Cucumber: a model angiosperm for mitochondrial transformation?

Plants possess three major genomes, carried in the chloroplast, mitochondrion, and nucleus. The chloroplast genomes of higher plants tend to be of similar sizes and structure. In contrast both the nuclear and mitochondrial genomes show great size differences, even among closely related species. The largest plant mitochondrial genomes exist in the genus Cucumis at 1500 to 2300 kilobases, over 100 times the sizes of the yeast or human mitochondrial genomes. Biochemical and molecular analyses have established that the huge Cucumis mitochondrial genomes are due to extensive duplication of short repetitive DNA motifs. The organellar genomes of almost all organisms are maternally transmitted and few methods exist to manipulate these important genomes. Although chloroplast transformation has been achieved, no routine method exists to transform the mitochondrial genome of higher plants. A mitochondrial-transformation system for a higher plant would allow geneticists to use reverse genetics to study mitochondrial gene expression and to establish the efficacy of engineered mitochondrial genes for the genetic improvement of the mitochondrial genome. Cucumber possesses three unique attributes that make it a potential model system for mitochondrial transformation of a higher plant. Firstly, its mitochondria show paternal transmission. Secondly, microspores possess relatively few, huge mitochondria. Finally, there exists in cucumber unique mitochondrial mutations conditioning strongly mosaic (msc) phenotypes. The msc phenotypes appear after regeneration of plants from cell culture and sort with specific rearranged and deleted regions in the mitochondrial genome. These mitochondrial deletions may be a useful genetic tool to develop selectable markers for mitochondrial transformation of higher plants.     

Dual effect of heparin on Fe2+-induced cardiolipin peroxidation: implications for peroxidation of cytochrome c oxidase bound cardiolipin

The effect of heparin on peroxidation of cardiolipin (CL) initiated by ferrous iron was studied in vitro using detergent-solubilized CL, liposomal CL, or CL bound to isolated cytochrome c oxidase (CcO). Heparin increased both the rate and the extent of CL peroxidation for detergent-solubilized CL and for CcO-bound CL. The effect of heparin was time- and concentration-dependent as monitored by the formation of conjugated dienes or thiobarbituric acid reactive substances. The results showed great similarity between the effect of heparin and the effect of certain iron chelators, such as ADP, on phospholipid peroxidation. Heparin increased the peroxidation of CcO-bound CL only when tertiary butyl hydroperoxide was also present. The enzyme activity of the resulting CcO complex decreased 25 %, in part due to peroxidation of functionally important CL. In contrast to peroxidation of detergent-solubilized CL, peroxidation of liposomal CL was inhibited by heparin, suggesting that the effect of heparin and ferrous iron depends on their proximity to the acyl chains of CL.     

Yeast mitochondrial biogenesis: a model system for humans?

Recently, our knowledge of yeast mitochondrial biogenesis has considerably progressed. This concerns the import machinery that guides preproteins synthesized on the cytoplasmic ribosomes through the mitochondrial outer and inner membranes, as well as the inner membrane insertion machinery of mitochondrially encoded polypeptides, or the proteins participating in the assembly and quality control of the respiratory complexes and ATP synthase. More recently, two new fields have emerged, biosynthesis of the iron-sulfur clusters and dynamics of the mitochondrion. Many of the newly discovered yeast proteins have homologues in human mitochondria. Thus, Saccharomyces cerevisiae has proven a particularly suitable simple organism for approaching the molecular bases of a growing number of human mitochondrial diseases caused by mutations in nuclear genes identified by positional cloning.     

Cys-labeling kinetics of membrane protein GlpG: a role for specific SDS binding and micelle changes?

Empirically, α-helical membrane protein folding stability in surfactant micelles can be tuned by varying the mole fraction MF^SDS of anionic (sodium dodecyl sulfate (SDS)) relative to nonionic (e.g., dodecyl maltoside (DDM)) surfactant, but we lack a satisfying physical explanation of this phenomenon. Cysteine labeling (CL) has thus far only been used to study the topology of membrane proteins, not their stability or folding behavior. Here, we use CL to investigate membrane protein folding in mixed DDM-SDS micelles. Labeling kinetics of the intramembrane protease GlpG are consistent with simple two-state unfolding-and-exchange rates for seven single-Cys GlpG variants over most of the explored MF^SDS range, along with exchange from the native state at low MF^SDS (which inconveniently precludes measurement of unfolding kinetics under native conditions). However, for two mutants, labeling rates decline with MF^SDS at 0–0.2 MF^SDS (i.e., native conditions). Thus, an increase in MF^SDS seems to be a protective factor for these two positions, but not for the five others. We propose different scenarios to explain this and find the most plausible ones to involve preferential binding of SDS monomers to the site of CL (based on computational simulations) along with changes in size and shape of the mixed micelle with changing MF^SDS (based on SAXS studies). These nonlinear impacts on protein stability highlights a multifaceted role for SDS in membrane protein denaturation, involving both direct interactions of monomeric SDS and changes in micelle size and shape along with the general effects on protein stability of changes in micelle composition.     

