Genetic control of leaf-blade morphogenesis by the <Emphasis Type="Italic">INSECATUS</Emphasis> gene in <Emphasis Type="Italic">Pisum sativum</Emphasis>

To understand the role of INSECATUS (INS) gene in pea, the leaf blades of wild-type, ins mutant and seven other genotypes, constructed by recombining ins with uni-tac, af, tl and mfp gene mutations, were quantitatively compared. The ins was inherited as a recessive mutant allele and expressed its phenotype in proximal leaflets of full size leaf blades. In ins leaflets, the midvein development was arrested in distal domain and a cleft was formed in lamina above this point. There was change in the identity of ins leaflets such that the intercalary interrupted midvein bore a leaf blade. Such adventitious blades in ins, ins tl and ins tl mfp were like the distal segment of respective main leaf blade. The ins phenotype was not seen in ins af and ins af uni-tac genotypes. There was epistasis of uni-tac over ins. The ins, tl and mfp mutations interacted synergistically to produce highly pronounced ins phenotype in the ins tl mfp triple mutant. The role(s) of INS in leaf-blade organogenesis are: positive regulation of vascular patterning in leaflets, repression of UNI activity in leaflet primordia for ectopic growth and in leaf-blade primordium for indeterminate growth of rachis, delimitation of proximal leaflet domain and together with TL and MFP homeostasis for meristematic activity in leaflet primordia. The variant apically bifid shape of the affected ins leaflets demonstrated that the leaflet shape is dependent on the venation pattern.     

Computational Design of a PAK1 Binding Protein

We describe a computational protocol, called DDMI, for redesigning scaffold proteins to bind to a specified region on a target protein. The DDMI protocol is implemented within the Rosetta molecular modeling program and uses rigid-body docking, sequence design, and gradient-based minimization of backbone and side-chain torsion angles to design low-energy interfaces between the scaffold and target protein. Iterative rounds of sequence design and conformational optimization were needed to produce models that have calculated binding energies that are similar to binding energies calculated for native complexes. We also show that additional conformation sampling with molecular dynamics can be iterated with sequence design to further lower the computed energy of the designed complexes. To experimentally test the DDMI protocol, we redesigned the human hyperplastic discs protein to bind to the kinase domain of p21-activated kinase 1 (PAK1). Six designs were experimentally characterized. Two of the designs aggregated and were not characterized further. Of the remaining four designs, three bound to the PAK1 with affinities tighter than 350 μM. The tightest binding design, named Spider Roll, bound with an affinity of 100 μM. NMR-based structure prediction of Spider Roll based on backbone and ¹³C^β chemical shifts using the program CS-ROSETTA indicated that the architecture of human hyperplastic discs protein is preserved. Mutagenesis studies confirmed that Spider Roll binds the target patch on PAK1. Additionally, Spider Roll binds to full-length PAK1 in its activated state but does not bind PAK1 when it forms an auto-inhibited conformation that blocks the Spider Roll target site. Subsequent NMR characterization of the binding of Spider Roll to PAK1 revealed a comparably small binding ‘on-rate’ constant (? 10⁵ M^− 1 s^− 1). The ability to rationally design the site of novel protein-protein interactions is an important step towards creating new proteins that are useful as therapeutics or molecular probes.     

Spontaneous plant regeneration in transformed roots and calli from Tylophora indica: changes in morphological phenotype and tylophorine accumulation associated with transformation by Agrobacterium rhizogenes

We examined the effects of genetic transformation by Agrobacterium rhizogenes on the production of tylophorine, a phenanthroindolizidine alkaloid, in the Indian medicinal plant, Tylophora indica. Transformed roots induced by the bacterium grew in axenic culture and produced shoots or embryogenic calli in the absence of hormone treatments. However, hormonal treatment was required to regenerate shoots in root explants of wild type control plants. Transformed plants showed morphological features typically seen in transgenic plants produced by A. rhizogenes, which include, short internodes, small and wrinkled leaves, more branches and numerous plagiotropic roots. Plants regenerated from transformed roots showed increased biomass accumulation (350–510% in the roots and 200–320% in the whole plants) and augmented tylophorine content (20–60%) in the shoots, resulting in a 160–280% increase in tylophorine production in different clones grown in vitro.     

