Systematic study on crystal-contact engineering of diphthine synthase: influence of mutations at crystal-packing regions on X-ray diffraction quality

It is well known that protein crystallizability can be influenced by site-directed mutagenesis of residues on the molecular surface of proteins, indicating that the intermolecular interactions in crystal-packing regions may play a crucial role in the structural regularity at atomic resolution of protein crystals. Here, a systematic examination was made of the improvement in the diffraction resolution of protein crystals on introducing a single mutation of a crystal-packing residue in order to provide more favourable packing interactions, using diphthine synthase from Pyrococcus horikoshii OT3 as a model system. All of a total of 21 designed mutants at 13 different crystal-packing residues yielded almost isomorphous crystals from the same crystallization conditions as those used for the wild-type crystals, which diffracted X-rays to 2.1 angstrom resolution. Of the 21 mutants, eight provided crystals with an improved resolution of 1.8 angstrom or better. Thus, it has been clarified that crystal quality can be improved by introducing a suitable single mutation of a crystal-packing residue. In the improved crystals, more intimate crystal-packing interactions than those in the wild-type crystal are observed. Notably, the mutants K49R and T146R yielded crystals with outstandingly improved resolutions of 1.5 and 1.6 angstrom, respectively, in which a large-scale rearrangement of packing interactions was unexpectedly observed despite the retention of the same isomorphous crystal form. In contrast, the mutants that provided results that were in good agreement with the designed putative structures tended to achieve only moderate improvements in resolution of up to 1.75 angstrom. These results suggest a difficulty in the rational prediction of highly effective mutations in crystal engineering.

Suitability of a point-contact SAW-based stiffness transducer for biomedical applications

Methods of diagnosis in Biomedical applications can be broadly divided into contact and non-contact based methods. So far, ultrasound based methods have been found to be most favorable for non-contact, non-invasive diagnosis, especially in the case of tissue stiffness analysis. We report here, the fabrication and characterization details of a new contact based transducer system for qualitative determination of the stiffnesses of non-piezoelectric substrates using the phenomenon of Surface Acoustic Waves (SAW). Preliminary trials to study the functionality of this system were carried out on various metallic and non-metallic substrates, and the results were found to be satisfactory. To confirm the suitability of this system for biomedical applications, similar trials have been conducted on tissue mimicking phantoms with varying degrees of stiffness.

The Critical Role of N- and C-Terminal Contact in Protein Stability and Folding of a Family 10 Xylanase under Extreme Conditions

Background: Stabilization strategies adopted by proteins under extreme conditions are very complex and involve various kinds of interactions. Recent studies have shown that a large proportion of proteins have their N- and C-terminal elements in close contact and suggested they play a role in protein folding and stability. However, the biological significance of this contact remains elusive. Methodology: In the present study, we investigate the role of N- and C-terminal residue interaction using a family 10 xylanase (BSX) with a TIM-barrel structure that shows stability under high temperature,alkali pH, and protease and SDS treatment. Based on crystal structure,an aromatic cluster was identified that involves Phe4, Trp6 and Tyr343 holding the Nand C-terminus together; this is a unique and important feature of this protein that might be crucial for folding and stabilityunder poly-extreme conditions. Conclusion: A series of mutants was created to disrupt this aromatic cluster formation and study the loss of stability and function under given conditions. While the deletions of Phe4 resulted in loss of stability, removal of Trp6 and Tyr343 affected in vivo folding and activity. Alanine substitution with Phe4, Trp6 and Tyr343 drastically decreased stability under all parameters studied. Importantly,substitution of Phe4 with Trp increased stability in SDS treatment.Mass spectrometry results of limited proteolysis further demonstrated that the Arg344 residue is highly susceptible to trypsin digestion in sensitive mutants such as DF4, W6A and Y343A, suggesting again that disruption of the Phe4-Trp6-Tyr343 (F-W-Y) cluster destabilizes the N-and C-terminal interaction. Our results underscore the importance of N- and C-terminal contact through aromatic interactions in protein folding and stability under extreme conditions, and these results may be useful to improve the stability of other proteins under suboptimal conditions.

The Nature of Learning about Customers in a Customer Service Setting - A Study of Frontline Contact Persons

The starting point of this thesis is the notion that in order for organisations to understand what customers value and how customers experience service, they need to learn about customers. The first and perhaps most important link in an organisation-wide learning process directed at customers is the frontline contact person. Service- and sales organisations can only learn about customers if the individual frontline contact persons learn about customers. Even though it is commonly recognised that learning about customers is the basis for an organisation’s success, few contributions within marketing investigate the fundamental nature of the phenomenon as it occurs in everyday customer service. Thus, what learning about customers is and how it takes place in a customer-service setting is an issue that is neglected in marketing research. In order to explore these questions, this thesis presents a socio-cultural approach to understanding learning about customers. Hence, instead of considering learning equal to cognitive processes in the mind of the frontline contact person or learning as equal to organisational information processing, the interactive, communication-based, socio-cultural aspect of learning about customers is brought to the fore. Consequently, the theoretical basis of the study can be found both in socio-cultural and practice-oriented lines of reasoning, as well as in the fields of service- and relationship marketing. As it is argued that learning about customers is an integrated part of everyday practices, it is also clear that it should be studied in a naturalistic and holistic way as it occurs in a customer-service setting. This calls for an ethnographic research approach, which involves direct, first-hand experience of the research setting during an extended period of time. Hence, the empirical study employs participant observations, informal discussions and interviews among car salespersons and service advisors at a car retailing company. Finally, as a synthesis of theoretically and empirically gained understanding, a set of concepts are developed and they are integrated into a socio-cultural model of learning about customers.

