Emotions and the Engineering of Adaptiveness in Complex Systems

A major challenge in the engineering of complex and critical systems is the management of change, both in the system and in its operational environment. Due to the growing of complexity in systems, new approaches on autonomy must be able to detect critical changes and avoid their progress towards undesirable states. We are searching for methods to build systems that can tune the adaptability protocols. New mechanisms that use system-wellness requirements to reduce the influence of the outer domain and transfer the control of uncertainly to the inner one. Under the view of cognitive systems, biological emotions suggests a strategy to configure value-based systems to use semantic self-representations of the state. A method inspired by emotion theories to causally connect to the inner domain of the system and its objectives of wellness, focusing on dynamically adapting the system to avoid the progress of critical states. This method shall endow the system with a transversal mechanism to monitor its inner processes, detecting critical states and managing its adaptivity in order to maintain the wellness goals. The paper describes the current vision produced by this work-in-progress.

Uncoupling of transfer of the presequence and unfolding of the mature domain in precursor translocation across the mitochondrial outer membrane

Translocation of mitochondrial precursor proteins across the mitochondrial outer membrane is facilitated by the translocase of the outer membrane (TOM) complex. By using site-specific photocrosslinking, we have mapped interactions between TOM proteins and a mitochondrial precursor protein arrested at two distinct stages, stage A (accumulated at 0°C) and stage B (accumulated at 30°C), in the translocation across the outer membrane at high resolution not achieved previously. Although the stage A and stage B intermediates were assigned previously to the forms bound to the cis site and the trans site of the TOM complex, respectively, the results of crosslinking indicate that the presequence of the intermediates at both stage A and stage B is already on the trans side of the outer membrane. The mature domain is unfolded and bound to Tom40 at stage B whereas it remains folded at stage A. After dissociation from the TOM complex, translocation of the stage B intermediate, but not of the stage A intermediate, across the inner membrane was promoted by the intermembrane-space domain of Tom22. We propose a new model for protein translocation across the outer membrane, where translocation of the presequence and unfolding of the mature domain are not necessarily coupled.

Single-pair fluorescence resonance energy transfer on freely diffusing molecules: Observation of Förster distance dependence and subpopulations

Photon bursts from single diffusing donor-acceptor labeled macromolecules were used to measure intramolecular distances and identify subpopulations of freely diffusing macromolecules in a heterogeneous ensemble. By using DNA as a rigid spacer, a series of constructs with varying intramolecular donor-acceptor spacings were used to measure the mean and distribution width of fluorescence resonance energy transfer (FRET) efficiencies as a function of distance. The mean single-pair FRET efficiencies qualitatively follow the distance dependence predicted by Förster theory. Possible contributions to the widths of the FRET efficiency distributions are discussed, and potential applications in the study of biopolymer conformational dynamics are suggested. The ability to measure intramolecular (and intermolecular) distances for single molecules implies the ability to distinguish and monitor subpopulations of molecules in a mixture with different distances or conformational states. This is demonstrated by monitoring substrate and product subpopulations before and after a restriction endonuclease cleavage reaction. Distance measurements at single-molecule resolution also should facilitate the study of complex reactions such as biopolymer folding. To this end, the denaturation of a DNA hairpin was examined by using single-pair FRET.

Horizontal gene transfer among genomes: The complexity hypothesis

Increasingly, studies of genes and genomes are indicating that considerable horizontal transfer has occurred between prokaryotes. Extensive horizontal transfer has occurred for operational genes (those involved in housekeeping), whereas informational genes (those involved in transcription, translation, and related processes) are seldomly horizontally transferred. Through phylogenetic analysis of six complete prokaryotic genomes and the identification of 312 sets of orthologous genes present in all six genomes, we tested two theories describing the temporal flow of horizontal transfer. We show that operational genes have been horizontally transferred continuously since the divergence of the prokaryotes, rather than having been exchanged in one, or a few, massive events that occurred early in the evolution of prokaryotes. In agreement with earlier studies, we found that differences in rates of evolution between operational and informational genes are minimal, suggesting that factors other than rate of evolution are responsible for the observed differences in horizontal transfer. We propose that a major factor in the more frequent horizontal transfer of operational genes is that informational genes are typically members of large, complex systems, whereas operational genes are not, thereby making horizontal transfer of informational gene products less probable (the complexity hypothesis).

