Well-defined Binding Site Research Articles

Molecular Design of Intercalation-Based Sensors. 2. Sensing of Carbon Dioxide in Functionalized Thin Films of Copper Octanediylbis(phosphonate)

Self-assembled thin films of layered copper alkanediylbis(phosphonates) retain the amine-specific intercalation chemistry of the corresponding microcrystalline solids. Aliphatic and aromatic amines bind in a 1:1 ratio to coordinatively unsaturated copper ions in anhydrous Cu 2(O3P(CH2)8PO3); and by selecting an amine with an appropriate functional tail group, a chemically and sterically well-defined interlamellar binding site for CO 2 is created. Powder X-ray diffraction, Fourier transform infrared spectroscopy, and solid-state NMR experiments were used to study the intercalation of 3-aminopropanol, (3-aminopropyl)methyldihydroxysilane, and p-xylylenediamine, and their reversible reaction with CO 2 to form carbonates and carbamates, respectively. By growing these films on the electrodes of a quartz crystal microbalance device, a sensor can be fabricated for monitoring CO 2 in gas streams at concentrations of 0.5-19% (v/v). A Henrian response (frequency change directly proportional to CO 2 partial pressure) was observed, and the time required for equilibration of these devices with CO2, using 5-layer films, was 3-4 min. Effective diffusion coefficients for CO2 in the films were determined using a dualtransport model and were found to be in the range (6-9) 10-9 cm 2 /s. Carbon dioxide, as both a reactant in and a product of largescale reactions, is the focus of much current research activity. Millions of tons of carbon dioxide are used every year in the carbonated beverage industry and for the production of carbonates, carboxylic acids, carbon monoxide, and urea, and it is a natural byproduct of ammonia production, grain fermentation, natural resevoirs, and chemical and petroleum operations. The need for carbon dioxide sensors for process control and especially for environmental monitoring is the driving force in this area of analytical research. Fast, quantitative, and reliable carbon dioxide sensors are also needed in anesthesiology and physiology, for measuring cardiovascular system exchange rates, and for determining the concentrations of blood gases during surgery. Most of the currently available sensors for carbon dioxide are

Secondary Structure Model of the Last Two Domains of Single-stranded RNA Phage Qβ

We have determined the nucleotide sequence of three positive single-stranded RNA coliphages and have used this information, together with the known sequences of the related phages Qβ and SP, to construct a secondary structure model for the two distal domains of Qβ RNA. The 3′ terminal domain, which is about 100 nucleotides long, contains most of the 3′ untranslated region and folds into four short, regular hairpins. The adjacent 3′ replicase domain contains about 1100 nucleotides. Hairpins in this protein-coding domain are much longer and more irregular than in the 3′ untranslated region. Both domains are defined by long-distance interactions. The secondary structure is not a collection of hairpin structures connected by single-stranded regions. Rather, the RNA stretches between the stem-loop structures are all involved in an extensive array of long-distance interactions that contract the molecule to a rigid structure in which all hairpins are predicted to have a fixed position with respect to each other. A general feature of the model is that helices tend to be organized in four-way junctions with little or no unpaired nucleotides between them. As a result, there is a potential for coaxial stacking of adjacent stems. The essential features of the model are supported by the SInuclease cleavage pattern.Viral RNA sequences are strongly constrained by their coding function. As a result, structural evolution by simple base-pair substitutionis not always possible, as this usually requires the juxtaposition of the codon wobble positions in stems. Rather, we often observe co-ordinate base substitutions that maintain the stem, but tend to change the position at which bulges or internal loops are found. Structures that differ in this way are apparently equally fit. Also, the relative position of hairpin loops can shift several nucleotides through an alignment based on maximal sequence identity i.e. amino acid homology. The fact that these structural irregularities do not occur at the 3′ untranslated region suggests indeed that the coding function of the RNA constrains the secondary structure. Hairpins with the stable tetraloop motif GNRA and UNCG or their complement are over-represented. This suggests their involvement in segregation of plus and minus strand. The genome of the coliphages contains a well-defined high affinity binding site for the coat protein, which serves to suppress replicase translation and also acts as a nucleation point in capsid formation. Close to the 3′ end we find an additional conserved helix that meets the described consensus criteria for coat-protein binding.