Conversion of type I 4:6 to 3:5 beta-turn types in human acidic fibroblast growth factor: effects upon structure, stability, folding, and mitogenic function

Human acidic fibroblast growth factor (FGF-1) is a member of the beta-trefoil superfold, a protein architecture that exhibits a characteristic threefold axis of structural symmetry. FGF-1 contains 11 beta-turns, the majority being type I 3:5; however, a type I 4:6 turn is also found at three symmetry-related locations. The relative uniqueness of the type I 4:6 turn in the FGF-1 structure suggests it may play a key role in the stability, folding, or function of the protein. To test this hypothesis a series of deletion mutations were constructed, the aim of which was to convert existing type I 4:6 turns at two locations into type I 3:5 turns. The results show it is possible to successfully substitute the type I 4:6 turn by a type I 3:5 turn with minimal impact upon protein stability or folding. Thus, these different turn structures, even though they differ in length, exhibit similar energetic properties. Additional sequence swapping mutations within the introduced type I 3:5 turns suggests that the turn sequence primarily affects stability but not turn structure (which appears dictated primarily by the local environment). Although the results suggest that a stable, foldable beta-trefoil protein may be designed utilizing a single turn type (type I 3:5), a type I 4:6 turn at turn 1 of FGF-1 appears essential for efficient mitogenic function.     

Snapshots of protein folding problem: implications of folding and misfolding studies

Deciphering the code that determines the three-dimensional structure of proteins and the ability to predict the final folded form of a protein is still elusive to molecular biophysists. In the case of several proteins a similar tertiary structure is not accompanied by any significant sequence similarity. The question now remains whether a code beyond the genetic code that describes the arrangement of the amino acid within a three dimensional protein structure. The available data undoubtedly demonstrates that the redundancy of this code must be tremendous. Several techniques such as nuclear magnetic resonance spectroscopy and laser detection techniques, coupled with fast initiation of the folding reaction, can now probe the folding events in milliseconds or even faster and provide highly relevant information. The thermodynamic analysis of the folding process and of kinetic intermediates opens whole new avenue of understanding. Breaking the protein folding code would enable scientists to look at a gene whose function is unknown and predict the three-dimensional structure of the protein it encodes. This would give them a very good idea of what the gene does. In this review we hope to bring together the information available about protein folding with particular emphasis on folding intermediate(s). Additionally, the practical consequences of the solution of the protein folding problem in medicine and biotechnology are also discussed.     

Mechanistic insights into the dual inhibition strategy for checking Leishmaniasis

Leishmaniasis (1) is an endemic disease mainly caused by the protozoan Leishmania donovani (Ld). Polyamines have been identified as essential organic compounds for the growth and survival of Ld. These are synthesized in Ld by polyamine synthesis pathway comprising of many enzymes such as ornithine decarboxylase (ODC), spermidine synthase (SS), and S-adenosylmethionine decarboxylase. Inhibition of these enzymes in Ld offers a viable prospect to check its growth and development. In the present work, we used computational approaches to search natural inhibitors against ODC and SS enzymes. We predicted three-dimensional structures of ODC and SS using comparative modeling and molecular dynamics (MD) simulations. Thousands of natural compounds were virtually screened against target proteins using high throughput approach. MD simulations were then performed to examine molecular interactions between the screened compounds and functional residues of the active sites of the enzymes. Herein, we report two natural compounds of dual inhibitory nature active against the two crucial enzymes of polyamine pathway of Ld. These dual inhibitors have the potential to evolve as lead molecules in the development of antileishmanial drugs. (1)These authors contributed equally.     

Influence of main channel structure on H(2)O(2) access to the heme cavity of catalase KatE of Escherichia coli

The main channel for H₂O₂ access to the heme cavity in large subunit catalases is twice as long as in small subunit catalases and is divided into two distinct parts. Like small subunit catalases, the 15 Å of the channel adjacent to the heme has a predominantly hydrophobic surface with only weak water occupancy, but the next 15 Å extending to the protein surface is hydrophilic and contains a complex water matrix in multiple passages. At the approximate junction of these two sections are a conserved serine and glutamate that are hydrogen bonded and associated with H₂O₂ in inactive variants. Mutation of these residues changed the dimensions of the channel, both enlarging and constricting it, and also changed the solvent occupancy in the hydrophobic, inner section of the main channel. Despite these structural changes and the prominent location of the residues in the channel, the variants exhibited less than a 2-fold change in the k_cat and apparent K_M kinetic constants. These results reflect the importance of the complex multi-passage structure of the main channel. Surprisingly, mutation of either the serine or glutamate to an aliphatic side chain interfered with heme oxidation to heme d.     

Quantized Water Access to the HIV-1 Protease Active Site as a Proposed Mechanism for Cooperative Mutations in Drug Affinity

The development of resistance to different drugs remains a major problem for a wide range of infections. In particular, combinations of specific mutations, which individually demonstrate no effect, exhibit significant cooperativity. Here we show that changes to the energy of ligand binding in different resistant HIV-1 proteases are correlated with the creation of water binding sites in the active site. This correlation is conserved across two drugs (ritonavir and lopinavir). We propose that individual mutations induce changes in flap packing that are insufficient to allow water binding but in combination allow access, leading to the observed cooperative resistance.