Understanding the Folding-Function Tradeoff in Proteins

When an amino-acid sequence cannot be optimized for both folding and function, folding can get compromised in favor of function. To understand this tradeoff better, we devise a novel method for extracting the “function-less” folding-motif of a protein fold from a set of structurally similar but functionally diverse proteins. We then obtain the β-trefoil folding-motif, and study its folding using structure-based models and molecular dynamics simulations. CompariA protein sequence serves two purpson with the folding of wild-type β-trefoil proteins shows that function affects folding in two ways: In the slower folding interleukin-1β, binding sites make the fold more complex, increase contact order and slow folding. In the faster folding hisactophilin, residues which could have been part of the folding-motif are used for function. This reduces the density of native contacts in functional regions and increases folding rate. The folding-motif helps identify subtle structural deviations which perturb folding. These may then be used for functional annotation. Further, the folding-motif could potentially be used as a first step in the sequence design of function-less scaffold proteins. Desired function can then be engineered into these scaffolds.     

Nitrate reductase-mediated synthesis of silver nanoparticles from AgNO3

Synthesis of silver nanoparticles using α-NADPH-dependent nitrate reductase and phytochelatin in vitro has been demonstrated for the first time. The silver ions were reduced in the presence of nitrate reductase, leading to the formation of a stable silver hydrosol 10–25 nm diam. and stabilized by the capping peptide. The nanoparticles were characterized by X-ray diffraction, transmission electron microscopy, X-ray photoelectron spectroscopy and UV-Vis absorption. These studies will help in designing a rational enzymatic strategy for the synthesis of nanomaterials of different chemical composition, shapes and sizes as well as their separation.     

A distinct complex of PRP19-related and trypanosomatid-specific proteins is required for pre-mRNA splicing in trypanosomes

The pre-mRNA splicing factor PRP19 is recruited into the spliceosome after forming the PRP19/CDC5L complex in humans and the Nineteen complex in yeast. Additionally, ‘PRP19-related’ proteins enter the spliceosome individually or in pre-assemblies that differ in these systems. The protistan family Trypanosomatidae, which harbors parasites such as Trypanosoma brucei, diverged early during evolution from opisthokonts. While introns are rare in these organisms, spliced leader trans splicing is an obligatory step in mRNA maturation. So far, ∼70 proteins have been identified as homologs of human and yeast splicing factors. Moreover, few proteins of unknown function have recurrently co-purified with splicing proteins. Here we silenced the gene of one of these proteins, termed PRC5, and found it to be essential for cell viability and pre-mRNA splicing. Purification of PRC5 combined with sucrose gradient sedimentation revealed a complex of PRC5 with a second trypanosomatid-specific protein, PRC3, and PRP19-related proteins SYF1, SYF3 and ISY1, which we named PRP19-related complex (PRC). Importantly, PRC and the previously described PRP19 complex are distinct from each other because PRC, unlike PRP19, co-precipitates U4 snRNA, which indicates that PRC enters the spliceosome prior to PRP19 and uncovers a unique pre-organization of these proteins in trypanosomes.     

Nano-Boehmite Induced Oxidative and Nitrosative Stress Responses in Vigna radiata L.

Journal of Plant Growth Regulation - Wide spectrum and increasing use of nano-sized aluminum oxyhydroxide (boehmite, nBhm) particles have left a risk of their environmental exposure and...     

Determining sensitive stages for learning to detect predators in larval bronzed frogs: Importance of alarm cues in learning

Successful survival and reproduction of prey organisms depend on their ability to detect their potential predators accurately and respond effectively with suitable defences. Predator detection can be innate or can be acquired through learning. We studied prey–predator interactions in the larval bronzed frogs (Sylvirana temporalis), which have the innate ability to detect certain predators. We conducted a series of experiments to determine if the larval S. temporalis rely solely on innate predator detection mechanisms or can also learn to use more specific cues such as conspecific alarm cues for the purpose. The results of our study clearly indicate that larval S. temporalis use both innate and learned mechanisms for predator detection. Predator-naïve tadpoles could detect kairomones alone as a potential threat and responded by reducing activity, suggesting an innate predator detection mechanism. Surprisingly, predator-naïve tadpoles failed to detect conspecific alarm cues as a potential threat, but learned to do so through experience. After acquiring the ability to detect conspecific alarm cues, they could associate novel predator cues with conspecific alarm cues. Further, post feeding stages of larval S. temporalis are sensitive for learning to detect conspecific alarm cues to label novel predators.     

The Molecular Mechanism of Domain Swapping of the C-Terminal Domain of the SARS-Coronavirus Main Protease

In three-dimensional domain swapping, two protein monomers exchange a part of their structures to form an intertwined homodimer, whose subunits resemble the monomer. Several viral proteins domain swap to increase their structural complexity or functional avidity. The main protease (M^pro) of the severe acute respiratory syndrome (SARS) coronavirus proteolyzes viral polyproteins and has been a target for anti-SARS drug design. Domain swapping in the α-helical C-terminal domain of M^pro (M^proC) locks M^pro into a hyperactive octameric form that is hypothesized to promote the early stages of viral replication. However, in the absence of a complete molecular understanding of the mechanism of domain swapping, investigations into the biological relevance of this octameric M^pro have stalled. Isolated M^proC can exist as a monomer or a domain-swapped dimer. Here, we investigate the mechanism of domain swapping of M^proC using coarse-grained structure-based models and molecular dynamics simulations. Our simulations recapitulate several experimental features of M^proC folding. Further, we find that a contact between a tryptophan in the M^proC domain-swapping hinge and an arginine elsewhere forms early during folding, modulates the folding route, and promotes domain swapping to the native structure. An examination of the sequence and the structure of the tryptophan containing hinge loop shows that it has a propensity to form multiple secondary structures and contacts, indicating that it could be stabilized into either the monomer- or dimer-promoting conformations by mutations or ligand binding. Finally, because all residues in the tryptophan loop are identical in SARS-CoV and SARS-CoV-2, mutations that modulate domain swapping may provide insights into the role of octameric M^pro in the early-stage viral replication of both viruses.     

The catalytic cycle for ribonucleotide incorporation by human DNA Pol λ

Although most DNA polymerases discriminate against ribonucleotide triphosphaets (rNTPs) during DNA synthesis, recent studies have shown that large numbers of ribonucleotides are incorporated into the eukaryotic nuclear genome. Here, we investigate how a DNA polymerase can stably incorporate an rNTP. The X-ray crystal structure of a variant of human DNA polymerase λ reveals that the rNTP occupies the nucleotide binding pocket without distortion of the active site, despite an unfavorable interaction between the 2'-O and Tyr505 backbone carbonyl. This indicates an energetically unstable binding state for the rNTP, stabilized by additional protein-nucleotide interactions. Supporting this idea is the 200-fold lower catalytic efficiency for rNTP relative to deoxyribonucleotide triphosphate (dNTP) incorporation, reflecting a higher apparent Km value for the rNTP. Furthermore, distortion observed in the structure of the post-catalytic product complex suggests that once the bond between the α- and β-phosphates of the rNTP is broken, the unfavorable binding state of the ribonucleotide cannot be maintained. Finally, structural and biochemical evaluation of dNTP insertion onto an ribonucleotide monophosphate (rNMP)-terminated primer indicates that a primer-terminal rNMP does not impede extension. The results are relevant to how ribonucleotides are incorporated into DNA in vivo, during replication and during repair, perhaps especially in non-proliferating cells when rNTP:dNTP ratios are high.