Buried Cavities Research Articles

Design of Ligand Binding to an Engineered Protein Cavity Using Virtual Screening and Thermal Up-shift Evaluation

Proteins could be used to carry and deliver small compounds. As a tool for designing ligand binding sites in protein cores, a three-step virtual screening method is presented that has been optimised using existing data on T4 lysozyme complexes and tested in a newly engineered cavity in flavodoxin. The method can pinpoint, in large databases, ligands of specific protein cavities. In the first step, physico-chemical filters are used to screen the library and discard a majority of compounds. In the second step, a flexible, fast docking procedure is used to score and select a smaller number of compounds as potential binders. In the third step, a finer method is used to dock promising molecules of the hit list into the protein cavity, and an optimised free energy function allows discarding the few false positives by calculating the affinity of the modelled complexes. To demonstrate the portability of the method, several cavities have been designed and engineered in the flavodoxin from Anabaena PCC 7119, and the W66F/L44A double mutant has been selected as a suitable host protein. The NCI database has then been screened for potential binders, and the binding to the engineered cavity of five promising compounds and three tentative non-binders has been experimentally tested by thermal up-shift assays and spectroscopic titrations. The five tentative binders (some apolar and some polar), unlike the three tentative non-binders, are shown to bind to the host mutant and, importantly, not to bind to the wild type protein. The three-step virtual screening method developed can thus be used to identify ligands of buried protein cavities. We anticipate that the method could also be used, in a reverse manner, to identify natural or engineerable protein cavities for the hosting of ligands of interest.

Progress in the Integration of planar and 3D Coils on LTCC by using photoimageable Inks

This paper presents some results of our investigations in the integration of coils in LTCC using the Fodel® Technology as line patterning technology. Planar as well as 3-D inductors were simulated, manufactured, measured and modeled. The structures were manufactured with different shapes, a line width from 30 μm to 70 μm, a space between turns from 100 μm and 200 μm and diameters of inner turns from 0.5 mm to 2 mm. The smallest and most critical inductors (0.5 mm diameter) were manufactured with vias, whose diameter were about 60 μm. The manufacturing of smaller coils was possible due to the increased resolution of Fodel® thick-film inks also in inner layers in combination with micro vias, which were produced with a multifunctional Laser. Combining these technologies with buried cavities and a symmetric turn arrangement we obtained inductors that can be used in a wider range of frequencies (up to 10 GHz).

Stabilization of Bacillus subtilis Lipase A by increasing the residual packing

Introduction of well-packed residues to the interior of a protein structure could be considered as a stabilization strategy since the reduction of buried cavities might stabilize protein structure. In this study, the less-packed residues with no water-contact were selected as target sites for increasing residual packing. When Lipase A from Bacillus subtilis (179 amino acids) was used as a model system, 43 less-packed residues were initially considered by analyzing their residual packing value and residual exposure ratio. Among the 43 residues, small amino acids such as GLY and ALA were chosen as target sites. Packing increases of ALA to VAL and GLY to ALA were estimated, by molecular modeling, to give 0.5368∼0.7433 kcal mol−1 stabilization. Mutants of Lipase A such as A38V, A75V, G80A, A105V A146V, and G172A were obtained via protein engineering. Thermostability assays revealed that A38V, G80A and G172V were the most stable mutants. This procedure for selecting the target residues for improved thermostability of Lipase A could be applied for improving the thermostability of other proteins and enzymes.

Transmission electron microscopy study of blisters in high-temperature annealed He and H co-implanted single-crystal silicon

Transmission electron microscopy has allowed us to draw the three-dimensional structure of blisters formed after high-temperature annealing of He-H co-implanted silicon by combining the unique capability of site-selective cross sectioning of the focused ion beam with conventional plan view images. It has been shown that blisters are formed by crystalline lamellae strongly bended, plastically deformed, and suspended over buried empty cavities. The volume of the protruding blister surface is almost equal to the buried empty volumes, thus suggesting a mechanism for blister formation based on H and He precipitation and migration of silicon atoms toward the surface.

Factors governing the protonation state of Zn-bound histidine in proteins: a DFT/CDM study.

