Origins of globular structure in proteins

AbstractSince natural proteins are the products of a long evolutionary process, the structural properties of present-day proteins should depend not only on physico-chemical constraints, but also on evolutionary constraints. Here we propose a model for protein evolution, in which membranes play a key role as a scaffold for supporting the gradual evolution from flexible polypeptides to well-folded proteins. We suggest that the folding process of present-day globular proteins is a relic of this putative evolutionary process. To test the hypothesis that membranes once acted as a cradle for the folding of globular proteins, extensive research on membrane proteins and the interactions of globular proteins with membranes will be required

Design of generic biosensors based on green fluorescent proteins with allosteric sites by directed evolution

AbstractProtein-engineering techniques have been adapted for the molecular design of biosensors that combine a molecular-recognition site with a signal-transduction function. The optical signal-transduction mechanism of green fluorescent protein (GFP) is most attractive, but hard to combine with a ligand-binding site. Here we describe a general method of creating entirely new molecular-recognition sites on GFPs. At the first step, a protein domain containing a desired molecular-binding site is inserted into a surface loop of GFP. Next, the insertional fusion protein is randomly mutated, and new allosteric proteins that undergo changes in fluorescence upon binding of target molecules are selected from the random library. We have tested this methodology by using TEM1 β-lactamase and its inhibitory protein as our model protein-ligand system. ‘Allosteric GFP biosensors’ constructed by this method may be used in a wide range of applications including biochemistry and cell biology

Comparison of the Frequency of Functional SH3 Domains with Different Limited Sets of Amino Acids Using mRNA Display

Although modern proteins consist of 20 different amino acids, it has been proposed that primordial proteins consisted of a small set of amino acids, and additional amino acids have gradually been recruited into the genetic code. This hypothesis has recently been supported by comparative genome sequence analysis, but no direct experimental approach has been reported. Here, we utilized a novel experimental approach to test a hypothesis that native-like globular proteins might be easily simplified by a set of putative primitive amino acids with retention of its structure and function than by a set of putative new amino acids. We performed in vitro selection of a functional SH3 domain as a model from partially randomized libraries with different sets of amino acids using mRNA display. Consequently, a library rich in putative primitive amino acids included a larger number of functional SH3 sequences than a library rich in putative new amino acids. Further, the functional SH3 sequences were enriched from the primitive library slightly earlier than from a randomized library with the full set of amino acids, while the function and structure of the selected SH3 proteins with the primitive alphabet were comparable with those from the 20 amino acid alphabet. Application of this approach to various combinations of codons in protein sequences may be useful not only for clarifying the precise order of the amino acid expansion in the early stages of protein evolution but also for efficiently creating novel functional proteins in the laboratory

Business valuation of location-specific infrastructure projects in data-poor regions

Thesis (S.M. in Urban Studies and Planning; and, S.M. in Real Estate Development)--Massachusetts Institute of Technology, Dept. of Urban Studies and Planning, 2000.Includes bibliographical references (leaves 56-57).This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.A methodology in determining the financial values (business values) of physical infrastructure projects is presented from the public point of view. The business valuation model in this thesis adopts three concepts of financial modeling, Monte Carlo simulation (probability-generated cash flow), Capital Asset Pricing Model, and Adjusted Present Value. Using this model, the business values of a hypothetical infrastructure project are simulated 1,000 times and the mean business value is analyzed in terms of patterns and magnitudes of the simulation. The results from the 1,000 simulations showed large differences between the value derived by this model and those by the traditional net present value method. Also, this model elucidated qualitative information on how levels of government’s financial support such as subsidies, tax incentives and revenue guarantees will affect the project’s business value by components. The model elucidated, as well, the qualitative information on how project’s contractual framework may affect the business value when private contractors bear key uncertain risks, such as demand changes and construction cost overruns.by Nobuhide Watanabe.S.M.in Urban Studies and Planning; and, S.M.in Real Estate Developmen

Transcriptome Analysis of WIPK/SIPK-Suppressed Plants Reveals Induction by Wounding of Disease Resistance-Related Genes Prior to the Accumulation of Salicylic Acid

Salicylic acid (SA) plays a key role in plant resistance to pathogens. Accumulation of SA is induced by wounding in tobacco plants in which the expression of WIPK and SIPK, two mitogen-activated protein kinases, is suppressed. Here, the mechanisms underlying the abnormal accumulation of SA in WIPK/SIPK-suppressed plants have been characterized. SA accumulation started around 12 h after wounding and was inhibited by cycloheximide (CHX), a protein synthesis inhibitor. SA accumulation, however, was enhanced several fold when leaf discs were transferred onto CHX after floating on water for >= 6 h. Temporal and spatial analyses of wound-induced and CHX-enhanced SA accumulation suggested that wounding induces activators for SA accumulation followed by the generation of repressors, and late CHX treatment inhibits the production of repressors more efficiently than that of activators. Microarray analysis revealed that the expression of many disease resistance-related genes, including N, a Resistance (R) gene for Tobacco mosaic virus and R gene-like genes, was up-regulated in wounded WIPK/SIPK-suppressed plants. Expression of the N gene and R gene-like genes peaked earlier than that of most other genes as well as SA accumulation, and was mainly induced in those parts of leaf discs where SA was highly accumulated. Moreover, wound-induced SA accumulation was decreased by the treatments which compromise the function of R proteins. These results indicate that signaling leading to the expression of disease resistance-related genes is activated by wounding in WIPK/SIPK-suppressed plants, and induction of R gene and R gene-like genes might lead to the biosynthesis of SA.ArticlePLANT AND CELL PHYSIOLOGY. 54(6):1005-1015 (2013)journal articl