Compatibility of the conformationally rigid CF3-Bpg side chain with the hydrophobic coiled-coil interface

3-(Trifluoromethyl)bicyclopent-[1.1.1]-1-yl glycine (CF₃-Bpg) has previously been established as a useful ¹⁹F NMR label to analyse the structures of oligomeric membrane-active peptides or transmembrane segments. To systematically examine the effect of side chain volume, conformational rigidity, and hydrophobicity of CF₃-Bpg in polypeptide environments the amino acid was incorporated into an established coiled-coil based screening system. A single substitution of either valine (position a16) or leucine (position d19) within the hydrophobic core of the heteromeric coiled coil has practically no effect on its structure. Despite its comparatively high hydrophobicity, however, the stiff and bulky side chain of CF₃-Bpg is not so well accommodated by the hydrophobic core as it leads to a more pronounced destabilization than observed for other, more polar fluorinated amino acids which carry more flexible side chains. CF₃-Bpg is therefore a useful ¹⁹F NMR label, though not for monitoring the stability of such helix–helix interactions.     

Statistical uncertainty and its propagation in the analysis of quantitative polymerase chain reaction data: Comparison of methods

Most methods for analyzing real-time quantitative polymerase chain reaction (qPCR) data for single experiments estimate the hypothetical cycle 0 signal y₀ by first estimating the quantification cycle (C_q) and amplification efficiency (E) from least-squares fits of fluorescence intensity data for cycles near the onset of the growth phase. The resulting y₀ values are statistically equivalent to the corresponding C_q if and only if E is taken to be error free. But uncertainty in E usually dominates the total uncertainty in y₀, making the latter much degraded in precision compared with C_q. Bias in E can be an even greater source of error in y₀. So-called mechanistic models achieve higher precision in estimating y₀ by tacitly assuming E = 2 in the baseline region and so are subject to this bias error. When used in calibration, the mechanistic y₀ is statistically comparable to C_q from the other methods. When a signal threshold y_q is used to define C_q, best estimation precision is obtained by setting y_q near the maximum signal in the range of fitted cycles, in conflict with common practice in the y₀ estimation algorithms.     

Multivariate data analysis in empirical research. A look on the bright side

The interpretive benefits of employing multivariate analysis methods on experimental data with more than one dependent variable are described heuristically and illustrated on a set of data from a simply designed experiment in physiological psychology. Multivariate analysis of variance (MANOVA) is performed on the 9 dependent variables contained in the sample data and on the four composites derived from a principal components analysis (PCA) of the variability of the nine. A linear discriminant analysis (LDA) is conducted following both MANOVA results, and 5 methods of determining the "important" dependent variables in the experimental-control group difference are presented and discussed in terms of the data at hand.     

Capture and analysis of quantitative proteomic data

Whilst the array of techniques available for quantitative proteomics continues to grow, the attendant bioinformatic software tools are similarly expanding in number. The data capture and analysis of such quantitative data is obviously crucial to the experiment and the methods used to process it will critically affect the quality of the data obtained. These tools must deal with a variety of issues, including identification of labelled and unlabelled peptide species, location of the corresponding MS scans in the experiment, construction of representative ion chromatograms, location of the true peptide ion chromatogram start and end, elimination of background signal in the mass spectrum and chromatogram and calculation of both peptide and protein ratios/abundances. A variety of tools and approaches are available, in part restricted by the nature of the experiment to be performed and available instrumentation. Currently, although there is no single consensus on precisely how to calculate protein and peptide abundances, many common themes have emerged which identify and reduce many of the key sources of error. These issues will be discussed, along with those relating to deposition of quantitative data. At present, mature data standards for quantitative proteomics are not yet available, although formats are beginning to emerge.     

A quantitative analysis of photometric data from aerial photographs for vegetation survey

This paper describes the application of quantitative density analysis to black and white aerial photographs for vegetation survey using a Quantimet 720 image analyser. The photometric data are analysed using both a supervised and an unsupervised classification strategy. Floristic data collected from an independent ground survey, are used to categorise those vegetation classes of interest against which the photometric data classifications are assessed. The preliminary results obtained suggest that broad classes of vegetation types may be distinguished automatically from their grey scale distribution patterns.     

Differential analysis of high-throughput quantitative genetic interaction data

Synthetic genetic arrays have been very effective at measuring genetic interactions in yeast in a high-throughput manner and recently have been expanded to measure quantitative changes in interaction, termed ''differential interactions'', across multiple conditions. Here, we present a strategy that leverages statistical information from the experimental design to produce a novel, quantitative differential interaction score, which performs favorably compared to previous differential scores. We also discuss the added utility of differential genetic-similarity in differential network analysis. Our approach is preferred for differential network analysis, and our implementation, written in MATLAB, can be found at http://chianti.ucsd.edu/~gbean/compute_differential_scores.m.     

Molecular motion of spin labeled side chains in alpha-helices: analysis by variation of side chain structure

Two single cysteine substitution mutants at helix surface sites in T4 lysozyme (D72C and V131C) have been modified with a series of nitroxide methanethiosulfonate reagents to investigate the structural and dynamical origins of their electron paramagnetic resonance spectra. The novel reagents include 4-substituted derivatives of either the pyrroline or pyrrolidine series of nitroxides. The spectral line shapes were analyzed as a function of side chain structure and temperature using a simulation method with a single order parameter and diffusion rates about three orthogonal axes as parameters. Taken together, the results provide strong support for an anisotropic motional model of the side chain, which was previously proposed from qualitative features of the spectra and crystal structures of spin labeled T4 lysozyme. Site-specific differences in apparent order parameter are interpreted in terms of backbone dynamics modes with characteristic correlation times in the nanosecond or faster time scale. The saturated 4-substituted pyrrolidine nitroxides are shown to be a suitable template for novel "functionalized" side chains designed to mimic salient features of the native side chains they replace.     