Current methods for site-directed structure generation.

There has been a rapid growth of interest in techniques for site-directed drug design, fueled by the increasing availability of structural models of proteins of therapeutic importance, and by studies reported in the literature showing that potent chemical leads can be obtained by these techniques. Structure generation programs offer the prospect of discovering highly original lead structures from novel chemical families. Due to the fact that this technique is more-or-less still in its infancy, there are no case studies available that demonstrate the use of structure generation programs for site-directed drug design. Such programs were first proposed in 1986, and became commercially available in early 1992. They have shown their ability to reproduce, or suggest reasonable alternatives for, ligands in well-defined binding sites. This brief review will discuss the recent advances that have been made in the field of site-directed structure generation.

Hydrogen adsorption on Mo(211): reconstruction and binding site conversion

The H/Mo(211) chemisorption system has been investigated over the temperature range 100–400 K using LEED, HREELS, and work function measurements. Above 1 ML, a transition from the (1 × 1) structure of the clean surface to a hydrogen induced (1 × 2) phase is observed and attributed to changes in the substrate periodicity. Vibrational spectroscopy suggests that below 1 ML hydrogen is bridge bonded and induces local distortions of the substrate. The transition to the (1 × 2) phase involves the ordering of these displacements and the occupation of three-fold sites partially populated by conversion of the bridge bonded species. This conversion is correlated with a sawtooth-like coverage dependence of the work function. In contrast to recent reports of delocalization on W(211), hydrogen is found to occupy well-defined binding sites at all coverages. Results for the present system are also compared with those for hydrogen adsorption on Mo(100), other bcc (211) substrates as well as the structurally similar fcc (110) surfaces.

Effect of zinc binding on the structure and stability of fibrolase, a fibrinolytic protein from snake venom.

Fibrolase is a metalloprotease with potential use as a fibrinolytic agent. Loss of the intrinsic zinc atom leads to a rapid decrease in enzymatic activity. Circular dichroism measurements indicate that there is a partial unfolding of an alpha-helical section of the protein concomitant with the loss of zinc. Removal of zinc can be affected by elevated temperatures, acidic pH values, and addition of chelating agents. At low molar concentrations, both ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) were found to remove zinc efficiently. Analysis of the sequence of fibrolase identified a segment which possessed a high degree of homology with the metal binding site of other zinc proteases, such as thermolysin and the collagenases. However, the putative zinc binding site in fibrolase lacks the additional glutamate ligand found in thermolysin and subtilisin. This sequence is also predicted to adopt an alpha-helical conformation. Together, these data indicate that there is a well-defined metal binding site in fibrolase and that metal binding is the most important factor governing the stability of this protein.

Multiple nuclear proteins bind upstream sequences in the promotor region of a T-cell receptor beta-chain variable-region gene: evidence for tissue specificity.

DNA-nuclear protein binding interactions were examined in the promoter region of a representative T-cell receptor Ti beta-chain variable-region gene by means of electrophoretic mobility-shift and DNase I-protection analysis. Within 175 bases upstream of the transcription initiation site, four protected regions ("footprints") were identified on the coding strand, at nucleotides -46 to -68 (I), -72 to -92 (II), -113 to -134 (III), and -136 to -175 (IV). Nuclear proteins (0.6 M NaCl fraction from a heparin-Sepharose column chromatography of nuclear extracts) of a variety of cell types produced footprints I, III, and IV and a fifth footprint (beyond nucleotide -200). In contrast, footprint II was produced only by T-cell extracts, although nuclear extracts of a transformed B-lymphoblastoid line produced a partial footprint in this region. Furthermore, footprint analysis of the noncoding strand showed that a continuous region of protection corresponding to the entire region of footprints I and II was generated, along with a DNase I-hypersensitive site, by nuclear proteins derived from T cells but not other cell types. Footprint I has the sequence structure of many well-defined protein-DNA binding sites. Nucleotide sequences in the region of footprint II bore no homology to known sequences, whereas those in the areas of footprints III and IV were similar to motifs within immunoglobulin and other enhancers. These findings may have implications for the tissue specificity of human Ti beta-chain gene transcription.

