Phylogenomic analysis of the duo: approaches to the origin of periodontitis.

is an oral human pathogen associated with the onset and progression of periodontitis, a chronic immune-inflammatory disease characterized by the destruction of the teeth-supporting tissue. belongs to the genus which is characterized by being composed of Gram-negative, asaccharolytic, non-spore-forming, non-motile, obligatory anaerobic species, inhabiting niches such as the oral cavity, urogenital tract, gastrointestinal tract and infected wound from different mammals including humans. Among the genus, stands out for its specificity in colonizing the human oral cavity and its keystone pathogen role in periodontitis pathogenesis.

Interfering with the Folding of Group A Streptococcal pili Proteins.

Gram-positive bacteria use their adhesive pili to attach to host cells during early stages of a bacterial infection. These extracellular hair-like appendages experience mechanical stresses of hundreds of picoNewtons; however, the presence of an internal isopeptide bond prevents the pilus protein from unfolding. Here, we describe a method to interfere with nascent pili proteins through a peptide that mimics one of the β-strands of the molecule.

A HaloTag-TEV genetic cassette for mechanical phenotyping of proteins from tissues.

Single-molecule methods using recombinant proteins have generated transformative hypotheses on how mechanical forces are generated and sensed in biological tissues. However, testing these mechanical hypotheses on proteins in their natural environment remains inaccesible to conventional tools. To address this limitation, here we demonstrate a mouse model carrying a HaloTag-TEV insertion in the protein titin, the main determinant of myocyte stiffness.

Single-Nucleotide Polymorphisms (SNP) Mining and Their Effect on the Tridimensional Protein Structure Prediction in a Set of Immunity-Related Expressed Sequence Tags (EST) in Atlantic Salmon ().

Single-nucleotide polymorphisms (SNPs) are single genetic code variations considered one of the most common forms of nucleotide modifications. Such SNPs can be located in genes associated to immune response and, therefore, they may have direct implications over the phenotype of susceptibility to infections affecting the productive sector. In this study, a set of immune-related genes ( [], integrin β2 (itβ2, also named ), [], [], []) were analyzed to identify SNPs by data mining.

The Mechanical Power of Titin Folding.

The delivery of mechanical power, a crucial component of animal motion, is constrained by the universal compromise between the force and the velocity of its constituent molecular systems. While the mechanisms of force generation have been studied at the single molecular motor level, there is little understanding of the magnitude of power that can be generated by folding proteins. Here, we use single-molecule force spectroscopy techniques to measure the force-velocity relation of folding titin domains that contain single internal disulfide bonds, a common feature throughout the titin I-band.

The power of the force: mechano-physiology of the giant titin.

Titin - the largest protein in the human body - spans half of the muscle sarcomere from the Z-disk to the M-band through a single polypeptide chain. More than 30 000 amino acid residues coded from a single gene (TTN, in humans Q8WZ42) form a long filamentous protein organized in individual globular domains concatenated in tandem. Owing to its location and close interaction with the other muscle filaments, titin is considered the third filament of muscle, after the thick-myosin and the thin-actin filaments.

Molecular strategy for blocking isopeptide bond formation in nascent pilin proteins.

Bacteria anchor to their host cells through their adhesive pili, which must resist the large mechanical stresses induced by the host as it attempts to dislodge the pathogens. The pili of gram-positive bacteria are constructed as a single polypeptide made of hundreds of pilin repeats, which contain intramolecular isopeptide bonds strategically located in the structure to prevent their unfolding under force, protecting the pilus from degradation by extant proteases and oxygen radicals. Here, we demonstrate the design of a short peptide that blocks the formation of the isopeptide bond present in the pilin Spy0128 from the human pathogen , resulting in mechanically labile pilin domains.

Proteins Breaking Bad: A Free Energy Perspective.

Protein aging may manifest as a mechanical disease that compromises tissue elasticity. As proved recently, while proteins respond to changes in force with an instantaneous elastic recoil followed by a folding contraction, aged proteins break bad, becoming unstructured polymers. Here, we explain this phenomenon in the context of a free energy model, predicting the changes in the folding landscape of proteins upon oxidative aging.

Mechanical Deformation Accelerates Protein Ageing.

A hallmark of tissue ageing is the irreversible oxidative modification of its proteins. We show that single proteins, kept unfolded and extended by a mechanical force, undergo accelerated ageing in times scales of minutes to days. A protein forced to be continuously unfolded completely loses its ability to contract by folding, becoming a labile polymer.

Multidomain proteins under force.

Advancements in single-molecule force spectroscopy techniques such as atomic force microscopy and magnetic tweezers allow investigation of how domain folding under force can play a physiological role. Combining these techniques with protein engineering and HaloTag covalent attachment, we investigate similarities and differences between four model proteins: I10 and I91-two immunoglobulin-like domains from the muscle protein titin, and two α + β fold proteins-ubiquitin and protein L. These proteins show a different mechanical response and have unique extensions under force.