Microperoxidase 11: a model system for porphyrin networks and heme–protein interactions

We measured the circular dichroism (CD) and absorption spectra of the B-band region of microperoxidase 11 (MP11) as a function of temperature and peptide concentration. At micromolar concentrations, small MP11 dimers or trimers lead to excitonic coupling between low-spin and high-spin heme groups, to which the NH₂ group of the MP11 N-terminal and H₂O are bound as a sixth ligand, respectively. These aggregates convert into monomers with hexacoordinated high-spin heme groups with increasing temperature. This transition can be described by a two-state model. Aggregation becomes more extended at 50 μM concentration and causes some B-band hyperchromism, which reflects a J-type arrangement of heme groups linked together in the aggregates formed. At near-millimolar concentration, the CD and absorption spectra of the B-band region suggest the existence of even more extended and thermally stable aggregates, which might involve μ-oxo dimers of the heme groups. The degree of aggregation at 50 and 500 μM concentration increases substantially if the sample is freed from most of its oxygen in a N₂ atmosphere. The CD spectrum of the monomeric high-spin species is reminiscent of that observed for the unfolded alkaline conformation of the intact protein. Finally, we investigated the binding of acetylmethionine (AcM) ligands to the heme at aggregation-supporting conditions (500 μM concentration). The data suggest that the ligand prevents any substantial aggregation. As a surprising result, our data reveal that AcM–MP11 complexes exhibit a high-spin/low-spin mixture, with the high-spin configuration being stabilized at high temperatures.     

Peptide 68–88 of apocytochrome c plays a crucial role in its insertion into membrane and binding to mitochondria

Apocytochrome c (Apocyt. c) is the precursor of cytochrome c. It is synthesized in the cytosol and posttranslationally imported into mitochondria. In order to determine the crucial sequence in apocyt. c translocation, deleted mutant and chemically synthesized peptides with different length were used. Obtained results showed that sequence 68–88 of apocyt. c plays a critical role in its insertion into membrane and binding to mitochondria.     

Data mining for protein–protein interactions in invertebrate model organisms

Well-annotated genome databases are available for many invertebrate species, notably the fruitfly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans. An adequate interpretation of this information at the biological level requires the exploration of the interactions between the gene products. Knowledge of protein interactions and the components of cell signalling pathways in the fly and worm are particularly valuable as hypotheses can be rapidly tested using the powerful genetic toolkits available. Invertebrates offer additional experimental advantages when attempting to characterise protein–protein interactions (PPIs). Their relatively small genome size compared to mammals helps to reduce missed interactions due to redundancy, and their function can be addressed using forward (mutants) and reverse (RNA interference) genetics. However, the researcher looking for evidence of PPIs for a protein of interest is faced with the challenge of extracting interaction data from sources that are highly varied, such as the results of microarray experiments in the unstructured text of research papers. This challenge is greatly reduced by a range of public databases of curated information, as well as publicly available, enhanced search engines, which can provide either direct experimental evidence for a PPI, or valuable clues for generating new hypotheses.     

Extended Parker–Sochacki method for Michaelis–Menten enzymatic reaction model

In this article, a new approach—namely, the extended Parker–Sochacki method (EPSM)—is presented for solving the Michaelis–Menten nonlinear enzymatic reaction model. The Parker–Sochacki method (PSM) is combined with a new resummation method called the Sumudu–Padé resummation method to obtain approximate analytical solutions for the model. The obtained solutions by the proposed approach are compared with the solutions of PSM and the Runge–Kutta numerical method (RKM). The comparison proves the practicality, efficiency, and correctness of the presented approach. It serves as a basis for solving other nonlinear biochemical reaction models in the future.     

