The nature of the excited state of the reaction center of photosystem II of green plants: A high-resolution fluorescence spectroscopy study

We studied the electronically excited state of the isolated reaction center of photosystem II with high-resolution fluorescence spectroscopy at 5 K and compared the obtained spectral features with those obtained earlier for the primary electron donor. The results show that there is a striking resemblance between the emitting and charge-separating states in the photosystem II reaction center, such as a very similar shape of the phonon wing with characteristic features at 19 and 80 cm−1, almost identical frequencies of a number of vibrational modes, a very similar double-Gaussian shape of the inhomogeneous distribution function, and relatively strong electron-phonon coupling for both states. We suggest that the emission at 5 K originates either from an exciton state delocalized over the inactive branch of the photosystem or from a fraction of the primary electron donor that is long-lived at 5 K. The latter possibility can be explained by a distribution of the free energy difference of the primary charge separation reaction around zero. Both possibilities are in line with the idea that the state that drives primary charge separation in the reaction center of photosystem II is a collective state, with contributions from all chlorophyll molecules in the central part of the complex.

A unified statistical framework for sequence comparison and structure comparison

We present an approach for assessing the significance of sequence and structure comparisons by using nearly identical statistical formalisms for both sequence and structure. Doing so involves an all-vs.-all comparison of protein domains [taken here from the Structural Classification of Proteins (scop) database] and then fitting a simple distribution function to the observed scores. By using this distribution, we can attach a statistical significance to each comparison score in the form of a P value, the probability that a better score would occur by chance. As expected, we find that the scores for sequence matching follow an extreme-value distribution. The agreement, moreover, between the P values that we derive from this distribution and those reported by standard programs (e.g., blast and fasta validates our approach. Structure comparison scores also follow an extreme-value distribution when the statistics are expressed in terms of a structural alignment score (essentially the sum of reciprocated distances between aligned atoms minus gap penalties). We find that the traditional metric of structural similarity, the rms deviation in atom positions after fitting aligned atoms, follows a different distribution of scores and does not perform as well as the structural alignment score. Comparison of the sequence and structure statistics for pairs of proteins known to be related distantly shows that structural comparison is able to detect approximately twice as many distant relationships as sequence comparison at the same error rate. The comparison also indicates that there are very few pairs with significant similarity in terms of sequence but not structure whereas many pairs have significant similarity in terms of structure but not sequence.

Evidence from molecular systematics for decreased avian diversification in the pleistocene Epoch.

Pleistocene glaciations have been suggested as major events influencing speciation rates in vertebrates. Avian paleontological studies suggest that most extant species evolved in the Pleistocene Epoch and that species' durations decreased through the Pleistocene because of heightened speciation rates. Molecular systematic studies provide another data base for testing these predictions. In particular, rates of diversification can be determined from molecular phylogenetic trees. For example, an increasing rate of speciation (but constant extinction) requires shorter intervals between successive speciation events on a phylogenetic tree. Examination of the cumulative distribution of reconstructed speciation events in mtDNA phylogenies of 11 avian genera, however, reveals longer intervals between successive speciation events as the present time is approached, suggesting a decrease in net diversification rate through the Pleistocene Epoch. Thus, molecular systematic studies do not indicate a pulse of Pleistocene diversification in passerine birds but suggest, instead, that diversification rates were lower in the Pleistocene than for the preceding period. Documented habitat shifts likely led to the decreased rate of diversification, although from molecular evidence we cannot discern whether speciation rates decreased or extinction rates increased.

Brush border myosin-I truncated in the motor domain impairs the distribution and the function of endocytic compartments in an hepatoma cell line.

Myosins I, a ubiquitous monomeric class of myosins that exhibits actin-based motor properties, are associated with plasma and/or vesicular membranes and have been suggested as players for trafficking events between cell surface and intracellular membranous structures. To investigate the function of myosins 1, we have transfected a mouse hepatoma cell line (BWTG3) with cDNAs encoding the chicken brush border myosin-I (BBMI) and two variants truncated in the motor domain. One variant is deleted of the first 446 amino acids and thereby lacks the ATP binding site, whereas the other is deleted of the entire motor domain and lacks the ATP and actin binding sites. We have observed (i) that significant amounts of the truncated variants are recovered with membrane fractions after cell fractionation, (ii) that they codistribute with a compartment containing alpha2-macroglobulin internalized for 30 min as determined by fluorescent microscopy, (iii) that the production of BBMI-truncated variants impairs the distribution of the acidic compartment and ligands internalized for 30 min, and (iv) that the production of the truncated variant containing the actin binding site decreases the rate of alpha2-macroglobulin degradation whereas the production of the variant lacking the ATP binding site and the actin binding site increases the rate of a2-macroglobulin degradation. These observations indicate that the two truncated variants have a dominant negative effect on the distribution and the function of the endocytic compartments. We propose that an unidentified myosin-I might contribute to the distribution of endocytic compartments in a juxtanuclear position and/or to the regulation of the delivery of ligands to the degradative compartment in BWTG3 cells.