NADH-quinone oxidoreductase: PSST subunit couples electron transfer from iron–sulfur cluster N2 to quinone

The proton-translocating NADH-quinone oxidoreductase (EC 1.6.99.3) is the largest and least understood enzyme complex of the respiratory chain. The mammalian mitochondrial enzyme (also called complex I) contains more than 40 subunits, whereas its structurally simpler bacterial counterpart (NDH-1) in Paracoccus denitrificans and Thermus thermophilus HB-8 consists of 14 subunits. A major unsolved question is the location and mechanism of the terminal electron transfer step from iron–sulfur cluster N2 to quinone. Potent inhibitors acting at this key region are candidate photoaffinity probes to dissect NADH-quinone oxidoreductases. Complex I and NDH-1 are very sensitive to inhibition by a variety of structurally diverse toxicants, including rotenone, piericidin A, bullatacin, and pyridaben. We designed (trifluoromethyl)diazirinyl[3H]pyridaben ([3H]TDP) as our photoaffinity ligand because it combines outstanding inhibitor potency, a suitable photoreactive group, and tritium at high specific activity. Photoaffinity labeling of mitochondrial electron transport particles was specific and saturable. Isolation, protein sequencing, and immunoprecipitation identified the high-affinity specifically labeled 23-kDa subunit as PSST of complex I. Immunoprecipitation of labeled membranes of P. denitrificans and T. thermophilus established photoaffinity labeling of the equivalent bacterial NQO6. Competitive binding and enzyme inhibition studies showed that photoaffinity labeling of the specific high-affinity binding site of PSST is exceptionally sensitive to each of the high-potency inhibitors mentioned above. These findings establish that the homologous PSST of mitochondria and NQO6 of bacteria have a conserved inhibitor-binding site and that this subunit plays a key role in electron transfer by functionally coupling iron–sulfur cluster N2 to quinone.

Binding of RNA template to a complex of HIV-1 reverse transcriptase/primer/template

HIV-1 reverse transcriptase (RT) catalyzes the synthesis of DNA from DNA or RNA templates. During this process, it must transfer its primer from one template to another RNA or DNA template. Binary complexes made of RT and a primer/template bind an additional single-stranded RNA molecule of the same nucleotide sequence as that of the DNA or RNA template. The additional RNA strand leads to a 10-fold decrease of the off-rate constant, koff, of RT from a primer/DNA template. In a binary complex of RT and a primer/template, the primer can be cross-linked to both the p66 and p51 subunits. Depending on the location of the photoreactive group in the primer, the distribution of the cross-linked primers between subunits is dependent on the nature of the template and of the additional single-stranded molecule. Greater cross-linking of the primer to p51 occurs with DNA templates, whereas cross-linking to p66 predominates with RNA templates. Excess single-stranded DNA shifts the distribution of cross-linking from p66 to p51 with RNA templates, and excess single-stranded RNA shifts the cross-linking from p51 to p66 with DNA templates. RT thus uses two primer/template binding modes depending on the nature of the template.

Preassembly of interleukin 2 (IL-2) receptor subunits on resting Kit 225 K6 T cells and their modulation by IL-2, IL-7, and IL-15: A fluorescence resonance energy transfer study

Assembly and mutual proximities of α, β, and γc subunits of the interleukin 2 receptors (IL-2R) in plasma membranes of Kit 225 K6 T lymphoma cells were investigated by fluorescence resonance energy transfer (FRET) using fluorescein isothiocyanate- and Cy3-conjugated monoclonal antibodies (mAbs) that were directed against the IL-2Rα, IL-2Rβ, and γc subunits of IL-2R. The cell-surface distribution of subunits was analyzed at the nanometer scale (2–10 nm) by FRET on a cell-by-cell basis. The cells were probed in resting phase and after coculture with saturating concentrations of IL-2, IL-7, and IL-15. FRET data from donor- and acceptor-labeled IL-2Rβ-α, γ-α, and γ-β pairs demonstrated close proximity of all subunits to each other in the plasma membrane of resting T cells. These mutual proximities do not appear to represent mAb-induced microaggregation, because FRET measurements with Fab fragments of the mAbs gave similar results. The relative proximities were meaningfully modulated by binding of IL-2, IL-7, and IL-15. Based on FRET analysis the topology of the three subunits at the surface of resting cells can be best described by a “triangular model” in the absence of added interleukins. IL-2 strengthens the bridges between the subunits, making the triangle more compact. IL-7 and IL-15 act in the opposite direction by opening the triangle possibly because they associate their private specific α receptors with the β and/or γc subunits of the IL-2R complex. These data suggest that IL-2R subunits are already colocalized in resting T cells and do not require cytokine-induced redistribution. This colocalization is significantly modulated by binding of relevant interleukins in a cytokine-specific manner.