We have performed systematic theoretical studies to elucidate the factors governing the His protonation/deprotonation state in Zn-binding sites, especially those containing the ubiquitous Zn-His-Asp/Glu triad. Specifically, we have addressed the following three questions: (1) How does the transfer of the Zn-bound His imidazole proton to the second-shell Asp/Glu carboxylate oxygen depend on the composition of the other first-shell ligands and the solvent accessibility of the metal-binding site? (2) Can any second-shell ligand with a proton acceptor group such as the backbone carbonyl oxygen also act as a proton acceptor? (3) What is the effect of the Asp/Glu in the Zn-His-Asp/Glu triad on the Zn-bound water protonation state? To address these questions, we used a combination of quantum mechanical and continuum dielectric methods to compute the free energies for deprotonating a Zn-bound imidazole/water in various Zn complexes. The calculations show that whether the Zn-bound His is protonated or deprotonated depends on (1) the solvent accessibility of the metal-binding site, and (2) the Lewis acid ability of Zn, which is indirectly determined by both the first- and the second-shell Zn ligands. The calculations also show that the effect of the Zn-His-Asp/Glu interaction on the nucleophilicity of the Zn-bound water depends on the solvent accessibility of the catalytic Zn site. Furthermore, they show that the Asp/Glu side chain in the Zn-His-Asp/Glu triad can increase the negative charge of its partner, His, and create an anionic hole that may stabilize a cation in buried cavities, provided that the Zn complex is cationic/neutral. The findings of this work are in accord with available experimental data.

Fabrication of SOI structures with buried cavities using Si wafer direct bonding and electrochemical etch-stop

This paper describes the fabrication of SOI structures with buried cavities using SDB and electrochemical etch-stop. These methods are suitable for thick membrane fabrication with accurate thickness, uniformity, and flatness. After a feed-through hole for supplied voltage and buried cavities was formed on a handle Si wafer with p-type, the handle wafer was bonded to an active Si wafer consisting of a p-type substrate with an n-type epitaxial layer corresponding to membrane thickness. The bonded pair was then thinned until electrochemical etch-stop occurred at the pn junction during electrochemical etchback. By using the SDB SOI structure with buried cavities, active membranes, which have a free standing structure with a dimension of 900×900 μm2, were fabricated. It is confirmed that the fabrication process of the SDB SOI structure with buried cavities is a powerful and versatile technology for new MEMS applications.

Unusual cavity shapes resulting from multistep mass transport controlled dissolution: Numerical simulation and experimental investigation with titanium using oxide film laser lithography

The shape evolution of cavities produced by multistep electrochemical micromachining is investigated both theoretically and experimentally. A boundary element code is used for 2D simulation of the shape evolution as a function of applied charge. A two-step process is simulated by assuming that metal dissolves from a small area at the bottom of a hemicylindrical groove protected by an insulating film. Similarly, the shape evolution in a three-step process is numerically simulated. The simulation shows that multistep isotropic etching can yield buried cavities with a narrow opening as well as large aspect ratio cavities. It also permits one to achieve aspect ratios larger than one. Electrochemical micromachining experiments were carried out with titanium using oxide film laser lithography (OFLL) for patterning the surface. Metal dissolution from the irradiated line features on the oxide was carried out in an electropolishing electrolyte starting from a flat surface or from preformed grooves. The resulting cavity shapes observed with a microscope corresponded well to the theoretical predictions. The experiments thus confirmed that isotropic etching involving two or three subsequent anodization–irradiation–dissolution steps can yield high aspect ratio cavities and partly occluded cavities. Possible implications of the present results for the shape evolution of corrosion pits are discussed.

Dynamic response of tunnels in stochastic soils by the boundary element method

A stochastic boundary element method (SBEM) is developed in this work for evaluating the dynamic response of underground openings excited by seismically induced, horizontally polarized shear waves under steady-state conditions. The surrounding geological medium is viewed as an elastic continuum exhibiting large randomness in its mechanical properties, which implies that the wave number of the propagating signal is a function of a random variable. Suitable Green's functions are proposed and used within the context of the SBEM formulation. More specifically, a series expansion for the Green's functions is employed, where the basis functions are orthogonal polynomials of a random argument (polynomial chaos). These are subsequently incorporated in the SBEM formulation, which employs the usual quadratic, isoparametric line elements for modeling the surfaces of the problem in question. Finally, this formulation is used for the solution of a few problems of engineering interest involving buried cavities (tunnels). We note that the present approach departs from earlier boundary element derivations based on perturbations, which are valid for ‘small’ amounts of randomness in the elastic continuum.

