Asymptotic and computational analysis of mistuning effects on the dynamics of bladed disks

Es bien conocido que las pequeñas imperfecciones existentes en los álabes de un rótor de turbomaquinaria (conocidas como “mistuning”) pueden causar un aumento considerable de la amplitud de vibración de la respuesta forzada y, por el contrario, tienen típicamente un efecto beneficioso en el flameo del rótor. Para entender estos efectos se pueden llevar a cabo estudios numéricos del problema aeroelástico completo. Sin embargo, el cálculo de “mistuning” usando modelos de alta resolución es una tarea difícil de realizar, ya que los modelos necesarios para describir de manera precisa el componente de turbomáquina (por ejemplo rotor) tienen, necesariamente, un número muy elevado de grados de libertad, y, además, es necesario hacer un estudio estadístico para poder explorar apropiadamente las distribuciones posibles de “mistuning”, que tienen una naturaleza aleatoria. Diferentes modelos de orden reducido han sido desarrollados en los últimos años para superar este inconveniente. Uno de estos modelos, llamado “Asymptotic Mistuning Model (AMM)”, se deriva de la formulación completa usando técnicas de perturbaciones que se basan en que el “mistuning” es pequeño. El AMM retiene sólo los modos relevantes para describir el efecto del mistuning, y permite identificar los mecanismos clave involucrados en la amplificación de la respuesta forzada y en la estabilización del flameo. En este trabajo, el AMM se usa para estudiar el efecto del “mistuning” de la estructura y de la amortiguación sobre la amplitud de la respuesta forzada. Los resultados obtenidos son validados usando modelos simplificados del rotor y también otros de alta definición. Además, en el marco del proyecto europeo FP7 "Flutter-Free Turbomachinery Blades (FUTURE)", el AMM se aplica para diseñar distribuciones de “mistuning” intencional: (i) una que anula y (ii) otra que reduce a la mitad la amplitud del flameo de un rotor inestable; y las distribuciones obtenidas se validan experimentalmente. Por último, la capacidad de AMM para predecir el comportamiento de flameo de rotores con “mistuning” se comprueba usando resultados de CFD detallados. Abstract It is well known that the small imperfections of the individual blades in a turbomachinery rotor (known as “mistuning”) can cause a substantial increase of the forced response vibration amplitude, and it also typically results in an improvement of the flutter vibration characteristics of the rotor. The understanding of these phenomena can be attempted just by performing numerical simulations of the complete aeroelastic problem. However, the computation of mistuning cases using high fidelity models is a formidable task, because a detailed model of the whole rotor has to be considered, and a statistical study has to be carried out in order to properly explore the effect of the random mistuning distributions. Many reduced order models have been developed in recent years to overcome this barrier. One of these models, called the Asymptotic Mistuning Model (AMM), is systematically derived from the complete bladed disk formulation using a consistent perturbative procedure that exploits the smallness of mistuning to simplify the problem. The AMM retains only the essential system modes that are involved in the mistuning effect, and it allows to identify the key mechanisms of the amplification of the forced response amplitude and the flutter stabilization. In this work, AMM methodolgy is used to study the effect of structural and damping mistuning on the forced response vibration amplitude. The obtained results are verified using a one degree of freedom model of a rotor, and also high fidelity models of the complete rotor. The AMM is also applied, in the frame of the European FP7 project “Flutter-Free Turbomachinery Blades (FUTURE)”, to design two intentional mistuning patterns: (i) one to complete stabilize an unstable rotor, and (ii) other to approximately reduce by half its flutter amplitude. The designed patterns are validated experimentally. Finally, the ability of AMM to predict the flutter behavior of mistuned rotors is checked against numerical, high fidelity CFD results.