Estimation of binding parameters for the protein–protein interaction using a site-directed spin labeling and EPR spectroscopy

Sensitivity of the electron paramagnetic resonance (CW EPR) to molecular tumbling provides potential means for studying processes of molecular association. It uses spin-labeled macromolecules, whose CW EPR spectra may change upon binding to other macromolecules. When a spin-labeled molecule is mixed with its liganding partner, the EPR spectrum constitutes a linear combination of spectra of the bound and unbound ligand (as seen in our example of spin-labeled cytochrome c ₂ interacting with cytochrome bc ₁ complex). In principle, the fraction of each state can be extracted by the numerical decomposition of the spectrum; however, the accuracy of such decomposition may often be compromised by the lack of the spectrum of the fully bound ligand, imposed by the equilibrium nature of molecular association. To understand how this may affect the final estimation of the binding parameters, such as stoichiometry and affinity of the binding, a series of virtual titration experiments was conducted. Our non-linear regression analysis considered a case in which only a single class of binding sites exists, and a case in which classes of both specific and non-specific binding sites co-exist. The results indicate that in both models, the error due to the unknown admixture of the unbound ligand component in the EPR spectrum causes an overestimation of the bound fraction leading to the bias in the dissociation constant. At the same time, the stoichiometry of the binding remains relatively unaffected, which overall makes the decomposition of the EPR spectrum an attractive method for studying protein–protein interactions in equilibrium. Our theoretical treatment appears to be valid for any spectroscopic techniques dealing with overlapping spectra of free and bound component.     

The potassium channel KcsA: a model protein in studying membrane protein oligomerization and stability of oligomeric assembly?

Many membrane proteins are functional as stable oligomers. An understanding of the conditions that elicit and enhance oligomerization is important in many therapeutics. In this regard, protein–protein and protein–lipid interactions play crucial roles in the assembly and stability of oligomeric complexes. Recent years have seen a rapid increase in the mechanistic information on the importance of cytoplasmic termini in determining subunit assembly and stability of oligomeric complexes. In addition, the role of specific protein–lipid interaction between anionic phospholipids and “hot spots” on the protein surface has also become evident in stabilizing oligomeric assemblies. This review focuses on several contemporary developments of membrane proteins that stabilize oligomers by taking the potassium channel KcsA as an exemplary ion channel.     

MuMoD: a Bayesian approach to detect multiple modes of protein–DNA binding from genome-wide ChIP data

High-throughput chromatin immunoprecipitation has become the method of choice for identifying genomic regions bound by a protein. Such regions are then investigated for overrepresented sequence motifs, the assumption being that they must correspond to the binding specificity of the profiled protein. However this approach often fails: many bound regions do not contain the ‘expected’ motif. This is because binding DNA directly at its recognition site is not the only way the protein can cause the region to immunoprecipitate. Its binding specificity can change through association with different co-factors, it can bind DNA indirectly, through intermediaries, or even enforce its function through long-range chromosomal interactions. Conventional motif discovery methods, though largely capable of identifying overrepresented motifs from bound regions, lack the ability to characterize such diverse modes of protein–DNA binding and binding specificities. We present a novel Bayesian method that identifies distinct protein–DNA binding mechanisms without relying on any motif database. The method successfully identifies co-factors of proteins that do not bind DNA directly, such as mediator and p300. It also predicts literature-supported enhancer–promoter interactions. Even for well-studied direct-binding proteins, this method provides compelling evidence for previously uncharacterized dependencies within positions of binding sites, long-range chromosomal interactions and dimerization.     

The binding interface of cytochrome c and cytochrome c₁ in the bc₁ complex: rationalizing the role of key residues

The interaction of cytochrome c with ubiquinol-cytochrome c oxidoreductase (bc₁ complex) has been studied for >30 years, yet many aspects remain unclear or controversial. We report the first molecular dynamic simulations of the cyt c-bc₁ complex interaction. Contrary to the results of crystallographic studies, our results show that there are multiple dynamic hydrogen bonds and salt bridges in the cyt c-c₁ interface. These include most of the basic cyt c residues previously implicated in chemical modification studies. We suggest that the static nature of x-ray structures can obscure the quantitative significance of electrostatic interactions between highly mobile residues. This provides a clear resolution of the discrepancy between the structural data and functional studies. It also suggests a general need to consider dynamic interactions of charged residues in protein-protein interfaces. In addition, a novel structural change in cyt c is reported, involving residues 21-25, which may be responsible for cyt c destabilization upon binding. We also propose a mechanism of interaction between cyt c₁ monomers responsible for limiting the binding of cyt c to only one molecule per bc₁ dimer by altering the affinity of the cytochrome c binding site on the second cyt c₁ monomer.