The lepidopteran mitochondrial control region: structure and evolution

For several species of lepidoptera, most of the approximately 350-bp mitochondrial control-region sequences were determined. Six of these species are in one genus, Jalmenus; are closely related; and are believed to have undergone recent rapid speciation. Recent speciation was supported by the observation of low interspecific sequence divergence. Thus, no useful phylogeny could be constructed for the genus. Despite a surprising conservation of control-region length, there was little conservation of primary sequences either among the three lepidopteran genera or between lepidoptera and Drosophila. Analysis of secondary structure indicated only one possible feature in common--inferred stem loops with higher-than-random folding energies-- although the positions of the structures in different species were unrelated to regions of primary sequence similarity. We suggest that the conserved, short length of control regions is related to the observed lack of heteroplasmy in lepidopteran mitochondrial genomes. In addition, determination of flanking sequences for one Jalmenus species indicated (i) only weak support for the available model of insect 12S rRNA structure and (ii) that tRNA translocation is a frequent event in the evolution of insect mitochondrial genomes.      

Exhaustive enumeration of protein conformations using experimental restraints.

We present an efficient new algorithm that enumerates all possible conformations of a protein that satisfy a given set of distance restraints. Rapid growth of all possible self-avoiding conformations on the diamond lattice provides construction of alpha-carbon representations of a protein fold. We investigated the dependence of the number of conformations on pairwise distance restraints for the proteins crambin, pancreatic trypsin inhibitor, and ubiquitin. Knowledge of between one and two contacts per monomer is shown to be sufficient to restrict the number of candidate structures to approximately 1,000 conformations. Pairwise RMS deviations of atomic position comparisons between pairs of these 1,000 structures revealed that these conformations can be grouped into about 25 families of structures. These results suggest a new approach to assessing alternative protein folds given a very limited number of distance restraints. Such restraints are available from several experimental techniques such as NMR, NOESY, energy transfer fluorescence spectroscopy, and crosslinking experiments. This work focuses on exhaustive enumeration of protein structures with emphasis on the possible use of NOESY-determined distance restraints.     

Regulation of apoptosis by the Ft1 protein, a new modulator of protein kinase B/Akt

The serine/threonine kinase protein kinase B (PKB)/Akt plays a central role in many cellular processes, including cell growth, glucose metabolism, and apoptosis. However, the identification and validation of novel regulators or effectors is key to future advances in understanding the multiple functions of PKB. Here we report the identification of a novel PKB binding protein, called Ft1, from a cDNA library screen using a green fluorescent protein-based protein-fragment complementation assay. We show that the Ft1 protein interacts directly with PKB, enhancing the phosphorylation of both of its regulatory sites by promoting its interaction with the upstream kinase PDK1. Further, the modulation of PKB activity by Ft1 has a strong effect on the apoptosis susceptibility of T lymphocytes treated with glucocorticoids. We demonstrate that this phenomenon occurs via a PDK1/PKB/GSK3/NF-ATc signaling cascade that controls the production of the proapoptotic hormone Fas ligand. The wide distribution of Ft1 in adult tissues suggests that it could be a general regulator of PKB activity in the control of differentiation, proliferation, and apoptosis in many cell types.     

A heterodimeric coiled-coil peptide pair selected in vivo from a designed library-versus-library ensemble

Novel heterodimeric coiled-coil pairs were selected simultaneously from two DNA libraries using an in vivo protein-fragment complementation assay with dihydrofolate reductase, and the best pair was biophysically characterized. We randomized the interface-flanking e and g positions to Gln, Glu, Arg or Lys, and the core a position to Asn or Val in both helices simultaneously, using trinucleotide codons in DNA synthesis. Selection cycles with three different stringencies yielded sets of coiled-coil pairs, of which 80 clones were statistically analyzed. Thereby, properties most crucial for successful heterodimerization could be distinguished from those mediating more subtle optimization. A strong bias towards an Asn pair in the core a position indicated selection for structural uniqueness, and a reduction of charge repulsions at the e/g positions indicated selection for stability. Increased stringency led to additional selection for heterospecificity by destabilizing the respective homodimers. Interestingly, the best heterodimers did not contain exclusively complementary charges. The dominant pair, WinZip-A1B1, proved to be at least as stable in vitro as naturally occurring coiled coils, and was shown to be dimeric and highly heterospecific with a K(D) of approximately 24 nM. As a result of having been selected in vivo it possesses all characteristics required for a general in vivo heterodimerization module. The combination of rational library design and in vivo selection presented here is a very powerful strategy for protein design, and it can reveal new structural relationships.     