SDB와 전기화학적 식각정지에 의한 마이크로 시스템용 매몰 공동을 갖는 SOI 구조의 제조

본 논문은 Si기판 직접접합기술과 전기화학적 식각정지를 이용하여 마이크로 시스템용 매몰 공동을 갖는 SOI 구조물의 일괄제조에 대한 새로운 공정기술에 관한 것이다. 저비용의 전기화학적 식각정지법으로 SOI의 정확한 두께를 제어하였다. 핸들링 기판 위에서 Si 이방성 습식식각으로 공동을 제조하였다. 산화막을 갖는 두 장의 Si기판을 직접접합한 후, 고온 열처리(<TEX>$1000^{\circ}C$</TEX>, 60분)를 시행하고 전기화학적 식각정지로 매몰 공동을 갖는 SDB SOI 구조를 박막화하였다. 제조된 SDB SOI 구조물 표면의 거칠기는 래핑과 폴리싱에 의한 기계적인 방법보다도 우수했다. 매몰 공동을 갖는 SDB SOI 구조는 새로운 마이크로 센서와 마이크로 엑츄에이터에 대단히 효과적이며 다양한 응용이 가능한 기판으로 사용될 것이다. This paper describes a new process technique for batch process of SOI(Si-on-Insulator) structures with buried cavities for MEMS(Micro Electro Mechanical System) applications by SDB(Si-wafer Direct Bonding) technology and electrochemical etch-stop. A low-cost electrochemical etch-stop method is used to control accurately the thickness of SOI. The cavities were made on the upper handling wafer by Si anisotropic etching. Two wafers are bonded with an intermediate insulating oxide layer. After high-temperature annealing(<TEX>$1000^{\circ}C$</TEX>, 60 min), the SDB SOI structure with buried cavities was thinned by electrochemical etch-stop. The surface of the fabricated SDB SOI structure have more roughness that of lapping and polishing by mechanical method. This SDB SOI structure with buried cavities will provide a powerful and versatile substrate for novel microsensors arid microactuators.

Helium/deuterium coimplanted silicon: A thermal desorption spectrometry investigation

Thermal desorption spectrometry has been applied to investigate the blistering and exfoliation phenomena which occur at the surface of a p-type (100) silicon wafer coimplanted with helium and deuterium. During the heat treatments in linear temperature ramp, an explosive emission of both gases occurs. The phenomenon is kinetically controlled with an effective activation energy of 1.3±0.2 eV. In addition, the desorption spectra present a second contribution, attributed to deuterium emission from buried cavities. Also in this case, the process is kinetically controlled with an effective activation energy of 1.9±0.3 eV. Thermal desorption spectrometry is a suitable technique to have information about various phenomena which occur during blistering and exfoliation.

Detection of buried dielectric cavities using the finite-difference time-domain method in conjunction with signal processing techniques

We address the problem of detecting low-dielectric contrast cavities buried deep in a lossy ground by using the finite-difference time-domain (FDTD) method in conjunction with signal processing techniques for extrapolation and object identification. It is well known that very low frequency probing is needed for deep penetration into the lossy ground, owing to a rapid decay of electromagnetic (EM) waves at higher frequencies. It is also recognized that numerical modeling using the FDTD method becomes very difficult, if not impossible, when the operating frequency becomes as low as 1 Hz. To circumvent this difficulty, we propose a hybrid approach in this paper that combines the FDTD method with signal processing techniques, e.g., rational function approximation and neural networks (NNs). Apart from the forward problem of modeling buried cavities, we also study the inverse scattering problem-that of estimating the depth of a buried object from the measured field values at the surface of the Earth or above. Numerical results for a buried prism are given to illustrate the application of the proposed technique.

GEOSCOPE and GEOLIDAR: Integrated Instruments for Underground Archaeological Investigations

Modern subsurface prospecting makes use of advanced technologies: coring, geophysical methods, and remote sensing. In the framework of this exciting progress, we have developed two integrated instruments for archaeological investigations. GEOSCOPE consists of a coring machine and a probe: the coring machine bores the soil while recording drilling parameters, the probe performs visual inspections and data acquisitions in the hole. GEOLIDAR is a laser range-finder conceived for the volumetric characterization of buried cavities by means of a motor-driven three-dimensional scan. The data are recorded and displayed to the archaeologist by a user-friendly computer interface. This paper describes the two systems, outlines the rationale that guided their conception, discusses the measurement accuracies and reports the first results.

