Dissection of complex protein dynamics in human thioredoxin.

Pressure effects on the structure and function of human thioredoxin

Protein surface hydration is fundamental to its structure, flexibility, dynamics, and function, but it has been challenging to disentangle their ultimate relationships. Here, we report our systematic characterization of hydration dynamics around a β-barrel protein, rat liver fatty acid-binding protein (rLFABP), to reveal the effect of different protein secondary structures on hydration water. We employed a tryptophan scan to the protein surface one at a time and examined a total of 17 different sites. We observed three types of hydration water relaxation with distinct time scales, from hundreds of femtoseconds to a hundred picoseconds. We also examined the anisotropy dynamics of the corresponding tryptophan side chains and observed two distinct relaxations from tens to hundreds of picoseconds. Integrating our previous findings on α-helical proteins, we conclude the following: (1) The hydration dynamics is highly heterogeneous around the protein surface of both α-helical and β-sheet proteins. The outer-layer water of the hydration shell behave like a bulk and relaxes in hundreds of femtoseconds. The inner-layer water collectively relaxes in two time scales, reorientation motions in a few picoseconds and network restructuring in tens to a hundred picoseconds. (2) The hydration dynamics are always faster than local protein relaxations and in fact drive the protein fluctuations on the picosecond time scale. (3) The hydration dynamics in general are more retarded around β-sheet structures than α-helical motifs. A thicker hydration shell and a more rigid interfacial hydration network are observed in the β-sheet protein. Overall, these findings elucidate the intimate relationship between water-protein interactions and dynamics on the ultrafast time scale.

Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein displaying a doubly constrained variable peptide loop. They bind specifically target proteins and interfere with their function. We have built a peptide aptamer library in a lentiviral expression system to isolate aptamers that inhibit cell proliferation in vitro. Using one of the isolated aptamers (R5G42) as a bait protein, we have performed yeast two-hybrid screening of cDNA libraries and identified calcineurin A as a target protein candidate. R5G42 bound calcineurin A in vitro and stimulated its phosphatase activity. When expressed transiently in human cells, R5G42 induced the dephosphorylation of BAD. We have identified an antiproliferative peptide aptamer that binds calcineurin and stimulates its activity. The use of this ligand may help elucidate the still elusive structural mechanisms of activation and inhibition of calcineurin. Our work illustrates the power of phenotypic screening of combinatorial protein libraries to interrogate the proteome and chart molecular regulatory networks.

Theoretical aspects of the effects of coupled coherent-incoherent dynamics on the primary processes of photosynthesis

Sorting nexin 9 (SNX9) functions in a complex with the GTPase dynamin-2 at clathrin-coated pits, where it provokes fission of vesicles to complete endocytosis. Here the SNX9.dynamin-2 complex binds to clathrin and adapter protein complex 2 (AP-2) that line these pits, and this occurs through interactions of the low complexity domain (LC4) of SNX9 with AP-2. Intriguingly, localization of the SNX9.dynamin-2 complex to clathrin-coated pits is blocked by interactions with the abundant glycolytic enzyme aldolase, which also binds to the LC4 domain of SNX9. The crystal structure of the LC4 motif of human SNX9 in complex with aldolase explains the biochemistry and biology of this interaction, where SNX9 binds near the active site of aldolase via residues 165-171 that are also required for the interactions of SNX9 with AP-2. Accordingly, SNX9 binding to aldolase is structurally precluded by the binding of substrate to the active site. Interactions of SNX9 with aldolase are far more extensive and differ from those of the actin-nucleating factor WASP with aldolase, indicating considerable plasticity in mechanisms that direct the functions of the aldolase as a scaffold protein.