Transgenic cattle produced by reverse-transcribed gene transfer in oocytes

A critical requirement for integration of retroviruses, other than HIV and possibly related lentiviruses, is the breakdown of the nuclear envelope during mitosis. Nuclear envelope breakdown occurs during mitotic M-phase, the envelope reforming immediately after cell division, thereby permitting the translocation of the retroviral preintegration complex into the nucleus and enabling integration to proceed. In the oocyte, during metaphase II (MII) of the second meiosis, the nuclear envelope is also absent and the oocyte remains in MII arrest for a much longer period of time compared with M-phase in a somatic cell. Pseudotyped replication-defective retroviral vector was injected into the perivitelline space of bovine oocytes during MII. We show that reverse-transcribed gene transfer can take place in an oocyte in MII arrest of meiosis, leading to production of offspring, the majority of which are transgenic. We discuss the implications of this mechanism both as a means of production of transgenic livestock and as a model for naturally occurring recursive transgenesis.

Gene Conversion of Major Histocompatibility Complex Genes in the Mouse Spermatogenesis is a Premeiotic Event

The molecular genetic mechanism of gene conversion in higher eukaryotes remains unknown. We find it of considerable interest to determine when during spermatogenesis gene conversion occurs. We have therefore purified pachytene spermatocytes and haploid spermatocytes from adult mice and analyzed these fractions for the presence of gene conversion products resulting from the transfer between the major histocompatibility complex class II genes Ebd and Abk in a polymerase chain reaction assay. We have further isolated spermatogenic cells from prepubescent mice and analyzed them for the presence of the same gene conversion products. We can detect gene conversion products in testis cells as early as in 8-d-old mice where the only existing spermatogenic cells are spermatogonia. The frequency of gene conversion products remains the same as the cells reach meiosis in 18-d-old mice, and is unchanged after meiosis is completed in haploid spermatocytes. Gene conversion of this specific fragment therefore appears to be a premeiotic event and, consequently, relies on genetic mechanisms other than normal meiotic recombination.

Femtosecond dynamics of DNA-mediated electron transfer

Diverse biophysical and biochemical studies have sought to understand electron transfer (ET) in DNA in part because of its importance to DNA damage and its repair. However, the dynamics and mechanisms of the elementary processes of ET in this medium are not fully understood and have been heavily debated. Two fundamental issues are the distance over which charge is transported and the time-scale on which the transport through the π-stack of the DNA base pairs may occur. With femtosecond resolution, we report direct observation in DNA of ultrafast ET, initiated by excitation of tethered ethidium (E), the intercalated electron acceptor (A); the electron donor (D) is 7-deazaguanine (Z), a modified base, placed at different, fixed distances from A. The ultrafast ET between these reactants in DNA has been observed with time constants of 5 ps and 75 ps and was found to be essentially independent of the D–A separation (10–17 Å). However, the ET efficiency does depend on the D–A distance. The 5-ps decay corresponds to direct ET observed from 7-deazaguanine but not guanine to E. From measurements of orientation anisotropies, we conclude that the slower 75-ps process requires the reorientation of E before ET, similar to E/nucleotide complexes in water. These results reveal the nature of ultrafast ET and its mechanism: in DNA, ET cannot be described as in proteins simply by a phenomenological parameter, β. Instead, the involvement of the base pairs controls the time scale and the degree of coherent transport.

Identification of the proton pathway in bacterial reaction centers: Inhibition of proton transfer by binding of Zn2+ or Cd2+

The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the light induced two-electron, two-proton reduction of a bound quinone molecule QB (the secondary quinone acceptor). A unique pathway for proton transfer to the QB site had so far not been determined. To study the molecular basis for proton transfer, we investigated the effects of exogenous metal ion binding on the kinetics of the proton-assisted electron transfer kAB(2) (QA−•QB−• + H+ → QA(QBH)−, where QA is the primary quinone acceptor). Zn2+ and Cd2+ bound stoichiometrically to the RC (KD ≤ 0.5 μM) and reduced the observed value of kAB(2) 10-fold and 20-fold (pH 8.0), respectively. The bound metal changed the mechanism of the kAB(2) reaction. In native RCs, kAB(2) was previously shown to be rate-limited by electron transfer based on the dependence of kAB(2) on the driving force for electron transfer. Upon addition of Zn2+ or Cd2+, kAB(2) became approximately independent of the electron driving force, implying that the rate of proton transfer was reduced (≥ 102-fold) and has become the rate-limiting step. The lack of an effect of the metal binding on the charge recombination reaction D+•QAQB−• → DQAQB suggests that the binding site is located far (>10 Å) from QB. This hypothesis is confirmed by preliminary x-ray structure analysis. The large change in the rate of proton transfer caused by the stoichiometric binding of the metal ion shows that there is one dominant site of proton entry into the RC from which proton transfer to QB−• occurs.

