Cytochrome c-Lipid Interactions: New Insights from Resonance Energy Transfer

Resonance energy transfer (RET) from anthrylvinyl-labeled phosphatidylcholine (AV-PC) or cardiolipin (AV-CL) to cytochrome c (cyt c) heme moiety was employed to assess the molecular-level details of protein interactions with lipid bilayers composed of PC with 2.5 (CL2.5), 5 (CL5), 10 (CL10), or 20 (CL20) mol % CL under conditions of varying ionic strength and lipid/protein molar ratio. Monte Carlo analysis of multiple data sets revealed a subtle interplay between 1), exchange of the neutral and acidic lipid in the protein-lipid interaction zone; 2), CL transition into the extended conformation; and 3), formation of the hexagonal phase. The switch between these states was found to be controlled by CL content and salt concentration. At ionic strengths ≥40 mM, lipid bilayers with CL fraction not exceeding 5 mol % exhibited the tendency to transform from lamellar to hexagonal phase upon cyt c adsorption, whereas at higher contents of CL, transition into the extended conformation seems to become thermodynamically favorable. At lower ionic strengths, deviations from homogeneous lipid distributions were observed only for model membranes containing 2.5 mol % CL, suggesting the existence of a certain surface potential critical for assembly of lipid lateral domains in protein-lipid systems that may subsequently undergo morphological transformations depending on ambient conditions. These characteristics of cyt c-CL interaction are of great interest, not only from the viewpoint of regulating cyt c electron transfer and apoptotic propensities, but also to elucidate the general mechanisms by which membrane functional activities can be modulated by protein-lipid interactions.     

The role of lipid-protein interactions in amyloid-type protein fibril formation

Structural transition of polypeptide chains into the beta-sheet state followed by amyloid fibril formation is the key characteristic of a number of the so-called conformational diseases. The multistep process of protein fibrillization can be modulated by a variety of factors, in particular by lipid-protein interactions. A wealth of experimental evidence provides support to the notion that amyloid fibril assembly and the toxicity of pre-fibrillar aggregates are closely related and are both intimately membrane associated phenomena. The present review summarizes the principal factors responsible for the enhancement of fibril formation in a membrane environment, viz. (i) structural transformation of polypeptide chain into a partially folded conformation, (ii) increase of the local concentration of a protein upon its membrane binding, (iii) aggregation-favoring orientation of the bound protein, and (iv) variation in the depth of bilayer penetration affecting the nucleation propensity of the membrane associated protein. The molecular mechanisms of membrane-mediated protein fibrillization are discussed. Importantly, the toxicity of lipid-induced pre-fibrillar aggregates is likely to have presented a very strong negative selection pressure in the evolution of amino acid sequences.     

Characterization of the molecular basis of the Drosophila mutations in carboxypeptidase D. Effect on enzyme activity and expression

Carboxypeptidase D (CPD) functions in the processing of proteins and peptides in the secretory pathway. Drosophila CPD is encoded by the silver gene (svr), which is differentially spliced to produce long transmembrane protein forms with three metallocarboxypeptidase (CP)-like domains and short soluble forms with a single CP domain. Many svr mutants have been reported, but the precise molecular defects have not been previously determined. In the present study, three mutant lines were characterized. svr (PG33) mutants do not survive past the early larval stage. These mutants have a P-element insertion within exon 1B upstream of the initiation ATG, which greatly reduces mRNA levels of all forms of CPD. Both svr (1) and svr (poi) mutants are viable, with a silvery body color and pointed wings. The wing shape is generally similar between these two mutants, although svr (poi) mutants have smaller wings. The svr (1) gene has a three-nucleotide deletion in exon 6, removing a leucine in a region of the protein predicted to function as a folding domain for the second CP-like domain. svr (poi) has a 1072-bp duplication of the gene that introduces a stop codon into the open reading frame, causing the truncation of the protein in the middle of the second CP-like domain. Both deletions eliminate enzyme activity of the second CP-like domain and appear to cause the misfolding of the protein. This greatly reduces the levels of the long forms of CPD protein but do not affect the levels of the short forms. Taken together, these findings suggest that lethal and viable svr alleles differ in which protein forms are affected. Flies that retain the short form are viable, whereas flies that are missing all forms of CPD do not survive past the early larval stages.     

Molecular dynamics simulations of lysozyme–lipid systems: probing the early steps of protein aggregation

Using the molecular dynamics simulation, the role of lipids in the lysozyme transition into the aggregation-competent conformation has been clarified. Analysis of the changes of lysozyme secondary structure upon its interactions with the model bilayer membranes composed of phosphatidylcholine and its mixtures with phosphatidylglycerol (10, 40, and 80 mol%) within the time interval of 100 ns showed that lipid-bound protein is characterized by the increased content of β-structures. Along with this, the formation of protein–lipid complexes was accompanied by the increase in the gyration radius and the decrease in RMSD of polypeptide chain. The results obtained were interpreted in terms of the partial unfolding of lysozyme molecule on the lipid matrix, with the magnitude of this effect being increased with increasing the fraction of anionic lipids. Based on the results of molecular dynamics simulation, a hypothetical model of the nucleation of lysozyme amyloid fibrils in a membrane environment was suggested.     

