Jaime Andrés Rivas-Pardo

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. Here, we show that mechanical unfolding of titin immunoglobulin (Ig) domains exposes buried cysteine residues, which then can be S-glutathionylated. S-glutathionylation of cryptic cysteines greatly decreases the mechanical stability of the parent Ig domain as well as its ability to fold. Both effects favor a more extensible state of titin. Furthermore, we demonstrate that S-glutathionylation of cryptic cysteines in titin mediates mechanochemical modulation of the elasticity of human cardiomyocytes. We propose that posttranslational modification of cryptic residues is a general mechanism to regulate tissue elasticity.

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 prove this novel technique by capturing the regiospecific reaction between a thiol and a vicinal disulfide bond. We designed a protein that includes a caged cysteine and a buried disulfide. The mechanical unfolding of this protein in the presence of an external nucleophile frees the single reactive cysteine residue, which now can cleave the target disulfide via a nucleophilic attack on either one of its two sulfur atoms. This produces two different and competing reaction pathways. We use single molecule force spectroscopy to monitor the cleavage of the disulfides, which extends the polypeptide by a magnitude unambiguously associated with each reaction pathway. This allowed us to measure, for the first time, the kinetics of disulfide bond isomerization in a protein.

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. We use these covalently anchored polyproteins to study the remarkable mechanical properties of HaloTag proteins. We show that the force that triggers unfolding of the HaloTag protein exhibits a 4-fold increase, from 131 to 491 pN, when the direction of the applied force is changed from the C-terminus to the N-terminus. Force-clamp experiments reveal that unfolding of the HaloTag protein is twice as sensitive to pulling force compared to protein L and refolds at a slower rate. We show how these properties allow for the long-term observation of protein folding-unfolding cycles at high forces, without interference from the HaloTag tether.

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. We use single-molecule techniques to demonstrate that unfolded titin Ig domains undergo a spontaneous stepwise folding contraction at forces below 10 pN, delivering up to 105 zJ of additional contractile energy, which is larger than the mechanical energy delivered by the power stroke of a myosin motor. Thus, it appears inescapable that folding of titin Ig domains is an important, but as yet unrecognized, contributor to the force generated by a contracting muscle.

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 Termococcus 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. Initial velocity studies indicate that catalysis follows a sequential ordered mechanism that correlates with the structural transitions experienced by the enzyme in solution and in the crystal state. The combined data allowed us to resolve the open-closed conformational transition that accounts for the complete reaction cycle and to identify the corresponding clusters of aminoacids residues responsible for it. These results provide molecular bases for a general mechanism conserved across the ADP-dependent kinase family.

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. However, in the presence of this substrate, an additional unfolding event is observed, intermediate 1*. Finally, in the presence of both substrates, the unfolding force of intermediates 1 and 1* increases as a consequence of the domain closure. These results show that SMFS can be used as a powerful experimental tool to investigate binding mechanisms of different enzymes with more than one ligand, expanding the repertoire of protocols traditionally used in enzymology.

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. In this work, we have studied the role of divalent metal cations on the catalysis and regulation of ADP-dependent glucokinases and phosphofructokinase from hyperthermophilic archaea by means of initial velocity assays as well as molecular dynamics simulations. The results show that a divalent cation is strictly necessary for the activity of these enzymes and they strongly suggest that the true substrate is the metal-nucleotide complex. Also, these enzymes are promiscuous in relation to their metal usage where the only considerations for metal assisted catalysis seem to be related to the ionic radii and coordination geometry of the cations. Molecular dynamics simulations strongly suggest that this metal is bound to the highly conserved NXXE motif, which constitutes one of the signatures of the ribokinase superfamily. Although free ADP cannot act as a phosphoryl donor it still can bind to these enzymes with a reduced affinity, stressing the importance of the metal in the proper binding of the nucleotide at the active site. Also, data show that the binding of a second metal to these enzymes produces a complex with a reduced catalytic constant. On the basis of these findings and considering evolutionary information for the ribokinase superfamily, we propose that the regulatory metal acts by modulating the energy difference between the protein-substrates complex and the reaction transition state, which could constitute a general mechanism for the metal regulation of the enzymes that belong this 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. Only Mn(2+) and Mg(2+) can fulfill this role binding to the same activating site but the affinity for Mn(2+) is 13-fold higher compared to that of Mg(2+). The role of the E190 residue, present in the highly conserved motif NXXE involved in Mg(2+) binding, is also evaluated in this behavior. The E190Q mutation drastically diminishes the kinetic affinity of this site for both cations. However, binding studies of free Mn(2+) and metal-Mant-ATP complex through EPR and FRET experiments between the ATP analog and Trp88, demonstrated that Mn(2+) as well as the metal-nucleotide complex bind with the same affinity to the wild type and E190Q mutant Pfk-2. These results suggest that this residue exert its role mainly kinetically, probably stabilizing the transition state and that the geometry of metal binding to E190 residue may be crucial to determine the catalytic competence.

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. Ageing rates vary among different proteins, but in all cases they lose their mechanical integrity. Random oxidative modification of cryptic side chains exposed by mechanical unfolding can be slowed by the addition of antioxidants such as ascorbic acid, or accelerated by oxidants. By contrast, proteins kept in the folded state and probed over week-long experiments show greatly reduced rates of ageing. We demonstrate a novel approach whereby protein ageing can be greatly accelerated: the constant unfolding of a protein for hours to days is equivalent to decades of exposure to free radicals under physiological conditions.

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 fromThermococcus litoralis (TlGK) relies on the highly conserved motifs NXXE (i.e. Asn‐Xaa‐Xaa‐Glu) and HXE (i.e. His‐Xaa‐Glu). Site‐directed mutagenesis of residues Glu279 (HXE) and Glu308 (NXXE) leads to enzymes with highly reduced catalytic rates. The replacement of Glu308 by Gln increased theK M for MgADP− and was activated by free Mg2+. On the other hand, HXE mutants did not affect theK M for MgADP−, were still inhibited by free Mg2+, and caused a large increase onK M for glucose and an 87‐fold weaker binding of glucose onto the non‐hydrolysableTlGK·AMP–AlF3 complex. Our findings put forward the fundamental role of the HXE motif in glucose binding during ternary complex formation.