The natural killer gene complex genetic locus Chok encodes Ly-49D, a target recognition receptor that activates natural killing

Previously, we established that natural killer (NK) cells from C57BL/6 (B6), but not BALB/c, mice lysed Chinese hamster ovary (CHO) cells, and we mapped the locus that determines this differential CHO-killing capacity to the NK gene complex on chromosome 6. The localization of Chok in the NK gene complex suggested that it may encode either an activating or an inhibitory receptor. Here, results from a lectin-facilitated lysis assay predicted that Chok is an activating B6 NK receptor. Therefore, we immunized BALB/c mice with NK cells from BALB.B6–Cmv1r congenic mice and generated a mAb, designated 4E4, that blocked B6-mediated CHO lysis. mAb 4E4 also redirected lysis of Daudi targets, indicating its reactivity with an activating NK cell receptor. Furthermore, only the 4E4+ B6 NK cell subset mediated CHO killing, and this lysis was abrogated by preincubation with mAb 4E4. Flow cytometric analysis indicated that mAb 4E4 specifically reacts with Ly-49D but not Ly-49A, B, C, E, G, H, or I transfectants. Finally, gene transfer of Ly-49DB6 into BALB/c NK cells conferred cytotoxic capacity against CHO cells, thus establishing that the Ly-49D receptor is sufficient to activate NK cells to lyse this target. Hence, Ly-49D is the Chok gene product and is a mouse NK cell receptor capable of directly triggering natural killing.

Unraveling the mechanism of the lactose permease of Escherichia coli

We studied the effect of pH on ligand binding in wild-type lactose permease or mutants in the four residues—Glu-269, Arg-302, His-322, and Glu-325—that are the key participants in H+ translocation and coupling between sugar and H+ translocation. Although wild-type permease or mutants in Glu-325 and Arg-302 exhibit marked decreases in affinity at alkaline pH, mutants in either His-322 or Glu-269 do not titrate. The results offer a mechanistic model for lactose/H+ symport. In the ground state, the permease is protonated, the H+ is shared between His-322 and Glu-269, Glu-325 is charge-paired with Arg-302, and substrate is bound with high affinity at the outside surface. Substrate binding induces a conformational change that leads to transfer of the H+ from His-322/Glu-269 to Glu-325 and reorientation of the binding site to the inner surface with a decrease in affinity. Glu-325 then is deprotonated on the inside because of rejuxtaposition with Arg-302. The His-322/Glu-269 complex then is reprotonated from the outside surface to reinitiate the cycle.

Dynamical principles in biological processes: A model of charge migration in proteins and DNA

The generalized master equations (GMEs) that contain multiple time scales have been derived quantum mechanically. The GME method has then been applied to a model of charge migration in proteins that invokes the hole hopping between local amino acid sites driven by the torsional motions of the floppy backbones. This model is then applied to analyze the experimental results for sequence-dependent long-range hole transport in DNA reported by Meggers et al. [Meggers, E., Michel-Beyerle, M. E., & Giese, B. (1998) J. Am. Chem. Soc. 120, 12950–12955]. The model has also been applied to analyze the experimental results of femtosecond dynamics of DNA-mediated electron transfer reported by Zewail and co-workers [Wan, C., Fiebig, T., Kelley, S. O., Treadway, C. R., Barton, J. K. & Zewail, A. H. (1999) Proc. Natl. Acad. Sci. USA 96, 6014–6019]. The initial events in the dynamics of protein folding have begun to attract attention. The GME obtained in this paper will be applicable to this problem.

Conformational changes between the active-site and regulatory light chain of myosin as determined by luminescence resonance energy transfer: The effect of nucleotides and actin

Myosin is thought to generate movement of actin filaments via a conformational change between its light-chain domain and its catalytic domain that is driven by the binding of nucleotides and actin. To monitor this change, we have measured distances between a gizzard regulatory light chain (Cys 108) and the active site (near or at Trp 130) of skeletal myosin subfragment 1 (S1) by using luminescence resonance energy transfer and a photoaffinity ATP-lanthanide analog. The technique allows relatively long distances to be measured, and the label enables site-specific attachment at the active-site with only modest affect on myosin’s enzymology. The distance between these sites is 66.8 ± 2.3 Å when the nucleotide is ADP and is unchanged on binding to actin. The distance decreases slightly with ADP-BeF3, (−1.6 ± 0.3 Å) and more significantly with ADP-AlF4 (−4.6 ± 0.2 Å). During steady-state hydrolysis of ATP, the distance is temperature-dependent, becoming shorter as temperature increases and the complex with ADP⋅Pi is favored over that with ATP. We conclude that the distance between the active site and the light chain varies as Acto-S1-ADP ≈ S1-ADP > S1-ADP-BeF3 > S1-ADP-AlF4 ≈ S1-ADP-Pi and that S1-ATP > S1-ADP-Pi. The changes in distance are consistent with a substantial rotation of the light-chain binding domain of skeletal S1 between the prepowerstroke state, simulated by S1-ADP-AlF4, and the post-powerstroke state, simulated by acto-S1-ADP.