Amino acids Thr56 and Thr58 are not essential for elongation factor 2 function in yeast

Yeast elongation factor 2 is an essential protein that contains two highly conserved threonine residues, T56 and T58, that could potentially be phosphorylated by the Rck2 kinase in response to environmental stress. The importance of residues T56 and T58 for elongation factor 2 function in yeast was studied using site directed mutagenesis and functional complementation. Mutations T56D, T56G, T56K, T56N and T56V resulted in nonfunctional elongation factor 2 whereas mutated factor carrying point mutations T56M, T56C, T56S, T58S and T58V was functional. Expression of mutants T56C, T56S and T58S was associated with reduced growth rate. The double mutants T56M/T58W and T56M/T58V were also functional but the latter mutant caused increased cell death and considerably reduced growth rate. The results suggest that the physiological role of T56 and T58 as phosphorylation targets is of little importance in yeast under standard growth conditions. Yeast cells expressing mutants T56C and T56S were less able to cope with environmental stress induced by increased growth temperatures. Similarly, cells expressing mutants T56M and T56M/T58W were less capable of adapting to increased osmolarity whereas cells expressing mutant T58V behaved normally. All mutants tested were retained their ability to bind to ribosomes in vivo. However, mutants T56D, T56G and T56K were under-represented on the ribosome, suggesting that these nonfunctional forms of elongation factor 2 were less capable of competing with wild-type elongation factor 2 in ribosome binding. The presence of nonfunctional but ribosome binding forms of elongation factor 2 did not affect the growth rate of yeast cells also expressing wild-type elongation factor 2.     

Phylogeography of a cryptic speciation continuum in Eurasian spadefoot toads (Pelobates)

Cryptic phylogeographic diversifications provide unique models to examine the role of phylogenetic divergence on the evolution of reproductive isolation, without extrinsic factors such as ecological and behavioural differentiation. Yet, to date very few comparative studies have been attempted within such radiations. Here, we characterize a new speciation continuum in a group of widespread Eurasian amphibians, the Pelobates spadefoot toads, by conducting multilocus (restriction site associated DNA sequencing and mitochondrial DNA) phylogenetic, phylogeographic and hybrid zone analyses. Within the P. syriacus complex, we discovered species‐level cryptic divergences (>5 million years ago [My]) between populations distributed in the Near‐East (hereafter P. syriacus sensu stricto [s.s.]) and southeastern Europe (hereafter P. balcanicus), each featuring deep intraspecific lineages. Altogether, we could scale hybridizability to divergence time along six different stages, spanning from sympatry without gene flow (P. fuscus and P. balcanicus, >10 My), parapatry with highly restricted hybridization (P. balcanicus and P. syriacus s.s., >5 My), narrow hybrid zones (~15 km) consistent with partial reproductive isolation (P. fuscus and P. vespertinus, ~3 My), to extensive admixture between Pleistocene and refugial lineages (≤2 My). This full spectrum empirically supports a gradual build up of reproductive barriers through time, reversible up until a threshold that we estimate at ~3 My. Hence, cryptic phylogeographic lineages may fade away or become reproductively isolated species simply depending on the time they persist in allopatry, and without definite ecomorphological divergence.     

Importance of individual amino acids in the Switch I region in eEF2 studied by functional complementation in S. cerevisiae

Elongation factor 2 (eEF2) is a member of the G-protein super family. G-proteins undergo conformational changes associated with binding of the guanosine nucleotide and hydrolysis of the bound GTP. These structural rearrangements affects the Switch I region (also known as the Effector loop). We have studied the role of individual amino acids in the Switch I region (amino acids 25-73) of S. cerevisiae eEF2 using functional complementation in yeast. 21 point mutations in the Switch I region were created by site-directed mutagenesis. Mutants K49R, E52Q, A53G, F55Y, K60R, Q63A, T68S, I69M and A73G were functional while mutants R54H, F55N, D57A, D57E, D57S, R59K, R59M, Q63E, R65A, R65N, T68A and T68M were inactive. Expression of mutants K49R, A53G, Q63A, I69M and A73G was associated with markedly decreased growth rates and yeast cells expressing mutants A53G and I69M became temperature sensitive. The functional capacity of eEF2 in which the major part Switch I (amino acids T56 to I69) was converted into the homologous sequence found in EF-G from E. coli was also studied. This protein chimera could functionally replace yeast eEF2 in vivo. Yeast cells expressing this mutant grew extremely slowly, showed increased cell death and became temperature sensitive. The ability of the mutant to replace authentic eEF2 in vivo indicates that the structural rearrangement of Switch I necessary for eEF2 function is similar in eukaryotes and bacteria. The effect of two point mutations in the P-loop was also studied. Mutant A25G but not A25V could functionally replace yeast eEF2 even if cells expressing the mutant grew slowly. The A25G mutation converted the consensus sequences AXXXXGK[T/S] in eEF2 to the corresponding motif GXXXXGK[T/S] found in all other G-proteins, suggesting that the alanine found in the P-loop of peptidyltranslocases are not essential for function.     

Chromium(VI) reduction in a membrane bioreactor with immobilized Pseudomonas cells

Chromate reduction was studied in a membrane bioreactor under action of Pseudomonas bacteria immobilized in agar–agar films on the surface of synthetic membrane. Immobilized cells are protected from the excessive toxic action at high chromate concentration that improves cell activity compared with free cells. Almost complete chromate reduction was observed at stepwise introducing of chromate in feed solution allowing maintenance of optimal chromate concentration. Reduction is suppressed by high metabolite concentrations, which reached on the sixth step of chromate adding in studied system. Cell ability to reduce chromate is restored after changing of feed and receiving solutions allowing remediation of Cr(VI)-contaminated water in semi-batch operation of membrane bioreactor.