Crystal structure of Taq DNA polymerase in complex with an inhibitory Fab: The Fab is directed against an intermediate in the helix-coil dynamics of the enzyme

We report the crystal structure of Thermus aquaticus DNA polymerase I in complex with an inhibitory Fab, TP7, directed against the native enzyme. Some of the residues present in a helical conformation in the native enzyme have adopted a γ turn conformation in the complex. Taken together, structural information that describes alteration of helical structure and solution studies that demonstrate the ability of TP7 to inhibit 100% of the polymerase activity of the enzyme suggest that the change in conformation is probably caused by trapping of an intermediate in the helix-coil dynamics of this helix by the Fab. Antibodies directed against modified helices in proteins have long been anticipated. The present structure provides direct crystallographic evidence. The Fab binds within the DNA binding cleft of the polymerase domain, interacting with several residues that are used by the enzyme in binding the primer:template complex. This result unequivocally corroborates inferences drawn from binding experiments and modeling calculations that the inhibitory activity of this Fab is directly attributable to its interference with DNA binding by the polymerase domain of the enzyme. The combination of interactions made by the Fab residues in both the polymerase and the vestigial editing nuclease domain of the enzyme reveal the structural basis of its preference for binding to DNA polymerases of the Thermus species. The orientation of the structure-specific nuclease domain with respect to the polymerase domain is significantly different from that seen in other structures of this polymerase. This reorientation does not appear to be antibody-induced and implies remarkably high relative mobility between these two domains.

The use of site-directed fluorophore labeling and donor–donor energy migration to investigate solution structure and dynamics in proteins

The use of molecular genetics for introducing fluorescent molecules enables the use of donor–donor energy migration to determine intramolecular distances in a variety of proteins. This approach can be applied to examine the overall molecular dimensions of proteins and to investigate structural changes upon interactions with specific target molecules. In this report, the donor–donor energy migration method is demonstrated by experiments with the latent form of plasminogen activator inhibitor type 1. Based on the known x-ray structure of plasminogen activator inhibitor type 1, three positions forming the corners of a triangle were chosen. Double Cys substitution mutants (V106C-H185C, H185C-M266C, and M266C-V106C) and corresponding single substitution mutants (V106C, H185C, and M266C) were created and labeled with a sulfhydryl specific derivative of BODIPY (=the D molecule). The side lengths of this triangle were obtained from analyses of the experimental data. The analyses account for the local anisotropic order and rotational motions of the D molecules, as well as for the influence of a partial DD-labeling. The distances, as determined from x-ray diffraction, between the Cα-atoms of the positions V106C–H185C, H185C–M266C, and M266C–V106C were 60.9, 30.8, and 55.1 Å, respectively. These are in good agreement with the distances of 54 ± 4, 38 ± 3, and 55 ± 3 Å, as determined between the BODIPY groups attached via linkers to the same residues. Although the positions of the D-molecules and the Cα-atoms physically cannot coincide, there is a reasonable agreement between the methods.

Identification of two distinct human SMC protein complexes involved in mitotic chromosome dynamics

The structural maintenance of chromosomes (SMC) family member proteins previously were shown to play a critical role in mitotic chromosome condensation and segregation in yeast and Xenopus. Other family members were demonstrated to be required for DNA repair in yeast and mammals. Although several different SMC proteins were identified in different organisms, little is known about the SMC proteins in humans. Here, we report the identification of four human SMC proteins that form two distinct heterodimeric complexes in the cell, the human chromosome-associated protein (hCAP)-C and hCAP-E protein complex (hCAP-C/hCAP-E), and the human SMC1 (hSMC1) and hSMC3 protein complex (hSMC1/hSMC3). The hCAP-C/hCAP-E complex is the human ortholog of the Xenopus chromosome-associated protein (XCAP)-C/XCAP-E complex required for mitotic chromosome condensation. We found that a second complex, hSMC1/hSMC3, is required for metaphase progression in mitotic cells. Punctate vs. diffuse distribution patterns of the hCAP-C/hCAP-E and hSMC1/hSMC3 complexes in the interphase nucleus indicate independent behaviors of the two complexes during the cell cycle. These results suggest that two distinct classes of SMC protein complexes are involved in different aspects of mitotic chromosome organization in human cells.