Chloroplast thioredoxin m from the green alga Chlamydomomas reinhardtii is very efficiently reduced in vitro and in vivo in the presence of photoreduced ferredoxin and a ferredoxin dependent ferredoxin-thioredoxin reductase. Once reduced, thioredoxin m has the capability to quickly activate the NADP malate dehydrogenase (EC 1.1.1.82) a regulatory enzyme involved in an energy-dependent assimilation of carbon dioxide in C4 plants. This activation is the result of the reduction of two disulfide bridges by thioredoxin m, that are located at the N- and C-terminii of the NADP malate dehydrogenase. The molecular structure of thioredoxin m was solved using NMR and compared to other known thioredoxins. Thioredoxin m belongs to the prokaryotic type of thioredoxin, which is divergent from the eukaryotic-type thioredoxins also represented in plants by the h (cytosolic) and f (chloroplastic) types of thioredoxins. The dynamics of the molecule have been assessed using (15)N relaxation data and are found to correlate well with regions of disorder found in the calculated NMR ensemble. The results obtained provide a novel basis to interpret the thioredoxin dependence of the activation of chloroplast NADP-malate dehydrogenase. The specific catalytic mechanism that takes place in the active site of thioredoxins is also discussed on the basis of the recent new understanding and especially in the light of the dual general acid-base catalysis exerted on the two cysteines of the redox active site. It is proposed that the two cysteines of the redox active site may insulate each other from solvent attack by specific packing of invariable hydrophobic amino acids.

Thioredoxin is the best representative enzyme of a group of proteins, widely distributed and possessing dithiol-disulfide oxidoreductase activity. We have constructed a cDNA library from messenger RNAs isolated from a lymphoblastoid B cell line (Epstein-Barr virus-immortalized normal human lymphocytes). Screening of this library with synthetic oligonucleotide probes, constructed from the NH2-terminal amino acid sequence of a protein produced by this line, allowed us to identify a full-length cDNA clone coding for human thioredoxin. The open reading frame (315 nucleotides long) codes for a protein of 104 amino acids (excluding the initial methionine). This protein possesses the highly conserved enzymatic active site common to plant and bacterial thioredoxins: Trp-Cys-Gly-Pro-Cys (amino acids 30-34). These data provide for the first time the complete primary sequence of a thioredoxin of mammalian origin. Recombinant human thioredoxin, expressed in Escherichia coli, possesses a dithiol-reducing enzymatic activity as assayed on mammalian and plant substrates. It is able to reduce the interchain disulfide bridges of murine pentameric IgM and porcin insulin and also to activate vegetal NADP-malate dehydrogenase. Studies of human thioredoxin mRNA expression and regulation in immunocompetent cells of human origin indicate that the protein is weakly expressed in resting lymphocytes and monocytes, but the level of human thioredoxin mRNA transcription is quite important in activated monocytes and established dividing human cell lines.

A new approach to theory and simulation of the non-Markovian dynamics of open quantum systems is presented. It is based on identification of a parameter which is uniformly bounded on wide time intervals: the occupation of the virtual cloud of quanta. By ‘virtual’ we denote those bath excitations which were emitted by the open system, but eventually will be reabsorbed before any measurement of the bath state. A useful property of the virtual cloud is that the number of its quanta is expected to saturate on long times, since physically this cloud is a (retarded) polarization of the bath around the system. Therefore, the joint state of open system and virtual cloud (we call it dressed state) can be accurately represented in a truncated basis of Fock states, on a wide time scale. At the same time, there can be an arbitrarily large number of the observable quanta (which survive up to measurement), especially if the open system is under driving. However, it turns out that the statistics of the bath-measurement outcomes is classical (in a suitable measurement basis): one can employ a Monte Carlo sampling of these outcomes. Therefore, it is possible to efficiently simulate the dynamics of the observable quantum field. In this work we consider the bath measurement with respect to the coherent states, which yields the Husimi function as the positive (quasi)probability distribution of the outcomes. The joint evolution of the dressed state and the corresponding outcome is called the dressed quantum trajectory. The Monte Carlo sampling of these trajectories yields a stochastic simulation method with promising convergence properties on wide time scales.

