate formation have been studied and show steady-state concentrations for intermediates between starting materials and the T = 3 shell, consistent with an assembly process in which all the steps are in equilibrium. These data have been used to derive a kinetic model of the assembly reaction that in turn allows us to determine the dominant assembly pathways explicitly, and to estimate the effect of the RNA on the free energy of association between the assembling protein subunits. The results reveal that there are only a small number of dominant assembly pathways, which vary depending on the relative ratios of RNA and protein. These results suggest that the genomic RNA plays significant roles in defining the precise assembly sub-pathway followed to create the final capsid.     

Non-native interactions play an effective role in protein folding dynamics

Systematic Monte Carlo simulations of simple lattice models show that the final stage of protein folding is an ordered process where native contacts get locked (i.e., the residues come into contact and remain in contact for the duration of the folding process) in a well‐defined order. The detailed study of the folding dynamics of protein‐like sequences designed as to exhibit different contact energy distributions, as well as different degrees of sequence optimization (i.e., participation of non‐native interactions in the folding process), reveals significant differences in the corresponding locking scenarios—the collection of native contacts and their average locking times, which are largely ascribable to the dynamics of non‐native contacts. Furthermore, strong evidence for a positive role played by non‐native contacts at an early folding stage was also found. Interestingly, for topologically simple target structures, a positive interplay between native and non‐native contacts is observed also toward the end of the folding process, suggesting that non‐native contacts may indeed affect the overall folding process. For target models exhibiting clear two‐state kinetics, the relation between the nucleation mechanism of folding and the locking scenario is investigated. Our results suggest that the stabilization of the folding transition state can be achieved through the establishment of a very small network of native contacts that are the first to lock during the folding process.     

Functional elucidation of a key contact between tRNA and the large ribosomal subunit rRNA during decoding

The selection of cognate tRNAs during translation is specified by a kinetic discrimination mechanism driven by distinct structural states of the ribosome. While the biochemical steps that drive the tRNA selection process have been carefully documented, it remains unclear how recognition of matched codon:anticodon helices in the small subunit facilitate global rearrangements in the ribosome complex that efficiently promote tRNA decoding. Here we use an in vitro selection approach to isolate tRNA^Trp miscoding variants that exhibit a globally perturbed tRNA tertiary structure. Interestingly, the most substantial distortions are positioned in the elbow region of the tRNA that closely approaches helix 69 (H69) of the large ribosomal subunit. The importance of these specific interactions to tRNA selection is underscored by our kinetic analysis of both tRNA and rRNA variants that perturb the integrity of this interaction.     

Influence of temperature on growth of Scenedesmus obliquus in diluted olive mill wastewater as culture medium

Scenedesmus obliquus can help to reduce the environmental impact of industrial olive mill wastewater from olive oil extraction in the three‐phase system. This work examines the effect of temperature changes (288–308 K) on algal growth, culture medium, and biochemical composition of S. obliquus. The maximum specific growth rate of 0.024 h^?1 occurred at an optimal temperature of 302.7 K. The apparent activation energies of cell growth and cell death were determined as 61.8 and 142.8 kJ/mol, respectively. At the end of culture the percentages of pigments, proteins, and carbohydrates were greater at the two ends of the temperature range studied, as it was also observed for the maximum elimination of biochemical oxygen demand (BOD₅). The mono‐ and polyunsaturated fatty acid content of the biomass was greater at the lowest temperature used (288 K).