Probing the minimal determinants of zinc binding with computational protein design

Structure-based protein design tests our understanding of the minimal determinants of protein structure and function. Previous studies have demonstrated that placing zinc binding amino acids (His, Glu, Asp or Cys) near each other in a folded protein in an arrangement predicted to be tetrahedral is often sufficient to achieve binding to zinc. However, few designs have been characterized with high-resolution structures. Here, we use X-ray crystallography, binding studies and mutation analysis to evaluate three alternative strategies for designing zinc binding sites with the molecular modeling program Rosetta. While several of the designs were observed to bind zinc, crystal structures of two designs reveal binding configurations that differ from the design model. In both cases, the modeling did not accurately capture the presence or absence of second-shell hydrogen bonds critical in determining binding-site structure. Efforts to more explicitly design second-shell hydrogen bonds were largely unsuccessful as evidenced by mutation analysis and low expression of proteins engineered with extensive primary and secondary networks. Our results suggest that improved methods for designing interaction networks will be needed for creating metal binding sites with high accuracy

A New Partitioning Around Medoids Algorithm

Kaufman & Rousseeuw (1990) proposed a clustering algorithm Partitioning Around Medoids (PAM) which maps a distance matrix into a specified number of clusters. A particularly nice property is that PAM allows clustering with respect to any specified distance metric. In addition, the medoids are robust representations of the cluster centers, which is particularly important in the common context that many elements do not belong well to any cluster. Based on our experience in clustering gene expression data, we have noticed that PAM does have problems recognizing relatively small clusters in situations where good partitions around medoids clearly exist. In this note, we propose to partition around medoids by maximizing a criteria Average Silhouette\u27\u27 defined by Kaufman & Rousseeuw. We also propose a fast-to-compute approximation of Average Silhouette\u27\u27. We implement these two new partitioning around medoids algorithms and illustrate their performance relative to existing partitioning methods in simulations

Computational Design of Zinc Binding Sites at Protein Interfaces and Enzyme Active Sites

Engineered proteins will continue to expand the molecular toolkit for applications in medicine, biotechnology, and basic research. While protein engineering efforts often use a parts list limited to the twenty amino acids, metal ions expand the parts list and are critical for the folding and function of 30-40% of known proteins. In particular, zinc ions are common as structural metal sites and catalytic metal sites. Thus, the work described here uses and develops computational methods to engineer structural zinc sites at protein interfaces and catalytic zinc sites at potential active sites. The first chapter discusses the design of a de novo zinc-mediated heterodimeric interaction that targets wild-type ubiquitin. Although zinc binding was successful, a lack of cooperativity resulted in a modest effect of zinc on ubiquitin binding affinity. The second chapter presents a de novo zinc-mediated homodimer as an alternative protein interface design strategy with more cooperative metal binding. Zinc binding improved the homodimer binding affinity by >100-fold, and crystal structures demonstrate moderate accuracy in the design of the zinc sites and the protein-protein interaction. The third chapter reveals the serendipitous discovery of de novo catalysis by this designed zinc-mediated homodimer. This discovery emphasizes the usefulness of protein interfaces for active site formation, the power of zinc for catalysis, and the modest rates achieved thus far in the field of de novo enzyme design. The fourth chapter introduces our efforts to purposefully design a new catalytic motif in a deeper protein cleft. Our approach differs from most enzyme design studies that instead rely on existing catalytic motifs and modify substrate-binding residues. A conformational change shown in the crystal structure of a designed zinc site in a TIM-barrel scaffold emphasizes the importance of second-shell hydrogen bonds to support the primary coordination shell for robust metal binding in deeper protein clefts. In summary, we have endeavored to better understand and more reliably engineer protein structure and function using a predictive computational approach, and as we improve our ability to design zinc sites in proteins, more sophisticated protein functions can be engineered for applied purposes.Doctor of Philosoph