Effect of circular cavity on maximum equivalent stress and stress intensity factor at a crack in buried pipeline

The effects of cavities on maximum equivalent stresses of buried pipelines are investigated in terms of two factors such as size and location of the underground cavities by using a finite element code. It has been found that the cavities affect maximum equivalent stresses of buried pipeline significantly in case that the diameters of cavities are larger than that of the pipeline. The variation of the stress intensity factors for a crack existing on the buried pipeline nearby cavities is also studied. The mode II stress intensity factor, KII, for a tilt crack located at the top portion of a buried pipeline is found to be influenced significantly regardless of the location of the underground cavities.

Crystal structure of the EF-hand parvalbumin at atomic resolution (0.91 A) and at low temperature (100 K). Evidence for conformational multistates within the hydrophobic core.

Several crystal structures of parvalbumin (Parv), a typical EF-hand protein, have been reported so far for different species with the best resolution achieving 1.5 A. Using a crystal grown under microgravity conditions, cryotechniques (100 K), and synchrotron radiation, it has now been possible to determine the crystal structure of the fully Ca2+-loaded form of pike (component pI 4.10) Parv.Ca2 at atomic resolution (0.91 A). The availability of such a high quality structure offers the opportunity to contribute to the definition of the validation tools useful for the refinement of protein crystal structures determined to lower resolution. Besides a better definition of most of the elements in the protein three-dimensional structure than in previous studies, the high accuracy thus achieved allows the detection of well-defined alternate conformations, which are observed for 16 residues out of 107 in total. Among them, six occupy an internal position within the hydrophobic core and converge toward two small buried cavities with a total volume of about 60 A3. There is no indication of any water molecule present in these cavities. It is probable that at temperatures of physiological conditions there is a dynamic interconversion between these alternate conformations in an energy-barrier dependent manner. Such motions for which the amplitudes are provided by the present study will be associated with a time-dependent remodeling of the void internal space as part of a slow dynamics regime (millisecond timescales) of the parvalbumin molecule. The relevance of such internal dynamics to function is discussed.

Anatomy of protein pockets and cavities: measurement of binding site geometry and implications for ligand design.

Identification and size characterization of surface pockets and occluded cavities are initial steps in protein structure-based ligand design. A new program, CAST, for automatically locating and measuring protein pockets and cavities, is based on precise computational geometry methods, including alpha shape and discrete flow theory. CAST identifies and measures pockets and pocket mouth openings, as well as cavities. The program specifies the atoms lining pockets, pocket openings, and buried cavities; the volume and area of pockets and cavities; and the area and circumference of mouth openings. CAST analysis of over 100 proteins has been carried out; proteins examined include a set of 51 monomeric enzyme-ligand structures, several elastase-inhibitor complexes, the FK506 binding protein, 30 HIV-1 protease-inhibitor complexes, and a number of small and large protein inhibitors. Medium-sized globular proteins typically have 10-20 pockets/cavities. Most often, binding sites are pockets with 1-2 mouth openings; much less frequently they are cavities. Ligand binding pockets vary widely in size, most within the range 10(2)-10(3)A3. Statistical analysis reveals that the number of pockets and cavities is correlated with protein size, but there is no correlation between the size of the protein and the size of binding sites. Most frequently, the largest pocket/cavity is the active site, but there are a number of instructive exceptions. Ligand volume and binding site volume are somewhat correlated when binding site volume is < or =700 A3, but the ligand seldom occupies the entire site. Auxiliary pockets near the active site have been suggested as additional binding surface for designed ligands (Mattos C et al., 1994, Nat Struct Biol 1:55-58). Analysis of elastase-inhibitor complexes suggests that CAST can identify ancillary pockets suitable for recruitment in ligand design strategies. Analysis of the FK506 binding protein, and of compounds developed in SAR by NMR (Shuker SB et al., 1996, Science 274:1531-1534), indicates that CAST pocket computation may provide a priori identification of target proteins for linked-fragment design. CAST analysis of 30 HIV-1 protease-inhibitor complexes shows that the flexible active site pocket can vary over a range of 853-1,566 A3, and that there are two pockets near or adjoining the active site that may be recruited for ligand design.