Dynamics and orientation of N+(CD3)3-bromoacetylcholine bound to its binding site on the nicotinic acetylcholine receptor

Dynamic and structural information has been obtained for an analogue of acetylcholine while bound to the agonist binding site on the nicotinic acetylcholine receptor (nAcChoR), using wide-line deuterium solid-state NMR. Analysis of the deuterium lineshape obtained at various temperatures from unoriented nAcChoR membranes labeled with deuterated bromoacetylcholine (BAC) showed that the quaternary ammonium group of the ligand is well constrained within the agonist binding site when compared with the dynamics observed in the crystalline solids. This motional restriction would suggest that a high degree of complementarity exists between the quaternary ammonium group of the ligand and the protein within the agonist binding site. nAcChoR membranes were uniaxially oriented by isopotential centrifugation as determined by phosphorous NMR of the membrane phospholipids. Analysis of the deuterium NMR lineshape of these oriented membranes enriched with the nAcChoR labeled with N+(CD3)3-BAC has enabled us to determine that the angle formed between the quaternary ammonium group of the BAC and the membrane normal is 42° in the desensitized form of the receptor. This measurement allows us to orient in part the bound ligand within the proposed receptor binding site.

The role of structure, energy landscape, dynamics, and allostery in the enzymatic function of myoglobin

The grail of protein science is the connection between structure and function. For myoglobin (Mb) this goal is close. Described as only a passive dioxygen storage protein in texts, we argue here that Mb is actually an allosteric enzyme that can catalyze reactions among small molecules. Studies of the structural, spectroscopic, and kinetic properties of Mb lead to a model that relates structure, energy landscape, dynamics, and function. Mb functions as a miniature chemical reactor, concentrating and orienting diatomic molecules such as NO, CO, O2, and H2O2 in highly conserved internal cavities. Reactions can be controlled because Mb exists in distinct taxonomic substates with different catalytic properties and connectivities of internal cavities.

Induced fit DNA recognition by a minor groove binding analogue of Hoechst 33258: fluctuations in DNA A tract structure investigated by NMR and molecular dynamics simulations

NMR analysis and molecular dynamics simulations of d(GGTAATTACC)2 and its complex with a tetrahydropyrimidinium analogue of Hoechst 33258 suggest that DNA minor groove recognition in solution involves a combination of conformational selection and induced fit, rather than binding to a preorganised site. Analysis of structural fluctuations in the bound and unbound states suggests that the degree of induced fit observed is primarily a consequence of optimising van der Waals contacts with the walls of the minor groove resulting in groove narrowing through: (i) changes in base step parameters, including increased helical twist and propeller twist; (ii) changes to the sugar–phosphate backbone conformation to engulf the bound ligand; (iii) suppression of bending modes at the TpA steps. In contrast, the geometrical arrangement of hydrogen bond acceptors on the groove floor appears to be relatively insensitive to DNA conformation (helical twist and propeller twist). We suggest that effective recognition of DNA sequences (in this case an A tract structure) appears to depend to a significant extent on the sequence being flexible enough to be able to adopt the geometrically optimal conformation compatible with the various binding interactions, rather than involving ‘lock and key’ recognition.

Mantle dynamics and seismic tomography

Three-dimensional imaging of the Earth's interior, called seismic tomography, has achieved breakthrough advances in the last two decades, revealing fundamental geodynamical processes throughout the Earth's mantle and core. Convective circulation of the entire mantle is taking place, with subducted oceanic lithosphere sinking into the lower mantle, overcoming the resistance to penetration provided by the phase boundary near 650-km depth that separates the upper and lower mantle. The boundary layer at the base of the mantle has been revealed to have complex structure, involving local stratification, extensive structural anisotropy, and massive regions of partial melt. The Earth's high Rayleigh number convective regime now is recognized to be much more interesting and complex than suggested by textbook cartoons, and continued advances in seismic tomography, geodynamical modeling, and high-pressure–high-temperature mineral physics will be needed to fully quantify the complex dynamics of our planet's interior.