Analysis of Longitudinal Marginal Structural Models

In this article we construct and study estimators of the causal effect of a time-dependent treatment on survival in longitudinal studies. We employ a particular marginal structural model (MSM), and follow a general methodology for constructing estimating functions in censored data models. The inverse probability of treatment weighted (IPTW) estimator is used as an initial estimator and the corresponding treatment-orthogonalized, one-step estimator is consistent and asymptotically linear when the treatment mechanism is consistently estimated. We extend these methods to handle informative censoring. A simulation study demonstrates that the the treatment-orthogonalized, one-step estimator is superior to the IPTW estimator in terms of efficiency. The proposed methodology is employed to estimate the causal effect of exercise on mortality in a longitudinal study of seniors in Sonoma County

Exploring interspecies sensemaking: dog tracking semiotics and multispecies ethnography

The domestic use of tracking technology with pets is on the rise, yet is under-researched. We investigate how tracking practices reconfigure human-dog relationships changing both humans and dogs. We question the sensemaking mechanisms by which both humans and dogs engage in context-based meaningful exchanges via the technology’s mediation. We show how an indexical semiotic perspective could inform the development of interspecies technology. Finally, we discuss the methodological issues raised by doing research with animals and propose an interspecies semiotics which integrates animal companions and animal researchers’ accounts into ethnographic observation

A Morphological Diagnostic for Dynamical Evolution of Wolf-Rayet Bubbles

We have observed H-alpha and [OIII] emission from eight of the most well defined Wolf-Rayet ring nebulae in the Galaxy. We find that in many cases the outermost edge of the [OIII] emission leads the H-alpha emission. We suggest that these offsets, when present, are due to the shock from the Wolf-Rayet bubble expanding into the circumstellar envelope. Thus, the details of the WR bubble morphology at H-alpha and [OIII] can then be used to better understand the physical condition and evolutionary stage of the nebulae around Wolf-Rayet stars, as well as place constraints on the nature of the stellar progenitor and its mass loss history.Comment: 11 pages, LaTex, 8 figures, accepted for publication in AJ, November 200

High-grade, Compact spectrometers for Earth observation from smallsats

The market for nano- and microsatellites is developing rapidly. There is a strong focus on 2D imaging of the Earth\u27s surface, with limited possibilities to obtain spectral information. More demanding applications, such as monitoring trace gases and aerosols, or water quality still require advanced spectral imaging instruments, which are large, heavy and expensive. In recent years TNO has investigated and developed different innovative designs to realize advanced spectrometers for space applications in a more compact and cost-effective manner. This offers multiple advantages: A compact instrument can be flown on a much smaller platform (nano- or microsatellite); a low-cost instrument opens up the possibility to fly multiple instruments in a satellite constellation, improving both global coverage and temporal sampling (e.g. multiple overpasses per day to study diurnal processes); in this way a constellation of low-cost instruments may provide added value to the larger scientific and operational satellite missions (e.g. the Copernicus Sentinel missions); and a small, lightweight spectrometer can easily be mounted on a high-altitude UAV (offering high spatial resolution). Moreover, a low-cost instrument may allow us to break through the \u27cost spiral\u27: lower cost will allow us to take more risk and thus progress faster. This may lead to a much faster development cycle than customary for current Earth-observation instruments. Finally, the TNO designs offer flexibility to tune the performance (spectral range, spectral resolution) of the spectrometer to a specific application. Thus, based on the same basic system design, these instruments offer a wide range of applications to a variety of clients, both inside and outside the scientific community using a quasi-recurrent instrument. In this presentation we will illustrate this innovative approach, using the most mature design of a hyperspectral imaging spectrometer (named \u27Tropolite\u27) as an example. Other, less developed, designs will be presented briefly. We will discuss the different design and manufacturing techniques that were used to realize these very compact and low-cost concepts. The first laboratory test results of a Tropolite breadboard will be presented and commented upon. Based on these test results the feasibility to use Tropolite for different applications (e.g. air quality, water quality, …) will be discussed further. Currently, efforts are being made to realize an in-orbit demonstration of Tropolite