A ligand-gated, hinged loop rearrangement opens a channel to a buried artificial protein cavity

Conformational changes that gate the access of substrates or ligands to an active site are important features of enzyme function. In this report, we describe an unusual example of a structural rearrangement near a buried artificial cavity in cytochrome c peroxidase that occurs on binding protonated benzimidazole. A hinged main-chain rotation at two residues (Pro 190 and Asn 195) results in a surface loop rearrangement that opens a large solvent-accessible channel for the entry of ligands to an otherwise inaccessible binding site. The trapping of this alternate conformational state provides a unique view of the extent to which protein dynamics can allow small molecule penetration into buried protein cavities.

Fabrication of SOI wafers with buried cavities using silicon fusion bonding and electrochemical etchback

This paper describes a new technique for batch fabrication of silicon-on-insulator (SOI) wafers for microelectromechanical systems (MEMS) applications by silicon wafer bonding techniques. The process permits the inclusion of buried cavities in the SOI wafers, providing a useful tool for sensor and actuator fabrication using the resulting wafers. A low-cost electrochemical etchback step is used to define accurately the thickness of the remaining single-crystal material even though the two wafers are bonded with an intermediate insulating oxide layer. The results presented include guidelines for backside contact definition which maximize the useful silicon area as a function of doping level. The final single-crystal silicon thickness is uniform to within 0.05 μm (standard deviation) and does not require any costly high-accuracy polishing steps.

Inverse problem for tripotential measures in the study of buried cavities

This paper presents a solution to the inverse electrical problem for the interpretation of apparent resistivity anomalies due to empty buried cavities of quasi-spherical shape when tripotential measures are carried out. The anomalies of the apparent resistivities ra,rb andrg,and the composed resistivitiesrmand rt were previously calculated for a sufficient class of spherical models of resistivity anomalies. Then, for the whole class of models, some functionals of spatial distribution of the apparent and composed resistivity were identified and analyzed. They represent the average characteristics of the anomalies and, depending in a simple way on the fundamental parameters of the sources of the anomalies (average diameter and depth), they allow reliable estimates to be determined. Among the studied functionals, those allowing the most stable and less biased estimates of the anomaly source parameters are identified by numerical simulations with random noise perturbed data. Finally the trend of standard deviation and bias of the estimates of the unknown parameters were analyzed by varying the source models and the set of functionals used for the inversion.

Crystal structure of recombinant triosephosphate isomerase from bacillus stearothermophilus. An analysis of potential thermostability factors in six isomerases with known three‐dimensional structures points to the importance of hydrophobic interactions

The structure of the thermostable triosephosphate isomerase (TIM) from Bacillus stearothermophilus complexed with the competitive inhibitor 2-phosphoglycolate was determined by X-ray crystallography to a resolution of 2.8 A. The structure was solved by molecular replacement using XPLOR. Twofold averaging and solvent flattening was applied to improve the quality of the map. Active sites in both the subunits are occupied by the inhibitor and the flexible loop adopts the "closed" conformation in either subunit. The crystallographic R-factor is 17.6% with good geometry. The two subunits have an RMS deviation of 0.29 A for 248 C alpha atoms and have average temperature factors of 18.9 and 15.9 A2, respectively. In both subunits, the active site Lys 10 adopts an unusual phi, psi combination. A comparison between the six known thermophilic and mesophilic TIM structures was conducted in order to understand the higher stability of B. stearothermophilus TIM. Although the ratio Arg/(Arg+Lys) is higher in B. stearothermophilus TIM, the structure comparisons do not directly correlate this higher ratio to the better stability of the B. stearothermophilus enzyme. A higher number of prolines contributes to the higher stability of B. stearothermophilus TIM. Analysis of the known TIM sequences points out that the replacement of a structurally crucial asparagine by a histidine at the interface of monomers, thus avoiding the risk of deamidation and thereby introducing a negative charge at the interface, may be one of the factors for adaptability at higher temperatures in the TIM family. Analysis of buried cavities and the areas lining these cavities also contributes to the greater thermal stability of the B. stearothermophilus enzyme. However, the most outstanding result of the structure comparisons appears to point to the hydrophobic stabilization of dimer formation by burying the largest amount of hydrophobic surface area in B. stearothermophilus TIM compared to all five other known TIM structures.

Formation of regolith-collapse sinkholes in southern Illinois: interpretation and identification of associated buried cavities

Formation of regolith-collapse sinkholes in southern Illinois: interpretation and identification of associated buried cavities