Folding protein models with a simple hydrophobic energy function: The fundamental importance of monomer inside/outside segregation

The present study explores a “hydrophobic” energy function for folding simulations of the protein lattice model. The contribution of each monomer to conformational energy is the product of its “hydrophobicity” and the number of contacts it makes, i.e., E(h⃗, c⃗) = −Σi=1N cihi = −(h⃗.c⃗) is the negative scalar product between two vectors in N-dimensional cartesian space: h⃗ = (h1, … , hN), which represents monomer hydrophobicities and is sequence-dependent; and c⃗ = (c1, … , cN), which represents the number of contacts made by each monomer and is conformation-dependent. A simple theoretical analysis shows that restrictions are imposed concomitantly on both sequences and native structures if the stability criterion for protein-like behavior is to be satisfied. Given a conformation with vector c⃗, the best sequence is a vector h⃗ on the direction upon which the projection of c⃗ − c̄⃗ is maximal, where c̄⃗ is the diagonal vector with components equal to c̄, the average number of contacts per monomer in the unfolded state. Best native conformations are suggested to be not maximally compact, as assumed in many studies, but the ones with largest variance of contacts among its monomers, i.e., with monomers tending to occupy completely buried or completely exposed positions. This inside/outside segregation is reflected on an apolar/polar distribution on the corresponding sequence. Monte Carlo simulations in two dimensions corroborate this general scheme. Sequences targeted to conformations with large contact variances folded cooperatively with thermodynamics of a two-state transition. Sequences targeted to maximally compact conformations, which have lower contact variance, were either found to have degenerate ground state or to fold with much lower cooperativity.

Structural distribution of stability in a thermophilic enzyme

Stability parameters for individual residues in Thermus thermophilus cysteine-free RNase H were determined by native state hydrogen exchange, thus providing a unique comparison of regional thermodynamics between thermophilic and mesophilic homologues. The general distribution of stability in the thermophilic protein is similar to that of its mesophilic homologue, with a proportional increase in stability for almost all residues. As a consequence, the residue-specific stabilities of the two proteins are remarkably similar under conditions where their global stabilities are the same. These results indicate that T. thermophilus RNase H is stabilized in a delocalized fashion, preserving a finely tuned balance of stabilizing interactions throughout the structure. Therefore, although protein stability can be altered by single amino acid substitution, evolution for optimal function may require more subtle and delocalized mechanisms.

Cannabinoid CB1 receptors and ligands in vertebrate retina: Localization and function of an endogenous signaling system

CB1, a cannabinoid receptor enriched in neuronal tissue, was found in high concentration in retinas of rhesus monkey, mouse, rat, chick, goldfish, and tiger salamander by using a subtype-specific polyclonal antibody. Immunolabeling was detected in the two synaptic layers of the retina, the inner and outer plexiform layers, of all six species examined. In the outer plexiform layer, CB1 was located in and/or on cone pedicles and rod spherules. Labeling was detected in some amacrine cells of all species and in the ganglion cells and ganglion cell axons of all species except fish. In addition, sparse labeling was found in the inner and/or outer segments of the photoreceptors of monkey, mouse, rat, and chick. Using GC/MS to detect possible endogenous cannabinoids, we found 3 nmol of 2-arachidonylglycerol per g of tissue, but no anandamide was detectable. Cannabinoid receptor agonists induced a dramatic reduction in the amplitude of voltage-gated L-type calcium channel currents in identified retinal bipolar cells. The presence and distribution of the CB1 receptor, the large amounts of 2-arachidonylglycerol found, and the effects of cannabinoids on calcium channel activity in bipolar cells suggest a substantive role for an endogenous cannabinoid signaling system in retinal physiology, and perhaps vision in general.

Heterogeneous Distribution of the Unusual Phospholipid Semilysobisphosphatidic Acid through the Golgi Complex

To investigate the distribution of lipids through the Golgi complex, we analyzed the envelopes of several viruses that assemble in different subcompartments of the Golgi, as well as subcellular fractions. Our results indicate that each Golgi subcompartment has a distinct phospholipid composition due mainly to differences in the relative amounts of semilysobisphosphatidic acid (SLBPA), sphingomyelin, phosphatidylserine, and phosphatidylinositol. Interestingly, SLBPA is enriched in the adjacent Golgi networks compared with the Golgi stack, and this enrichment varies with cell type. The heterogeneous distribution of SLBPA through the Golgi complex suggests it may play an important role in the structure and/or function of this organelle.