A HaloTag Anchored Ruler for Week-Long Studies of Protein Dynamics.

Under physiological conditions, protein oxidation and misfolding occur with very low probability and on long times scales. Single-molecule techniques provide the ability to distinguish between properly folded and damaged proteins that are otherwise masked in ensemble measurements. However, at physiological conditions these rare events occur with a time constant of several hours, inaccessible to current single-molecule approaches.

Work Done by Titin Protein Folding Assists Muscle Contraction.

Current theories of muscle contraction propose that the power stroke of a myosin motor is the sole source of mechanical energy driving the sliding filaments of a contracting muscle. These models exclude titin, the largest protein in the human body, which determines the passive elasticity of muscles. Here, we show that stepwise unfolding/folding of titin immunoglobulin (Ig) domains occurs in the elastic I band region of intact myofibrils at physiological sarcomere lengths and forces of 6-8 pN.

Dissecting the functional roles of the conserved NXXE and HXE motifs of the ADP-dependent glucokinase from Thermococcus litoralis.

The activity of the ADP-dependent glucokinase from Thermococcus litoralis (TlGK) relies on the highly conserved motifs NXXE (i.e. Asn-Xaa-Xaa-Glu) and HXE (i.

Identifying sequential substrate binding at the single-molecule level by enzyme mechanical stabilization.

Enzyme-substrate binding is a dynamic process intimately coupled to protein structural changes, which in turn changes the unfolding energy landscape. By the use of single-molecule force spectroscopy (SMFS), we characterize the open-to-closed conformational transition experienced by the hyperthermophilic adenine diphosphate (ADP)-dependent glucokinase from Thermococcus litoralis triggered by the sequential binding of substrates. In the absence of substrates, the mechanical unfolding of TlGK shows an intermediate 1, which is stabilized in the presence of Mg·ADP(-), the first substrate to bind to the enzyme.

S-glutathionylation of cryptic cysteines enhances titin elasticity by blocking protein folding.

The giant elastic protein titin is a determinant factor in how much blood fills the left ventricle during diastole and thus in the etiology of heart disease. Titin has been identified as a target of S-glutathionylation, an end product of the nitric-oxide-signaling cascade that increases cardiac muscle elasticity. However, it is unknown how S-glutathionylation may regulate the elasticity of titin and cardiac tissue.

Nanomechanics of HaloTag tethers.

The active site of the Haloalkane Dehydrogenase (HaloTag) enzyme can be covalently attached to a chloroalkane ligand providing a mechanically strong tether, resistant to large pulling forces. Here we demonstrate the covalent tethering of protein L and I27 polyproteins between an atomic force microscopy (AFM) cantilever and a glass surface using HaloTag anchoring at one end and thiol chemistry at the other end. Covalent tethering is unambiguously confirmed by the observation of full length polyprotein unfolding, combined with high detachment forces that range up to ∼2000 pN.

Crystal structure, SAXS and kinetic mechanism of hyperthermophilic ADP-dependent glucokinase from Thermococcus litoralis reveal a conserved mechanism for catalysis.

ADP-dependent glucokinases represent a unique family of kinases that belong to the ribokinase superfamily, being present mainly in hyperthermophilic archaea. For these enzymes there is no agreement about the magnitude of the structural transitions associated with ligand binding and whether they are meaningful to the function of the enzyme. We used the ADP-dependent glucokinase from Thermococcus litoralis as a model to investigate the conformational changes observed in X-ray crystallographic structures upon substrate binding and to compare them with those determined in solution in order to understand their interplay with the glucokinase function.

Direct observation of disulfide isomerization in a single protein.

Photochemical uncaging techniques use light to release active molecules from otherwise inert compounds. Here we expand this class of techniques by demonstrating the mechanical uncaging of a reactive species within a single protein. We proved this novel technique by capturing the regiospecific reaction between a thiol and a vicinal disulfide bond.

Catalytic and regulatory roles of divalent metal cations on the phosphoryl-transfer mechanism of ADP-dependent sugar kinases from hyperthermophilic archaea.

In some archaea, glucose degradation proceeds through a modified version of the Embden-Meyerhof pathway where glucose and fructose-6-P phosphorylation is carried out by kinases that use ADP as the phosphoryl donor. Unlike their ATP-dependent counterparts these enzymes have been reported as non-regulated. Based on the three dimensional structure determination of several ADP-dependent kinases they can be classified as members of the ribokinase superfamily.

Divalent metal cation requirements of phosphofructokinase-2 from E. coli. Evidence for a high affinity binding site for Mn2+.

The reaction catalyzed by E. coli Pfk-2 presents a dual-cation requirement. In addition to that chelated by the nucleotide substrate, an activating cation is required to obtain full activity of the enzyme.