A gadolinium(III) nitronyl nitroxide linear chain with a complex spin structure

A new nitronyl nitroxide radical, 2-(4-carboxy-phenyl)-4,4,5,5-tetra-methyl-4,5-dihydro-1H-imidazol-1-oxyl-3-oxide, was used to prepare a linear chain system containing Gd(II) ions and these radicals. With this radical which has two sets of coordinating oxygen atoms, it was possible to build a very peculiar spin system with a complex magnetic structure.     

A centralized model for creating shared,standardized, microsatellite data that simplifies inter-laboratory collaboration

We demonstrate an efficient model for standardizing microsatellite DNA data among laboratories studying Oncorhynchus mykiss. Eight laboratories standardized 13 microsatellite loci following allele nomenclature of a central laboratory (average inter-laboratory genotyping concordance >98%). Following this central model, we have currently standardized 298 alleles from throughout the species native range. Although we focus here on O. mykiss, our experiences and recommendation apply equally to other broadly distributed species that may benefit from multi-laboratory collaborative data collection.     

Synthesis and cytotoxicity of oligomycin A derivatives modified in the side chain

A novel way of chemical modification of the macrolide antibiotic oligomycin A (1) at the side chain was developed. Mesylation of 1 with methane sulfonyl chloride in the presence of 4-dimethylaminopyridine produced 33-O-mesyl oligomycin in 56% yield. Reactions of this intermediate with sodium azide produced the key derivative 33-azido-33-deoxy-oligomycin A in 60% yield. 1,3-Dipolar cycloaddition reaction with propiolic acid, methyl ester of propiolic acid, and phenyl acetylene resulted in 33-deoxy-33-(1,2,3-triazol-1-yl)oligomycin A derivatives substituted at N4 of the triazole cycle. The mesylated oligomycin A and 33-deoxy-33-azidooligomycin A did not inhibit F₀F₁ ATFase ATPase; however, 33-azido-33-deoxy-oligomycin A and the derivatives containing 4-phenyltriazole, 4-methoxycarbonyl-triazole and 3-dimethylaminoethyl amide of carboxyltriazole substituents demonstrated a high cytotoxicity against K562 leukemia and HCT116 human colon carcinoma cell lines whereas non-malignant skin fibroblasts were less sensitive to these compounds. Novel series of oligomycin A derivatives allow for the search of intracellular molecules beyond F₀F₁ ATP synthase relevant to the cytotoxic properties of this perspective chemical class.     

A new approach to the rapid determination of protein side chain conformations

Two efficient algorithms have been developed which allow amino acid side chain conformations to be optimized rapidly for a given peptide backbone conformation. Both these approaches are based on the assumption that each side chain can be represented by a small number of rotameric states. These states have been obtained by a dynamic cluster analysis of a large data base of known crystallographic structures. Successful applications of these algorithms to the prediction of known protein conformations are presented.     

A general method for identifying correct solutions in the quantitative analysis of gene conversion data

Past attempts to obtain values for meiotic parameters relating to hybrid DNA formation and the correction of mismatched bases in hybrid DNA have not given unique solutions unless various simplifying assumptions were made. A method is given for identifying correct sets of solutions after calculating the frequency of hybrid DNA formation at a heterozygous site and using the fact that closely linked sites within a locus have very similar hybrid DNA formation frequencies. The method is illustrated with simulated data and Sordaria fimicola data; it can also show up incorrect assumptions in analysis. A method is suggested for assessing the importance of double-strand gaps in producing conversions.     

Application of pattern recognition techniques to the analysis of protein crystal structure data. I. Characteristics of per-residue side chain contact frequency distributions

A statistical study of amino acid side chain contact interactions was carried out using a data set based on 36 protein structures. For each type of amino acid, a distribution of per-residue inter-side-chain contacts was obtained, over the observed span of zero to 11 contacts per residue. Significant observations included the following: 1) The mean number of inter-side-chain contacts is proportional to side chain surface area with the exception of Lys and Arg. 2) The mean number of contacts was greater for amino acids in beta-sheet relative to alpha-helical regions. 3) The more polar or surface-loving amino acids exhibited non-normal distributions, whereas distributions for the non-polar or interior-loving amino acids fell within accepted limits of normality.     

Studies on the Biosynthesis of the ergosterol side chain

1. A convenient synthesis of 24-methylene[23,25-(3)H(3)]dihydrolanosterol is described. 2. A general anaerobic-aerobic method for the incorporation of sterols into whole yeast cells is also described and illustrated by experiments with (3)H-labelled lanosterol. 3. The method was used to convert labelled 24-methylene-dihydrolanosterol into ergosterol, in good yield, by Saccharomyces cerevisiae. 4. Degradation of the biosynthetic ergosterol provided confirmation of the conversion, which supports the proposed mechanism for the biosynthesis of the ergosterol side chain. 5. Mechanisms for the further conversion of the 24-methylene side chain into the ergosterol side chain are discussed and it was shown that a compound, [3alpha-(3)H(1)]-ergost-7-en-3beta-ol, with a fully saturated side chain, can also be efficiently incorporated into ergosterol. 6. This result was confirmed by a procedure involving formation of the 5,8-epidioxide and subsequently the 5,8-epidioxy-22,23-epoxide of the biosynthetic ergosterol.