Mandelbrot and Taylor (1967), Clark (1972) and Allais (1973) have introduced the concept of time deformation to model the subjective view of time held by market participants. In these models, time speeds up when the information flow rises. This information flow can be measured by the interest rate (Allais), the transaction volume (Mandelbrot and Taylor) or the volatility (Clark). These new time scales were shown to fit quite adequately the properties of the time series. Rather than the physical time, they are thus the “natural” or relevant time scale in which to measure the price generating process. More recently, Stock (1988) shows that in such time scale prices could be linearly related to the underlying fundamentals. Stock also demonstrates that this time scale could capture the conditional heteroskedasticity exhibited by the time series. With the changing volume of transactions at the intradaily frequencies, the use of such time scales seems even more relevant (Dacorogna et al., 1993b; Ghysels and Jasiak, 1994).

Thioredoxin is a ubiquitous dithiol oxidoreductase found in many organisms and involved in numerous biochemical processes. Human thioredoxin-like protein (hTRXL) is differentially expressed at different development stages of human fetal cerebrum and belongs to an expanding family of thioredoxins. We have solved the crystal structure of the recombinant N-terminal catalytic domain (hTRXL-N) of hTRXL in its oxidized form at 2.2-A resolution. Although this domain shares a similar three-dimensional structure with human thioredoxin (hTRX), a unique feature of hTRXL-N is the large number of positively charged residues distributed around the active site, which has been implicated in substrate specificity. Furthermore, the hTRXL-N crystal structure is monomeric while hTRX is dimeric in its four crystal structures (reduced, oxidized, C73S and C32S/C35S mutants) reported to date. As dimerization is the key regulatory factor in hTRX, the positive charge and lack of dimer formation of hTRXL-N suggest that it could interact with the acidic amino-acid rich C-terminal region, thereby suggesting a novel regulation mechanism.

Thioredoxin (TRX) is a small multifunctional protein with a redox-active dithiol/disulfide in the conserved active site. Human TRX was originally identified as a cytokine-like factor in virus-transformed cells. The TRX family of proteins share the active site sequence: -Cys-Xxx-Xxx-Cys-. Intracellularly TRX scavenges reactive oxygen species and regulates various signal transduction pathways in cellular activation and apoptosis. TRX is induced and released from cells in response to oxidative stress. TRX levels in plasma or serum are good markers for oxidative stress. Extracellularly, TRX shows anti-inflammatory effects. Circulating TRX in plasma inhibits neutrophil extravasation into the inflammatory sites. More recently we found that TRX inhibits the expression and release of macrophage migration inhibitory factor (MIF), which is a classical proinflammatory cytokine and a member of TRX family. Administration of recombinant TRX protein may become a novel therapeutic strategy for acute inflammatory disorders.

Glutathione transferases are detoxification enzymes that catalyze the addition of glutathione (GSH) to a wide variety of hydrophobic compounds. Although this group of enzymes has been extensively characterized by crystallographic studies, little is known about their dynamic properties. This study investigates the role of protein dynamics in the mechanism of a human class mu enzyme (GSTM2-2) by characterizing the motional properties of the unliganded enzyme, the enzyme-substrate (GSH) complex, an enzyme-product complex [S-(2,4-dinitrobenzyl)glutathione, GSDNB], and an enzyme-inhibitor complex (S-1-hexylglutathione, GSHEX). The kinetic on- and off-rates for these ligands are 10-20-fold lower than the diffusion limit, suggesting dynamic conformational heterogeneity of the active site. The off-rate of GSDNB is similar to the turnover number for its enzymatic formation, suggesting that product release is rate-limiting when 1-chloro-2,4-dinitrobenzene is the substrate. The dynamic properties of GSTM2-2 were investigated over a wide range of time scales using (15)N nuclear spin relaxation, residual dipolar couplings, and amide hydrogen-deuterium exchange rates. These data show that the majority of the protein backbone is rigid on the nanosecond to picosecond time scale for all forms of the enzyme. The presence of motion on the millisecond to microsecond time scale was detected for a small number of residues within the active site. These motions are likely to play a role in facilitating substrate binding and product release. The residual dipolar couplings also show that the conformation of the active site region is more open in solution than in the crystalline environment, further enhancing ligand accessibility to the active site. Amide hydrogen-deuterium exchange rates indicate a reduction in the dynamic properties of several residues near the active site due to the binding of ligand. GSH binding reduces the exchange rate of a number of residues in proximity to its binding site, while GSHEX causes a reduction in amide-exchange rates throughout the entire active site region. The location of the dinitrobenzene (DNB) ring in the GSDNB-GSTM2-2 complex was modeled using chemical shift changes that occur when GSDNB binds to the enzyme. The DNB ring makes a number of contacts with hydrophobic residues in the active site, including Met108. Replacement of Met108 with Ala increases the turnover number of the enzyme by a factor of 1.7.