An Experimental Study to Show the Effect of Thermal Stress on Thermal Contact Conductance at Sub-megapascal Contact Pressures

Experimental studies are presented to show the effect of thermal stresses on thermal contact conductance (TCC) at low contact pressures. It is observed that in a closed contact assembly, contact pressure acting on the interface changes with the changing temperature of contact members. This change in contact pressure consequently causes variations in the TCC of the junction. A relationship between temperature change and the corresponding magnitude of developed thermal stress in a contact assembly is determined experimentally. Inclusion of a term called temperature dependent load correction factor is suggested in the theoretical model for TCC to make it capable of predicting TCC values more accurately in contact assemblies that experience large temperature fluctuations. [DOI: 10.1115/1.4001615]

Deformation and friction of MoS2 particles in liquid suspensions used to lubricate sliding contact

Tribology of a well known solid lubricant molybdenum disulphide is studied here in water and oil medium, over a large range of contact dimensions. Lateral force microscopy is used to identify the deformation modes, intra-crystalline slip, plastic grooving, fragmentation and fracture, of single particles The medium and agglomeration were found to dictate the deformation mode Steel on steel tribology lubricated by suspensions of these particles in liquid media was conducted over a range of contact pressure and sliding velocity. A scrutiny of the frictional data with the aid of Raman spectroscopy to identify the transfer film, suggested that the particle size, as it is at contact, is an important tribological parameter Ultrasonication of the suspension and dispersion of the particle by surfactants were used to control the apriori particle size fed into the suspension.Correspondence of friction data of the gently sonicated suspension with that of the ultrasonicated suspension with dispersants indicated the importance of liquid ingestion by these particles as it controls their mode of deformation and consequent tribology. (C) 2010 Elsevier B V All rights reserved.

Thermal gap conductance at low contact pressures (<1 MPa): Effect of gold plating and plating thickness

Thermal contact conductance (TCC) measurements are made on bare and gold plated (<= 0.5 mu m) oxygen free high conductivity (OFHC) Cu and brass contacts in vacuum, nitrogen, and argon environments. It is observed that the TCC in gaseous environment is significantly higher than that in vacuum due to the enhanced thermal gap conductance. It is found that for a given contact load and gas pressure, the thermal gap conductance for bare OFHC Cu contacts is higher than that for gold plated contacts. It is due to the difference in the molecular weights of copper and gold, which influences the exchange of kinetic energy between the gas molecules and contact surfaces. Furthermore, the gap conductance is found to increase with increasing thickness of gold plating. Topography measurements and scanning electron microscopy (SEM) analysis of contact surfaces revealed that surfaces become smoother with increasing gold plating thickness, thus resulting in smaller gaps and consequently higher gap conductance. (C) 2010 Elsevier Ltd. All rights reserved.

Les fleuves dans l'œuvre romanesque de Jean-Marie Gustave Le Clézio

Relying on Merleau-Ponty's phenomenology of perception and on Mircea Eliade's works on the Sacred and the Profane, this study explores the river as a perceptual space and as the sacred Center in a cosmic vision of the world in twelve of Jean-Marie Gustave Le Clézio's fictional works, from The Interrogation (1963) to Revolutions (2003). In the first chapter, after introducing the field of study, I discuss the relation between the radical subjectivity and the evasiveness of perceiving subjects in Le Clézio's fiction. Next are some thoughts on the relation between Merleau-Ponty's and Le Clézio's ideas. The second chapter studies the river as an experience in the text, first as a topographical space, then as a sound world. The investigations move on to its water as a visual and a tactile phenomenon. Then follows the human use of the river, the (absence of) baths, and the river as a traveling space. The chapter closes with the study of the metaphorical use of the word, occurring mainly in urban space and for phenomena in the sky. The third chapter is organized around the river as the Center of the world in a religious cosmogony, where the river represents the origin of the world and of the human race. The core analysis shows how the middle of the river is a symbolic space of a new beginning. As a sacred space, the river abolishes time as the object of contemplation and as relative immobility from the point of view of a person drifting downstream. The functions of a new beginning and of abolition of time are combined in the symbolic immersions in the water. Finally, the dissertation explores other symbolical spaces, such as the unknown destination of the drift, and the river as the Center of a utopia. The chapter closes with the existential agony as a result of the elimination of the Center in the urban environment. In the final chapter, the river is compared to other watercourses : the creek, the brook and the rapids. The river is more of a spatial entity, whereas the actual water is more important in the smaller watercourses. The river is more common than the other watercourses as a topographical element in the landscape, whereas the minor watercourses invite the characters to a closer contact with their element, in immersions and in drinking their water. Finally, the work situates the rivers in a broader context of different fictional spaces in Le Clézio's text.

Superconducting gap in Nb seen by point contact spectroscopy

The nonlinear current voltage characteristics of a point contact convey information about various excitations in the metal. We have made a poin~ contact study on a superconductor to see the band gap and on a normal metal to see Ihe transport characteristics.

Evolution of and preconditions for generation of corrugations on a plane surface in rolling contact

A hard roller under normal load is driven by the flat surface of a soft disc. Corrugations are generated on the disc when certain surface morphological, load, speed and mechanical property-oriented conditions are met. The evolutionary process of corrugation generation and the preconditions necessary for it are investigated morphologically and mechanically for four disc materials: mild steel, brass, PTFE and PMMA.