A Mutant of Arp2p Causes Partial Disassembly of the Arp2/3 Complex and Loss of Cortical Actin Function in Fission Yeast

The Arp2/3 complex is an essential component of the yeast actin cytoskeleton that localizes to cortical actin patches. We have isolated and characterized a temperature-sensitive mutant of Schizosaccharomyces pombe arp2 that displays a defect in cortical actin patch distribution. The arp2+ gene encodes an essential actin-related protein that colocalizes with actin at the cortical actin patch. Sucrose gradient analysis of the Arp2/3 complex in the arp2-1 mutant indicated that the Arp2p and Arc18p subunits are specifically lost from the complex at restrictive temperature. These results are consistent with immunolocalization studies of the mutant that show that Arp2-1p is diffusely localized in the cytoplasm at restrictive temperature. Interestingly, Arp3p remains localized to the cortical actin patch under the same restrictive conditions, leading to the hypothesis that loss of Arp2p from the actin patch affects patch motility but does not severely compromise its architecture. Analysis of the mutant Arp2 protein demonstrated defects in ATP and Arp3p binding, suggesting a possible model for disruption of the complex.

The Dynamin-like Protein DLP1 Is Essential for Normal Distribution and Morphology of the Endoplasmic Reticulum and Mitochondria in Mammalian Cells

The dynamin family of large GTPases has been implicated in vesicle formation from both the plasma membrane and various intracellular membrane compartments. The dynamin-like protein DLP1, recently identified in mammalian tissues, has been shown to be more closely related to the yeast dynamin proteins Vps1p and Dnm1p (42%) than to the mammalian dynamins (37%). Furthermore, DLP1 has been shown to associate with punctate vesicles that are in intimate contact with microtubules and the endoplasmic reticulum (ER) in mammalian cells. To define the function of DLP1, we have transiently expressed both wild-type and two mutant DLP1 proteins, tagged with green fluorescent protein, in cultured mammalian cells. Point mutations in the GTP-binding domain of DLP1 (K38A and D231N) dramatically changed its intracellular distribution from punctate vesicular structures to either an aggregated or a diffuse pattern. Strikingly, cells expressing DLP1 mutants or microinjected with DLP1 antibodies showed a marked reduction in ER fluorescence and a significant aggregation and tubulation of mitochondria by immunofluorescence microscopy. Consistent with these observations, electron microscopy of DLP1 mutant cells revealed a striking and quantitative change in the distribution and morphology of mitochondria and the ER. These data support very recent studies by other authors implicating DLP1 in the maintenance of mitochondrial morphology in both yeast and mammalian cells. Furthermore, this study provides the first evidence that a dynamin family member participates in the maintenance and distribution of the ER. How DLP1 might participate in the biogenesis of two presumably distinct organelle systems is discussed.

Agrin in Alzheimer’s disease: Altered solubility and abnormal distribution within microvasculature and brain parenchyma

Agrin is a heparan sulfate proteoglycan that is widely expressed in neurons and microvascular basal lamina in the rodent and avian central nervous system. Agrin induces the differentiation of nerve-muscle synapses, but its function in either normal or diseased brains is not known. Alzheimer’s disease (AD) is characterized by loss of synapses, changes in microvascular architecture, and formation of neurofibrillary tangles and senile plaques. Here we have asked whether AD causes changes in the distribution and biochemical properties of agrin. Immunostaining of normal, aged human central nervous system revealed that agrin is expressed in neurons in multiple brain areas. Robust agrin immunoreactivity was observed uniformly in the microvascular basal lamina. In AD brains, agrin is highly concentrated in both diffuse and neuritic plaques as well as neurofibrillary tangles; neuronal expression of agrin also was observed. Furthermore, patients with AD had microvascular alterations characterized by thinning and fragmentation of the basal lamina. Detergent extraction and Western blotting showed that virtually all the agrin in normal brain is soluble in 1% SDS. In contrast, a large fraction of the agrin in AD brains is insoluble under these conditions, suggesting that it is tightly associated with β-amyloid. Together, these data indicate that the agrin abnormalities observed in AD are closely linked to β-amyloid deposition. These observations suggest that altered agrin expression in the microvasculature and the brain parenchyma contribute to the pathogenesis of AD.

Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function

During the aging process, mammals lose up to a third of their skeletal muscle mass and strength. Although the mechanisms underlying this loss are not entirely understood, we attempted to moderate the loss by increasing the regenerative capacity of muscle. This involved the injection of a recombinant adeno-associated virus directing overexpression of insulin-like growth factor I (IGF-I) in differentiated muscle fibers. We demonstrate that the IGF-I expression promotes an average increase of 15% in muscle mass and a 14% increase in strength in young adult mice, and remarkably, prevents aging-related muscle changes in old adult mice, resulting in a 27% increase in strength as compared with uninjected old muscles. Muscle mass and fiber type distributions were maintained at levels similar to those in young adults. We propose that these effects are primarily due to stimulation of muscle regeneration via the activation of satellite cells by IGF-I. This supports the hypothesis that the primary cause of aging-related impairment of muscle function is a cumulative failure to repair damage sustained during muscle utilization. Our results suggest that gene transfer of IGF-I into muscle could form the basis of a human gene therapy for preventing the loss of muscle function associated with aging and may be of benefit in diseases where the rate of damage to skeletal muscle is accelerated.

Controlling small guanine–nucleotide-exchange factor function through cytoplasmic RNA intramers

ADP-ribosylation factor (ARF) GTPases and their regulatory proteins have been implicated in the control of diverse biological functions. Two main classes of positive regulatory elements for ARF have been discovered so far: the large Sec7/Gea and the small cytohesin/ARNO families, respectively. These proteins harbor guanine–nucleotide-exchange factor (GEF) activity exerted by the common Sec7 domain. The availability of a specific inhibitor, the fungal metabolite brefeldin A, has enabled documentation of the involvement of the large GEFs in vesicle transport. However, because of the lack of such tools, the biological roles of the small GEFs have remained controversial. Here, we have selected a series of RNA aptamers that specifically recognize the Sec7 domain of cytohesin 1. Some aptamers inhibit guanine–nucleotide exchange on ARF1, thereby preventing ARF activation in vitro. Among them, aptamer M69 exhibited unexpected specificity for the small GEFs, because it does not interact with or inhibit the GEF activity of the related Gea2-Sec7 domain, a member of the class of large GEFs. The inhibitory effect demonstrated in vitro clearly is observed as well in vivo, based on the finding that M69 produces similar results as a dominant-negative, GEF-deficient mutant of cytohesin 1: when expressed in the cytoplasm of T-cells, M69 reduces stimulated adhesion to intercellular adhesion molecule-1 and results in a dramatic reorganization of F-actin distribution. These highly specific cellular effects suggest that the ARF-GEF activity of cytohesin 1 plays an important role in cytoskeletal remodeling events of lymphoid cells.

In vivo restoration of laminin 5 β3 expression and function in junctional epidermolysis bullosa

The blistering disorder, lethal junctional epidermolysis bullosa (JEB), can result from mutations in the LAMB3 gene, which encodes laminin 5 β3 (β3). Appropriate expression of LAMβ3 in JEB skin tissue could potentially ameliorate the symptoms of the underlying disease. To explore the utility of this therapeutic approach, primary keratinocytes from six unrelated JEB patients were transduced with a retroviral vector encoding β3 and used to regenerate human skin on severe combined immunodeficient (SCID) mice. Tissue regenerated from β3-transduced JEB keratinocytes produced phenotypically normal skin characterized by sustained β3 expression and the formation of hemidesmosomes. Additionally, β3 gene transfer corrected the distribution of a number of important basement membrane zone proteins including BPAG2, integrins β4/β1, and laminins α3/γ2. Skin produced from β3-negative (β3[−]) JEB cells mimicked the hallmarks of the disease state and did not exhibit any of the aforementioned traits. Therefore, by effecting therapeutic gene transfer to β3-deficient primary keratinocytes, it is possible to produce healthy, normal skin tissue in vivo. These data support the utility of gene therapy for JEB and highlight the potential for gene delivery in the treatment of human genetic skin disease.

Activation-dependent changes in receptor distribution and dendritic morphology in hippocampal neurons expressing P2X2-green fluorescent protein receptors

ATP-gated P2X2 receptors are widely expressed in neurons, but the cellular effects of receptor activation are unclear. We engineered functional green fluorescent protein (GFP)-tagged P2X2 receptors and expressed them in embryonic hippocampal neurons, and report an approach to determining functional and total receptor pool sizes in living cells. ATP application to dendrites caused receptor redistribution and the formation of varicose hot spots of higher P2X2-GFP receptor density. Redistribution in dendrites was accompanied by an activation-dependent enhancement of the ATP-evoked current. Substate-specific mutant T18A P2X2-GFP receptors showed no redistribution or activation-dependent enhancement of the ATP-evoked current. Thus fluorescent P2X2-GFP receptors function normally, can be quantified, and reveal the dynamics of P2X2 receptor distribution on the seconds time scale.