Application of protein-fragment complementation assays in cell biology

We have developed a general experimental strategy that enables the quantitative detection of dynamic protein-protein interactions in intact living cells, based on protein-fragment complementation assays (PCAs). In this method, protein-protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. Here we discuss the application of PCA to different aspects of cell biology.     

raf RBD and ubiquitin proteins share similar folds, folding rates and mechanisms despite having unrelated amino acid sequences

Recent experimental and theoretical studies in protein folding suggest that the rates and underlying mechanisms by which proteins attain the native state are largely determined by the topological complexity of a specific fold rather than by the fine details of the amino acid sequences. However, such arguments are based upon the examination of a limited number of protein folds. To test this view, we sought to investigate whether proteins belonging to the ubiquitin superfamily display similar folding behavior. To do so, we compared the folding-unfolding transitions of mammalian ubiquitin (mUbi) with those of its close yeast homologue (yUbi), and to those of the structurally related Ras binding domain (RBD) of the serine/threonine kinase raf that displays no apparent sequence homology with the ubiquitin family members. As demonstrated for mUbi [Krantz, B. A., and Sosnick, T. R. (2000) Biochemistry 39, 11696-11701], we show that a two-state transition model with no burst phase intermediate can describe folding of both yUbi and raf RBD. We further demonstrate that (1) all three proteins refold at rates that are within 1 order of magnitude (1800, 1100, and 370 s(-1) for mUbi, raf RBD, and yUbi, respectively), (2) both mUbi and raf RBD display similar refolding heterogeneity, and (3) the folding free energy barriers of both mUbi and raf RBD display a similar temperature dependence and sensitivity to a stabilizing agent or to mutations of a structurally equivalent central core residue. These findings are consistent with the view that rates and mechanisms for protein folding depend mostly on the complexity of the native structure topology rather than on the fine details of the amino acid sequence.     

Characterization of a neuregulin-related gene, Don-1, that is highly expressed in restricted regions of the cerebellum and hippocampus.

Members of the epidermal growth factor family of receptors have long been implicated in the pathogenesis of various tumors, and more recently, apparent roles in the developing heart and nervous system have been described. Numerous ligands that activate these receptors have been isolated. We report here on the cloning and initial characterization of a second ligand for the erbB family of receptors. This factor, which we have termed Don-1 (divergent of neuregulin 1), has structural similarity with the neuregulins. We have isolated four splice variants, two each from human and mouse, and have shown that they are capable of inducing tyrosine phosphorylation of erbB3, erbB4, and erbB2. In contrast to those of neuregulin, high levels of expression of Don-1 are restricted to the cerebellum and dentate gyrus in the adult brain and to fetal tissues.     

Mechanics,Structure and Function of Biopolymer Condensates

The spontaneous nature of biopolymer phase separation in cells entails that the resulting condensates can be thermodynamic machines, which, in the process of condensing, can take on distinct forms themselves and deform neighboring cellular structures. We introduce here general notions of material and mechanical properties of protein condensates with an emphasis on how molecular arrangements and intermolecular interaction within condensates determine their ability to do work on their surroundings. We further propose functional implications of these concepts to cellular and subcellular morphology and biogenesis.     

Combined monte carlo and molecular dynamics simulation of fully hydrated dioleyl and palmitoyl-oleyl phosphatidylcholine lipid bilayers

We have applied a new equilibration procedure for the atomic level simulation of a hydrated lipid bilayer to hydrated bilayers of dioleyl-phosphatidylcholine (DOPC) and palmitoyl-oleyl phosphatidylcholine (POPC). The procedure consists of alternating molecular dynamics trajectory calculations in a constant surface tension and temperature ensemble with configurational bias Monte Carlo moves to different regions of the configuration space of the bilayer in a constant volume and temperature ensemble. The procedure is applied to bilayers of 128 molecules of POPC with 4628 water molecules, and 128 molecules of DOPC with 4825 water molecules. Progress toward equilibration is almost three times as fast in central processing unit (CPU) time compared with a purely molecular dynamics (MD) simulation. Equilibration is complete, as judged by the lack of energy drift in 200-ps runs of continuous MD. After the equilibrium state was reached, as determined by agreement between the simulation volume per lipid molecule with experiment, continuous MD was run in an ensemble in which the lateral area was restrained to fluctuate about a mean value and a pressure of 1 atm applied normal to the bilayer surface. Three separate continuous MD runs, 200 ps in duration each, separated by 10,000 CBMC steps, were carried out for each system. Properties of the systems were calculated and averaged over the three separate runs. Results of the simulations are presented and compared with experimental data and with other recent simulations of POPC and DOPC. Analysis of the hydration environment in the headgroups supports a mechanism by which unsaturation contributes to reduced transition temperatures. In this view, the relatively horizontal orientation of the unsaturated bond increases the area per lipid, resulting in increased water penetration between the headgroups. As a result the headgroup-headgroup interactions are attenuated and shielded, and this contributes to the lowered transition temperature.