We study the influence of delayed optical feedback from a short external cavity (EC) on the emission dynamics of vertical-cavity surface-emitting semiconductor lasers (VCSELs). We find the emergence of pulse packages (PP) exhibiting characteristics which originate from the interplay of time-delay induced dynamics and polarization mode (PM) competition. We provide detailed analysis of the polarization dynamics applying complementary cross-correlation and spectral analysis techniques. The analysis reveals an interplay of the dynamics linked to the hierarchy of time scales present in the laser system: the fast time scale of the EC delay, the slower time scale of the PP, and the slow time scale of the polarization mode competition. For an analysis of the dynamics we provide a toolbox of methods adapted to the different relevant time scales and temporal variations of the dynamics. This complementary view unveils distinct changes in the relative oscillation phase of the two PM for increasing the injection current on both the fast and the slow time scales. Our results emphasize the significance of the polarization mode competition for PP dynamics in VCSELs, contrasting the observed behavior to what was reported for edge emitters.

Homodimeric H(+)-pyrophosphatase (H(+)-PPase; EC 3.6.1.1) is a unique enzyme playing a pivotal physiological role in pH homeostasis of organisms. This novel H(+)-PPase supplies energy at the expense of hydrolyzing metabolic byproduct, pyrophosphate (PP(i)), for H(+) translocation across membrane. The functional unit for the translocation is considered to be a homodimer. Its putative active site on each subunit consists of PP(i) binding motif, Acidic I and II motifs, and several essential residues. In this investigation structural mapping of these vital regions was primarily determined utilizing single molecule fluorescence resonance energy transfer. Distances between two C termini and also two N termini on homodimeric subunits of H(+)-PPase are 49.3 + or - 4.0 and 67.2 + or - 5.7 A, respectively. Furthermore, putative PP(i) binding motifs on individual subunits are found to be relatively far away from each other (70.8 + or - 4.8 A), whereas binding of potassium and substrate analogue led them to closer proximity. Moreover, substrate analogue but not potassium elicits significant distance variations between two Acidic I motifs and two His-622 residues on homodimeric subunits. Taken together, this study provides the first quantitative measurements of distances between various essential motifs, residues, and putative active sites on homodimeric subunits of H(+)-PPase. A working model is accordingly proposed elucidating the distance variations of dimeric H(+)-PPase upon substrate binding.

Homing endonucleases, like restriction enzymes, cleave double-stranded DNA at specific target sites. The cleavage mechanism(s) utilized by LAGLIDADG endonucleases have been difficult to elucidate; their active sites are divergent, and only one low resolution cocrystal structure has been determined. Here we report two high resolution structures of the dimeric I-CreI homing endonuclease bound to DNA: a substrate complex with calcium and a product complex with magnesium. The bound metals in both complexes are verified by manganese anomalous difference maps. The active sites are positioned close together to facilitate cleavage across the DNA minor groove; each contains one metal ion bound between a conserved aspartate (Asp 20) and a single scissile phosphate. A third metal ion bridges the two active sites. This divalent cation is bound between aspartate residues from the active site of each subunit and is in simultaneous contact with the scissile phosphates of both DNA strands. A metal-bound water molecule acts as the nucleophile and is part of an extensive network of ordered water molecules that are positioned by enzyme side chains. These structures illustrate a unique variant of a two-metal endonuclease mechanism is employed by the highly divergent LAGLIDADG enzyme family.