Symmetry, stability, and dynamics of multidomain and multicomponent protein systems

Symmetry is commonly observed in many biological systems. Here we discuss representative examples of the role of symmetry in structural molecular biology. Point group symmetries are observed in many protein oligomers whose three-dimensional atomic structures have been elucidated by x-ray crystallography. Approximate symmetry also occurs in multidomain proteins. Symmetry often confers stability on the molecular system and results in economical usage of basic components to build the macromolecular structure. Symmetry is also associated with cooperativity. Mild perturbation from perfect symmetry may be essential in some systems for dynamic functions.

Intrinsic compressibility and volume compression in solvated proteins by molecular dynamics simulation at high pressure.

Constant pressure and temperature molecular dynamics techniques have been employed to investigate the changes in structure and volumes of two globular proteins, superoxide dismutase and lysozyme, under pressure. Compression (the relative changes in the proteins' volumes), computed with the Voronoi technique, is closely related with the so-called protein intrinsic compressibility, estimated by sound velocity measurements. In particular, compression computed with Voronoi volumes predicts, in agreement with experimental estimates, a negative bound water contribution to the apparent protein compression. While the use of van der Waals and molecular volumes underestimates the intrinsic compressibilities of proteins, Voronoi volumes produce results closer to experimental estimates. Remarkably, for two globular proteins of very different secondary structures, we compute identical (within statistical error) protein intrinsic compressions, as predicted by recent experimental studies. Changes in the protein interatomic distances under compression are also investigated. It is found that, on average, short distances compress less than longer ones. This nonuniform contraction underlines the peculiar nature of the structural changes due to pressure in contrast with temperature effects, which instead produce spatially uniform changes in proteins. The structural effects observed in the simulations at high pressure can explain protein compressibility measurements carried out by fluorimetric and hole burning techniques. Finally, the calculation of the proteins static structure factor shows significant shifts in the peaks at short wavenumber as pressure changes. These effects might provide an alternative way to obtain information concerning compressibilities of selected protein regions.

Ultra-fast excited state dynamics in green fluorescent protein: multiple states and proton transfer.

The green fluorescent protein (GFP) of the jellyfish Aequorea Victoria has attracted widespread interest since the discovery that its chromophore is generated by the autocatalytic, posttranslational cyclization and oxidation of a hexapeptide unit. This permits fusion of the DNA sequence of GFP with that of any protein whose expression or transport can then be readily monitored by sensitive fluorescence methods without the need to add exogenous fluorescent dyes. The excited state dynamics of GFP were studied following photo-excitation of each of its two strong absorption bands in the visible using fluorescence upconversion spectroscopy (about 100 fs time resolution). It is shown that excitation of the higher energy feature leads very rapidly to a form of the lower energy species, and that the excited state interconversion rate can be markedly slowed by replacing exchangeable protons with deuterons. This observation and others lead to a model in which the two visible absorption bands correspond to GFP in two ground-state conformations. These conformations can be slowly interconverted in the ground state, but the process is much faster in the excited state. The observed isotope effect suggests that the initial excited state process involves a proton transfer reaction that is followed by additional structural changes. These observations may help to rationalize and motivate mutations that alter the absorption properties and improve the photo stability of GFP.

A structural basis for a phosphoramide mustard-induced DNA interstrand cross-link at 5'-d(GAC).

Phosphoramide mustard-induced DNA interstrand cross-links were studied both in vitro and by computer simulation. The local determinants for the formation of phosphoramide mustard-induced DNA interstrand cross-links were defined by using different pairs of synthetic oligonucleotide duplexes, each of which contained a single potentially cross-linkable site. Phosphoramide mustard was found to cross-link dG to dG at a 5'-d(GAC)-3'. The structural basis for the formation of this 1,3 cross-link was studied by molecular dynamics and quantum chemistry. Molecular dynamics indicated that the geometrical proximity of the binding sites also favored a 1,3 dG-to-dG linkage over a 1,2 dG-to-dG linkage in a 5'-d(GCC)-3' sequence. While the enthalpies of 1,2 and 1,3 mustard cross-linked DNA were found to be very close, a 1,3 structure was more flexible and may therefore be in a considerably higher entropic state.