Accurate prediction of protein folding rates from sequence and sequence‐derived residue flexibility and solvent accessibility

Protein folding rates vary by several orders of magnitude and they depend on the topology of the fold and the size and composition of the sequence. Although recent works show that the rates can be predicted from the sequence, allowing for high‐throughput annotations, they consider only the sequence and its predicted secondary structure. We propose a novel sequence‐based predictor, PFR‐AF, which utilizes solvent accessibility and residue flexibility predicted from the sequence, to improve predictions and provide insights into the folding process. The predictor includes three linear regressions for proteins with two‐state, multistate, and unknown (mixed‐state) folding kinetics. PFR‐AF on average outperforms current methods when tested on three datasets. The proposed approach provides high‐quality predictions in the absence of similarity between the predicted and the training sequences. The PFR‐AF's predictions are characterized by high (between 0.71 and 0.95, depending on the dataset) correlation and the lowest (between 0.75 and 0.9) mean absolute errors with respect to the experimental rates, as measured using out‐of‐sample tests. Our models reveal that for the two‐state chains inclusion of solvent‐exposed Ala may accelerate the folding, while increased content of Ile may reduce the folding speed. We also demonstrate that increased flexibility of coils facilitates faster folding and that proteins with larger content of solvent‐exposed strands may fold at a slower pace. The increased flexibility of the solvent‐exposed residues is shown to elongate folding, which also holds, with a lower correlation, for buried residues. Two case studies are included to support our findings. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Oviposition Marking Behavior of <Emphasis Type="Italic">Diachasma alloeum</Emphasis>, (Hymenoptera: Braconidae), Parasitizing <Emphasis Type="Italic">Rhagoletis pomonella</Emphasis>, (Diptera: Tephritidae)

Diachasma alloeum (Muesebeck) (Hymenoptera: Braconidae) is a solitary larval endoparasitoid attacking Rhagoletis (Diptera: Tephritidae) species. Rhagoletis pomonella (Walsh) mark the surface of fruit after oviposition with an oviposition marking pheromone (OMP) which deters conspecific female flies. Herein we demonstrate that female D. alloeum wasps reared from either apple or hawthorn race R. pomenella larvae also deposit an OMP that reduces oviposition by conspecific female wasps. Significantly fewer wasps accepted fruit that had received prior wasp oviposition and OMP or OMP alone without oviposition compared with control fruit for a minimum of 7 days on both fruit types. Rinsing fruit with a 50% ethanol solution appeared to remove the OMP rendering fruit more acceptable for oviposition than marked fruit that was not rinsed. Wasps of each host race were able to detect and avoid the OMP of the sister race and fruit substrate type did not affect wasp response to the pheromone. The possibility of an internal marker deposited during oviposition is also discussed.     

Septins enforce morphogenetic events during sexual reproduction and contribute to virulence of Cryptococcus neoformans

Septins are conserved, cytoskeletal GTPases that contribute to cytokinesis, exocytosis, cell surface organization and vesicle fusion by mechanisms that are poorly understood. Roles of septins in morphogenesis and virulence of a human pathogen and basidiomycetous yeast Cryptococcus neoformans were investigated. In contrast to a well‐established paradigm in S. cerevisiae, Cdc3 and Cdc12 septin homologues are dispensable for growth in C. neoformans yeast cells at 24°C but are essential at 37°C. In a bilateral cross between septin mutants, cells fuse but the resulting hyphae exhibit morphological abnormalities, including lack of properly fused specialized clamp cells and failure to produce spores. Interestingly, post‐mating hyphae of the septin mutants have a defect in nuclear distribution. Thus, septins are essential for the development of spores, clamp cell fusion and also play a specific role in nuclear dynamics in hyphae. In the post‐mating hyphae the septins localize to discrete sites in clamp connections, to the septa and the bases of the initial emerging spores. Strains lacking CDC3 or CDC12 exhibit significantly reduced virulence in a Galleria mellonella model of infection. Thus, C. neoformans septins are vital to morphology of the hyphae and contribute to virulence.     

Two CDC42 paralogues modulate Cryptococcus neoformans thermotolerance and morphogenesis under host physiological conditions

The precise regulation of morphogenesis is a key mechanism by which cells respond to a variety of stresses, including those encountered by microbial pathogens in the host. The polarity protein Cdc42 regulates cellular morphogenesis throughout eukaryotes, and we explore the role of Cdc42 proteins in the host survival of the human fungal pathogen Cryptococcus neoformans. Uniquely, C. neoformans has two functional Cdc42 paralogues, Cdc42 and Cdc420. Here we investigate the contribution of each paralogue to resistance to host stress. In contrast to non‐pathogenic model organisms, C. neoformans Cdc42 proteins are not required for viability under non‐stress conditions but are required for resistance to high temperature. The paralogues play differential roles in actin and septin organization and act downstream of C. neoformans Ras1 to regulate its morphogenesis sub‐pathway, but not its effects on mating. Cdc42, and not Cdc420, is upregulated in response to temperature stress and is required for virulence in a murine model of cryptococcosis. The C. neoformans Cdc42 proteins likely perform complementary functions with other Rho‐like GTPases to control cell polarity, septin organization and hyphal transitions that allow survival in the environment and in the host.     