Specificity of RNA and coat protein interaction in alfalfa mosaic virus and related viruses

Well-defined coat protein binding sites were found to be present on the genomic RNAs of AlMV and TSV. In view of the regulatory importance of these sites in virus multiplication, the possibility that nonrelated proteins also were able to interact with these sites was investigated. The coat proteins of TSV, BMV, CMV, and SBMV recognize specific sites on AlMV RNA 1. The significance of these sites in virus multiplication is discussed. No specific binding sites were found with TMV coat protein or the nonviral proteins ovalbumin, myoglobin, and lysozyme. Moreover, the ability of AlMV coat protein to recognize specific sites on the heterologous RNAs of TSV, BMV, bacteriophage MS2, and Escherichia coli was tested. No specific sites were found with MS2-RNA. However, specific binding sites were found with BMV-RNA 3 and, unexpectedly, with E. coli 16 S ribosomal RNA. From these data it was concluded that binding of AlMV and TSV coat proteins to their genomic RNAs is a specific process. However, the binding of coat protein to BMV-RNAs and to ribosomal RNAs may result from secondary folding that may have been conserved for other purposes throughout evolution of the RNAs.

Small-angle neutron scattering study of the association between porcine pancreatic colipase and taurodeoxycholate micelles

Small-angle neutron scattering studies have shown the association of porcine colipase with bile salts micelles to be a lateral one. The molecular structure parameters of the individual components were determined first. A radius of gyration of 13.9 Å is found for colipase, which implies a non-spherical shape for this molecule. The size of taurodeoxycholate micelles is controlled by the ionic strength of the solution. In 0.15 m-NaCl their volume is comparable to that of colipase; they are elongated with an axial ratio of about 2. At higher ionic strengths the elongation of the micelles increases. In 0.15 m-NaCl the complex is found to be an association of one colipase molecule with a volume of detergent corresponding to that of one free micelle. The contrast variation study of the radius of gyration shows that in the complex the centre of masses of the protein and of the detergent are well-separated: a distance between 29 and 45 Å has been estimated. The value of the radius of gyration of the complex at high contrast, and the agreement between the contrast variation analysis and a straightforward application of the parallel axes theorem indicate that the complex is formed by the juxtaposition of the protein and a preformed micelle, which has approximately the same size and shape as a free micelle. There is only one localized surface contact between the protein and the micelle, which implies that colipase possesses a relatively well-defined binding site.

Three-Dimensional Structure of tRNA

This chapter explores that correlation between molecular structure and biological function is one of the main aims of present day biology. The detailed mechanism of spelling out the genetic message is closely linked to subcellular structures, and molecular tools of the utmost refinement are required. The chapter reviews that tRNA's play the key role in the transmission of genetic information from DNA into protein structure. During these processes, tRNA's are specifically charged with their appropriate amino acids by enzymes that can act only on well-defined, undenatured, three-dimensional structures. tRNA's further interact with the well-defined binding sites of the ribosomal subunits. They interact with the properly aligned messenger. They carry amino acids and peptides into their correct respective positions for the formation of peptide bonds. All these highly specific nucleic acid protein interactions and nucleic acid-nucleic acid interactions cannot be understood without the existence of a well-defined three-dimensional structure of tRNA. It also provides an overview on the three independent approaches, that is, small-angle X-ray scattering, viscosity measurement, and enzymatic degradation. It concludes that tRNA has a structure of higher order that is more compact and more stable than a loose combination of helical segments. There is a tertiary structure for tRNA.