Protein Function Prediction by an ARTMAP Neural Network

Accurate prediction of protein functions solely from its amino acid sequence is 7 of paramount importance, particularly in the development of new drugs. An 8 ARTMAP neural network (NN) is employed to predict a protein’s function 9 based only on its amino-acid (AA) sequence. For our protein database, a Gene 10 Ontology-based search against the UniProt/SwissProt database for “DNA se-11 quence-specific binding proteins”. The search complement set was also re-12 trieved. For training and testing, various size datasets were generated. Datasets 13 were generated either by random sampling from the existing categories or by 14 classifying the proteins first into sub-groups based on a similarity measure and 15 then randomly sampling from each sub-group. Our NN’s performance with the 16 latter method performed better than with the former method in every size da-17 taset. Our NN has been successful in predicting the function of a protein from its 18 AA sequence by extracting a shared sequence-specific feature that is linked to 19 specific DNA binding proteins. This result is of major importance in structural 20 biology and biomedicine as it can provide a basis of the development of highly 21 specific tools for genome modification and gene therapy

The rotation-coupled sliding of EcoRV

It has been proposed that certain type II restriction enzymes (REs), such as EcoRV, track the helical pitch of DNA as they diffuse along DNA, a so-called rotation-coupled sliding. As of yet, there is no direct experimental observation of this phenomenon, but mounting indirect evidence gained from single-molecule imaging of RE–DNA complexes support the hypothesis. We address this issue by conjugating fluorescent labels of varying size (organic dyes, proteins and quantum dots) to EcoRV, and by fusing it to the engineered Rop protein scRM6. Single-molecule imaging of these modified EcoRVs sliding along DNA provides us with their linear diffusion constant (D1), revealing a significant size dependency. To account for the dependence of D1 on the size of the EcoRV label, we have developed four theoretical models describing different types of motion along DNA and find that our experimental results are best described by rotation-coupled sliding of the protein. The similarity of EcoRV to other type II REs and DNA binding proteins suggests that this type of motion could be widely preserved in other biological contexts

α-Helices in the Type III Secretion Effectors: A Prevalent Feature with Versatile Roles

We are grateful for support for equipment from the French Government Programme Investissements d’Avenir France BioImaging (FBI, N◦ANR-10-INSB-04-01) and the French gouvernement (Agence Nationale de la Recherche) Investissement d’Avenir programme, Laboratoire d’Excellence “Integrative Biology of Emerging Infectious Diseases” (ANR-10-LABX-62-IBEID)International audienceType III Secretion Systems (T3SSs) are multicomponent nanomachines located at the cell envelope of Gram-negative bacteria. Their main function is to transport bacterial proteins either extracellularly or directly into the eukaryotic host cell cytoplasm. Type III Secretion effectors (T3SEs), latest to be secreted T3S substrates, are destined to act at the eukaryotic host cell cytoplasm and occasionally at the nucleus, hijacking cellular processes through mimicking eukaryotic proteins. A broad range of functions is attributed to T3SEs, ranging from the manipulation of the host cell’s metabolism for the benefit of the bacterium to bypassing the host’s defense mechanisms. To perform this broad range of manipulations, T3SEs have evolved numerous novel folds that are compatible with some basic requirements: they should be able to easily unfold, pass through the narrow T3SS channel, and refold to an active form when on the other side. In this review, the various folds of T3SEs are presented with the emphasis placed on the functional and structural importance of α-helices and helical domains

Dynamic Characterization of the Human Heme Nitric Oxide/Oxygen (HNOX) Domain under the Influence of Diatomic Gaseous Ligands

Soluble guanylate cyclase (sGC) regulates numerous physiological processes. The β subunit Heme Nitric Oxide/Oxygen (HNOX) domain makes this protein sensitive to small gaseous ligands. The structural basis of the activation mechanism of sGC under the influence of ligands (NO, O2, CO) is poorly understood. We examine the effect of different ligands on the human sGC HNOX domain. HNOX systems with gaseous ligands were generated and explored using Molecular Dynamics (MD). The distance between heme Fe2+ and histidine in the NO-ligated HNOX (NO-HNOX) system is larger compared to the O2, CO systems. NO-HNOX rapidly adopts the conformation of the five-group metal coordination system. Loops α, β, γ and helix-f exhibit increased mobility and different hydrogen bond networks in NO-HNOX compared to the other systems. The removal of His from the Fe coordination sphere in NO-HNOX is assisted by interaction of the imidazole ring with the surrounding residues which in turn leads to the release of signaling helix-f and activation of the sGC enzyme. Insights into the conformational dynamics of a human sGC HNOX domain, especially for regions which are functionally critical for signal transduction, are valuable in the understanding of cardiovascular diseases

Variations of the NodB Architecture Are Attuned to Functional Specificities into and beyond the Carbohydrate Esterase Family 4

Enzymes of the carbohydrate esterase family 4 (CE4) deacetylate a broad range of substrates, including linear, branched and mesh-like polysaccharides. Although they are enzymes of variable amino acid sequence length, they all comprise the conserved catalytic domain NodB. NodB carries the metal binding and active site residues and is characterized by a set of conserved sequence motifs, which are linked to the deacetylation activity. Besides a non-structured, flexible peptide of variable length that precedes NodB, several members of the CE4 family contain additional domains whose function or contribution to substrate specificity are not efficiently characterized. Evidence suggests that CE4 family members comprising solely the NodB domain have developed features linked to a variety of substrate specificities. To understand the NodB-based substrate diversity within the CE4 family, we perform a comparative analysis of all NodB domains structurally characterized so far. We show that amino acid sequence variations, topology diversities and excursions away from the framework structure give rise to different NodB domain classes associated with different substrate specificities and particular functions within and beyond the CE4 family. Our work reveals a link between specific NodB domain characteristics and substrate recognition. Thus, the details of the fold are clarified, and the structural basis of its variations is deciphered and associated with function. The conclusions of this work are also used to make predictions and propose specific functions for biochemically/enzymatically uncharacterized NodB-containing proteins, which have generally been considered as putative CE4 deacetylases. We show that some of them probably belong to different enzymatic families

Tolerance to d-amphetamine: Behavioral specificity

In a Y-maze exploratory task mice tend to enter that compartment which was least recently visited (spontaneous alternation). Low doses of d-amphetamine (1.

Purification, crystallization, X-ray diffraction analysis and phasing of an engineered single-chain PvuII restriction endonuclease

PvuII is the first type II restriction endonuclease to be converted from its wild-type homodimeric form into an enzymatically active single-chain variant. The enzyme was crystallized and phasing was successfully performed by molecular replacement