Oleanolic acid and ursolic acid affect peptidoglycan metabolism in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis>

The plant pentacyclic triterpenoids, oleanolic and ursolic acids, inhibit the growth and survival of many bacteria, particularly Gram-positive species, including pathogenic ones. The effect of these compounds on the facultative human pathogen Listeria monocytogenes was examined. Both acids affected cell morphology and enhanced autolysis of the bacterial cells. Autolysis of isolated cell walls was inhibited by oleanolic acid, but the inhibitory activity of ursolic acid was less pronounced. Both compounds inhibited peptidoglycan turnover and quantitatively affected the profile of muropeptides obtained after digestion of peptidoglycan with mutanolysin. These results suggest that peptidoglycan metabolism is a cellular target of oleanolic and ursolic acids.     

Biosynthesis of salvinorin A proceeds via the deoxyxylulose phosphate pathway

Salvinorin A, a neoclerodane diterpenoid, isolated from the Mexican hallucinogenic plant Salvia divinorum, is a potent kappa-opioid receptor agonist. Its biosynthetic route was studied by NMR and HR-ESI-MS analysis of the products of the incorporation of [1-(13)C]-glucose, [Me-(13)C]-methionine, and [1-(13)C;3,4-(2)H2]-1-deoxy-D-xylulose into its structure. While the use of cuttings and direct-stem injection were unsuccessful, incorporation of (13)C into salvinorin A was achieved using in vitro sterile culture of microshoots. NMR spectroscopic analysis of salvinorin A (2.7 mg) isolated from 200 microshoots grown in the presence of [1-(13)C]-glucose established that this pharmacologically important diterpene is biosynthesized via the 1-deoxy-D-xylulose-5-phosphate pathway, instead of the classic mevalonic acid pathway. This was confirmed further in plants grown in the presence of [1-(13)C;3,4-(2)H2]-1-deoxy-D-xylulose. In addition, analysis of salvinorin A produced by plants grown in the presence of [Me-(13)C]-methionine indicates that methylation of the C-4 carboxyl group is catalyzed by a type III S-adenosyl-L-methionine-dependent O-methyltransferase.     

Regulatory role of cathepsin D in apoptosis

Cathepsin D (CTSD, EC 3.4.23.5) is well known aspartyl protease. Among different role in cell physiology, a new function of this enzyme is examined. Cathepsin D is an important regulator of apoptotic pathways in cells. It acts at different stage of intrinsic and extrinsic pathway of apoptosis. Cathepsin D can either induce apoptosis in presence of cytotoxic factors, but in certain studies an inhibitory role in apoptosis was also reviewed. Detailed review of involvement of cathepsin D in cell apoptosis is a purpose of this paper.     

Toward rational protein crystallization: A Web server for the design of crystallizable protein variants

Growing well-diffracting crystals constitutes a serious bottleneck in structural biology. A recently proposed crystallization methodology for "stubborn crystallizers" is to engineer surface sequence variants designed to form intermolecular contacts that could support a crystal lattice. This approach relies on the concept of surface entropy reduction (SER), i.e., the replacement of clusters of flexible, solvent-exposed residues with residues with lower conformational entropy. This strategy minimizes the loss of conformational entropy upon crystallization and renders crystallization thermodynamically favorable. The method has been successfully used to crystallize more than 15 novel proteins, all stubborn crystallizers. But the choice of suitable sites for mutagenesis is not trivial. Herein, we announce a Web server, the surface entropy reduction prediction server (SERp server), designed to identify mutations that may facilitate crystallization. Suggested mutations are predicted based on an algorithm incorporating a conformational entropy profile, a secondary structure prediction, and sequence conservation. Minor considerations include the nature of flanking residues and gaps between mutation candidates. While designed to be used with default values, the server has many user-controlled parameters allowing for considerable flexibility. Within, we discuss (1) the methodology of the server, (2) how to interpret the results, and (3) factors that must be considered when selecting mutations. We also attempt to benchmark the server by comparing the server's predictions with successful SER structures. In most cases, the structure yielding mutations were easily identified by the SERp server. The server can be accessed at http://www.doe-mbi.ucla.edu/Services/SER.